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RANG AND DALE’S

Pharmacology
 CHEMICAL TRANSMISSION AND DRUG ACTION IN THE CENTRAL NERVOUS SYSTEM 28

RANG AND DALE’S

Pharmacology
NINTH EDITION

JAMES M. RITTER DPhil FRCP HonFBPhS FMedSci

Emeritus Professor of Clinical Pharmacology, King’s College London
Fellow Commoner, Trinity Hall, Senior Physician Advisor CUC (GSK), Addenbrooke’s Hospital
Cambridge, United Kingdom

ROD FLOWER PhD LLD DSc HonFBPhS FMedSci FRS

Emeritus Professor of Pharmacology
Bart’s and the London School of Medicine
Queen Mary, University of London
London, United Kingdom

GRAEME HENDERSON PhD, FRSB, HonFBPhS

Professor of Pharmacology
University of Bristol
Bristol, United Kingdom

YOON KONG LOKE MBBS MD FRCP FBPhS

Professor of Medicine and Pharmacology
Norwich Medical School, University of East Anglia
Norwich, United Kingdom

DAVID MacEWAN PhD FRSB FBPhS SFHEA

Professor of Molecular Pharmacology/Toxicology & Head of Department
Department of Molecular and Clinical Pharmacology
University of Liverpool
Liverpool, United Kingdom

HUMPHREY P. RANG MB BS MSc MA DPhil HonFBPhS FMedSci FRS

Emeritus Professor of Pharmacology
University College London
London, United Kingdom

For additional online content visit StudentConsult.com

Edinburgh London New York Oxford Philadelphia St Louis Sydney 2020

© 2020, Elsevier Ltd. All rights reserved.

First edition 1987

Second edition 1991
Third edition 1995
Fourth edition 1999
Fifth edition 2003
Sixth edition 2007
Seventh edition 2012
Eighth edition 2016

The right of James M. Ritter, Rod Flower, Graeme Henderson, Yoon Kong Loke, David MacEwan, and
Humphrey P. Rang to be identified as authors of this work has been asserted by them in accordance with
the Copyright, Designs and Patents Act 1988.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
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This book and the individual contributions contained in it are protected under copyright by the
Publisher (other than as may be noted herein).

Potential Competing Financial Interests Statements for Rang and Dale 9E (2014–2018)

HPR: has no competing financial interests to declare.

JMR: has received salary from Quintiles and GSK.
GH: has no competing financial interests to declare.
YKL: has received funding from Polpharma and Thame Pharmaceuticals.
DJM: has no competing financial interests to declare.
RJF: serves as a board member for Antibe Therapeutics.

Notices
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and using any information, methods, compounds or experiments described herein. Because of rapid
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 Rang and Dale’s Pharmacology
Ninth Edition Preface
In this edition, as in its predecessors, we set out to explain the World Health Organization’s list of recommended
what drugs do in terms of the mechanisms by which they act. international non-proprietary names (rINN). Sometimes
This entails analysis not only at the cellular and molecular these conflict with the familiar names of drugs (e.g. the
level, where knowledge and techniques are advancing endogenous mediator prostaglandin I2 – the standard name
rapidly, but also at the level of physiological mechanisms in the scientific literature – becomes ‘epoprostenol’ – a name
and pathological disturbances. Pharmacology has its roots unfamiliar to most scientists – in the rINN list). In these
in therapeutics, where the aim is to ameliorate the effects cases, we generally adopt conventional scientific nomen-
of disease, so we have attempted to make the link between clature. Sometimes English and American usage varies (as
effects at the molecular and cellular level and the range of with adrenaline/epinephrine and noradrenaline/norepine-
beneficial and adverse effects that humans experience when phrine). Adrenaline and noradrenaline are the official names
drugs are used for therapeutic or other reasons. Therapeutic in EU member states and are used in this book.
agents have a high rate of obsolescence. In the decade 2008 Drug action can be understood only in the context of
to 2017, 301 new drugs gained regulatory approval for what else is happening in the body. So, at the beginning
use as therapeutic agents. The majority exploit the same of most chapters, we briefly discuss the physiological and
molecular targets as drugs already in use. Knowledge of biochemical processes relevant to the action of the drugs
the mechanisms of action of the class of drugs to which described in that chapter. We have included the chemical
a new agent belongs provides a good starting point for structures of drugs only where this information helps in
understanding and using a new compound intelligently. understanding their pharmacological and pharmacokinetic
Significantly, however, one-third of these new arrivals characteristics, since chemical structures are readily available
are ‘first-in-class’ drugs. That is, they act on novel molecular for reference online.
targets not previously exploited for therapeutic purposes, The overall organisation of the book has been retained,
and are therefore likely to produce effects not previously with sections covering: (1) the general principles of drug
described. Not all will succeed clinically, but some will action; (2) the chemical mediators and cellular mechanisms
stimulate the development of improved follow-up com- with which drugs interact in producing their therapeutic
pounds of the same type. Furthermore, about a quarter of effects; (3) the action of drugs on specific organ systems; (4)
the new compounds are ‘biopharmaceuticals’ – mainly the action of drugs on the nervous system; (5) the action of
proteins produced by bioengineering rather than synthetic drugs used to treat infectious diseases and cancer; and (6) a
chemistry. These are growing in importance as therapeutic range of special topics such as adverse effects, non-medical
agents, and generally have characteristics somewhat different uses of drugs, etc. This organisation reflects our belief that
from conventional drugs and are covered in a revised chapter. drug action needs to be understood, not just as a description
The very high rate of innovation in drug discovery is a recent of the effects of individual drugs and their uses, but as a
– and very welcome – change, due in large part to the rapid chemical intervention that perturbs the network of chemical
advances in molecular and cell biology that have stemmed and cellular signalling that underlies the function of any
from the sequencing of the human genome in 2003. We have living organism. In addition to updating each chapter, we
tried to strike a balance between the need to keep up with have added new material on biopharmaceuticals, and on
these modern developments and the danger of information personalised medicine, topics of particular current interest.
overload. Our emphasis is on explaining the general principles Additional current material on cognition-enhancing drugs
underlying drug action, which apply to old and new alike, has been included in Chapter 48.
and to describe in more detail the actions and mechanisms Despite the fact that pharmacology, like other branches
of familiar, established drugs, while including references of biomedical science, advances steadily, with the acquisition
that cover modern and future developments. of new information, the development of new concepts and
Pharmacology is a lively scientific discipline in its own the introduction of new drugs for clinical use, we have
right, with an importance beyond that of providing a basis avoided making the ninth edition any longer than its
for the use of drugs in therapy, and we aim to provide a predecessor by cutting out dated and obsolete material,
good background, not only for future doctors but also for and have made extensive use of small print text to cover
scientists in other disciplines who need to understand how more specialised and speculative information that is not
drugs act. We have, therefore, where appropriate, described essential to understanding the key message, but will, we
how drugs are used as probes for elucidating cellular and hope, be helpful to students seeking to go into greater
physiological functions, to improve our understanding of depth. In selecting new material for inclusion, we have
how the human body functions normally and what goes taken into account not only new agents but also recent
wrong with it in disease, even when the compounds have extensions of basic knowledge that presage further drug
no clinical use. Besides therapeutic applications, drugs have development. And, where possible, we have given a brief
other impacts on society, which we cover in chapters on outline of new treatments in the pipeline. Reference lists
psychoactive drugs, drug abuse, and the use of drugs in sport. are largely restricted to guidance on further reading, together
Names of drugs and related chemicals are established with review articles that list key original papers.
through usage and sometimes there is more than one name Finally, we hope that we have conveyed something of
in common use. For prescribing purposes, it is important our own enthusiasm for the science and importance of
to use standard names, and we follow, as far as possible, pharmacology in the modern world. xv
 RANG AND DALE’S PHARMACOLOGY NINTH EDITION PREFACE

We would like to put on record our appreciation of the

ACKNOWLEDGEMENTS team at Elsevier who worked on this edition: Alexandra
We are grateful to many colleagues who have helped us Mortimer (content strategist), Trinity Hutton (content
with comments and suggestions, and would particularly development specialist), Joanna Souch (project manager),
like to thank the following for their help and advice in the Nichole Beard (illustration manager).
preparation of this edition: Dr Steve Alexander, Professor
Emma Baker, Dr Barbara Jennings, Professor Eamonn Kelly, London, 2018
Professor Munir Pirmohamed and Professor Emma Rob- Humphrey P. Rang
inson. We would also like to thank Dr Christine Edmead James M. Ritter
for her work on the self-assessment questions which are Rod Flower
available as additional material on the online edition of Graeme Henderson
this book. David MacEwan
Yoon Kong Loke

xvi
 GENERAL PRINCIPLES SECTION 1

What is pharmacology? 1
doses. Botulinum toxin (Ch. 14) provides a striking example:
OVERVIEW it is the most potent poison known in terms of its lethal
dose, but is widely used both medically and cosmetically.
In this introductory chapter we explain how phar- General aspects of harmful effects of drugs are considered
macology came into being and evolved as a scientific in Chapter 58. Toxicology is the study of toxic effects of
discipline, and describe the present-day structure chemical substances (including drugs), and toxicological
of the subject and its links to other biomedical sciences. testing is undertaken on new chemical entities during their
The structure that has emerged forms the basis of development as potential medicinal products (Ch. 60), but
the organisation of the rest of the book. Readers in the subject is not otherwise covered in this book.
a hurry to get to the here-and-now of pharmacology
can safely skip this chapter.
ORIGINS AND ANTECEDENTS

WHAT IS A DRUG? Pharmacology can be defined as the study of the effects of

drugs on the function of living systems. As a science, it
For the purposes of this book, a drug can be defined as a was born in the mid-19th century, one of a host of new
chemical substance of known structure, other than a nutrient or biomedical sciences based on principles of experimentation
an essential dietary ingredient,1 which, when administered to a rather than dogma that came into being in that remarkable
living organism, produces a biological effect. period. Long before that – indeed from the dawn of civilisa-
A few points are worth noting. Drugs may be synthetic tion – herbal remedies were widely used, pharmacopoeias
chemicals, chemicals obtained from plants or animals, or were written, and the apothecaries’ trade flourished.
products of biotechnology (biopharmaceuticals). A medicine However, nothing resembling scientific principles was
is a chemical preparation, which usually, but not necessarily, applied to therapeutics, which was known at that time as
contains one or more drugs, administered with the intention materia medica.2 Even Robert Boyle, who laid the scientific
of producing a therapeutic effect. Medicines usually contain foundations of chemistry in the middle of the 17th century,
other substances (excipients, stabilisers, solvents, etc.) besides was content, when dealing with therapeutics (A Collection
the active drug, to make them more convenient to use. To of Choice Remedies, 1692), to recommend concoctions of
count as a drug, the substance must be administered as such, worms, dung, urine and the moss from a dead man’s skull.
rather than released by physiological mechanisms. Many The impetus for pharmacology came from the need to
substances, such as insulin or thyroxine, are endogenous improve the outcome of therapeutic intervention by doctors,
hormones but are also drugs when they are administered who were at that time skilled at clinical observation and
intentionally. Many drugs are not used commonly in diagnosis but broadly ineffectual when it came to treatment.3
medicine but are nevertheless useful research tools. The Until the late 19th century, knowledge of the normal and
definition of drug also covers toxins, which again are not abnormal functioning of the body was too rudimentary to
usually administered in the clinic but nonetheless are critical provide even a rough basis for understanding drug effects;
pharmacological tools. In everyday parlance, the word drug at the same time, disease and death were regarded as
is often associated with psychoactive substances and addic- semi-sacred subjects, appropriately dealt with by authoritar-
tion – unfortunate negative connotations that tend to bias ian, rather than scientific, doctrines. Clinical practice often
uninformed opinion against any form of chemical therapy. displayed an obedience to authority and ignored what
In this book we focus mainly on drugs used for therapeutic appear to be easily ascertainable facts. For example, cinchona
purposes but also describe psychoactive drugs and provide bark was recognised as a specific and effective treatment
important examples of drugs used as experimental tools. for malaria, and a sound protocol for its use was laid down
Poisons fall strictly within the definition of drugs, and by Lind in 1765. In 1804, however, Johnson declared it to
indeed ‘all drugs are poisons… it is only the dose which be unsafe until the fever had subsided, and he recommended
makes a thing poison’ (an aphorism credited to Paracelsus, instead the use of large doses of calomel (mercurous
a 16th century Swiss physician); conversely, poisons may be chloride) in the early stages – a murderous piece of advice
effective therapeutic agents when administered in sub-toxic that was slavishly followed for the next 40 years.

1 2
Like most definitions, this one has its limits. For example, there are a The name persists today in some ancient universities, being attached to
number of essential dietary constituents, such as iron and various chairs of what we would call clinical pharmacology.
3
vitamins, that are used as medicines. Furthermore, some biological Oliver Wendell Holmes, an eminent physician, wrote in 1860: ‘[I]
products (e.g. epoietin) show batch-to-batch variation in their chemical firmly believe that if the whole materia medica, as now used, could be
constitution that significantly affects their properties. There is also the sunk to the bottom of the sea, it would be all the better for mankind
study of pharmaceutical-grade nutrients or ‘nutraceuticals’. and the worse for the fishes’ (see Porter, 1997). 1
1 SECTION 1 General Principles

The motivation for understanding what drugs can and by Chain and Florey during the Second World War, based
cannot do came from clinical practice, but the science could on the earlier work of Fleming.
be built only on the basis of secure foundations in physiol- These few well-known examples show how the growth
ogy, pathology and chemistry. It was not until 1858 that of synthetic chemistry, and the resurgence of natural product
Virchow proposed the cell theory. The first use of a structural chemistry, caused a dramatic revitalisation of therapeutics
formula to describe a chemical compound was in 1868. in the first half of the 20th century. Each new drug class
Bacteria as a cause of disease were discovered by Pasteur that emerged gave pharmacologists a new challenge, and
in 1878. Previously, pharmacology hardly had the legs to it was then that pharmacology really established its identity
stand on, and we may wonder at the bold vision of Rudolf and its status among the biomedical sciences.
Buchheim, who created the first pharmacology institute In parallel with the exuberant proliferation of therapeutic
(in his own house) in Estonia in 1847. molecules – driven mainly by chemistry – which gave phar-
In its beginnings, before the advent of synthetic organic macologists so much to think about, physiology was also
chemistry, pharmacology concerned itself exclusively with making rapid progress, particularly in relation to chemical
understanding the effects of natural substances, mainly mediators, which are discussed in depth throughout this
plant extracts – and a few (mainly toxic) chemicals such book. Many hormones, neurotransmitters and inflammatory
as mercury and arsenic. An early development in chemistry mediators were discovered in this period, and the realisa-
was the purification of active compounds from plants. tion that chemical communication plays a central role in
Friedrich Sertürner, a young German apothecary, purified almost every regulatory mechanism that our bodies possess
morphine from opium in 1805. Other substances quickly immediately established a large area of common ground
followed, and, even though their structures were unknown, between physiology and pharmacology, for interactions
these compounds showed that chemicals, not magic or vital between chemical substances and living systems were exactly
forces, were responsible for the effects that plant extracts what pharmacologists had been preoccupied with from the
produced on living organisms. Early pharmacologists outset. Indeed, these fields have developed hand-in-hand
focused most of their attention on such plant-derived drugs as wherever there is either a physiological or pathological
as quinine, digitalis, atropine, ephedrine, strychnine and mechanism, pharmacology could be there to exploit it with
others (many of which are still used today and will have a drug. The concept of ‘receptors’ for chemical mediators,
become old friends by the time you have finished reading first proposed by Langley in 1905, was quickly taken up by
this book).4 pharmacologists such as Clark, Gaddum, Schild and others,
and is a constant theme in present-day pharmacology (as you
will soon discover as you plough through the next two chap-
PHARMACOLOGY IN THE 20TH AND ters). The receptor concept, and the technologies developed
21ST CENTURIES from it, have had a massive impact on drug discovery and
therapeutics. Biochemistry also emerged as a distinct science
Beginning in the 20th century, the fresh wind of synthetic early in the 20th century, and the discovery of enzymes and
chemistry began to revolutionise the pharmaceutical the delineation of biochemical pathways provided yet another
industry, and with it the science of pharmacology. New framework for understanding drug effects. The picture of
synthetic drugs, such as barbiturates and local anaesthetics, pharmacology that emerges from this brief glance at history
began to appear, and the era of antimicrobial chemotherapy (Fig. 1.1) is of a subject evolved from ancient prescientific
began with the discovery by Paul Ehrlich in 1909 of arsenical therapeutics, involved in commerce from the 17th century
compounds for treating syphilis. Around the same time, onwards, and which gained respectability by donning the
William Blair-Bell was world renowned for his pioneering trappings of science as soon as this became possible in the
work at Liverpool in the treatment of breast cancers with mid-19th century. Pharmacology grew rapidly in partnership
another relatively poisonous agent, lead colloid mixtures. with the evolution of organic chemistry and other biomedical
The thinking was that yes, drugs were toxic, but they were sciences, and was quick to assimilate the dramatic advances
slightly more toxic to a microbe or cancer cell. This early in molecular and cell biology in the late 20th century. Signs
chemotherapy has laid the foundations for much of the of its carpetbagger past still cling to pharmacology, for the
antimicrobial and anticancer therapies still used today. pharmaceutical industry has become very big business and
Further breakthroughs came when the sulfonamides, the much pharmacological research nowadays takes place in a
first antibacterial drugs, were discovered by Gerhard commercial environment, a rougher and more pragmatic
Domagk in 1935, and with the development of penicillin place than academia.5 No other biomedical ‘ology’ is so close
to Mammon.
ALTERNATIVE THERAPEUTIC PRINCIPLES
4
A handful of synthetic substances achieved pharmacological Modern medicine relies heavily on drugs as the main
prominence long before the era of synthetic chemistry began. Diethyl
ether, first prepared as ‘sweet oil of vitriol’ in the 16th century, and tool of therapeutics. Other therapeutic procedures, such
nitrous oxide, prepared by Humphrey Davy in 1799, were used to liven
up parties before being introduced as anaesthetic agents in the mid-19th
5
century (see Ch. 42). Amyl nitrite (see Ch. 21) was made in 1859 and Some of our most distinguished pharmacological pioneers made their
can claim to be the first ‘rational’ therapeutic drug; its therapeutic effect careers in industry: for example, Henry Dale, who laid the foundations
in angina was predicted on the basis of its physiological effects – a true of our knowledge of chemical transmission and the autonomic nervous
‘pharmacologist’s drug’ and the smelly forerunner of the system (Ch. 13); George Hitchings and Gertrude Elion, who described
nitrovasodilators that are widely used today. Aspirin (Ch. 27), the most the antimetabolite principle and produced the first effective anticancer
widely used therapeutic drug in history, was first synthesised in 1853, drugs (Ch. 57); and James Black, who introduced the first
with no therapeutic application in mind. It was rediscovered in 1897 in β-adrenoceptor and histamine H2-receptor antagonists (Chs 15 and 31).
the laboratories of the German company Bayer, who were seeking a less It is no accident that in this book, where we focus on the scientific
toxic derivative of salicylic acid. Bayer commercialised aspirin in 1899 principles of pharmacology, most of our examples are products of
2 and made a fortune. industry, not of nature.
 What is pharmacology? 1
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Fig. 1.1 The development of pharmacology.

as surgery, diet, exercise, psychological treatments etc., are terms, detected by objective means, and influenced benefi-
also important, of course, as is deliberate non-intervention, cially by appropriate chemical or physical interventions.
but none is so widely applied as drug-based therapeutics. They focus instead mainly on subjective malaise, which
Before the advent of science-based approaches, repeated may be disease-associated or not. Abandoning objectivity
attempts were made to construct systems of therapeutics, in defining and measuring disease goes along with a similar
many of which produced even worse results than pure departure from scientific principles in assessing therapeutic
empiricism. One of these was allopathy, espoused by James efficacy and risk, with the result that principles and practices
Gregory (1735–1821). The favoured remedies included can gain acceptance without satisfying any of the criteria
bloodletting, emetics and purgatives, which were used until of validity that would convince a critical scientist, and that
the dominant symptoms of the disease were suppressed. are required by law to be satisfied before a new drug can
Many patients died from such treatment, and it was in be introduced into therapy. Demand for ‘alternative’
reaction against it that Hahnemann introduced the practice therapies by the general public, alas, has little to do with
of homeopathy in the early 19th century. The implausible demonstrable efficacy.6
guiding principles of homeopathy are:
THE EMERGENCE OF BIOTECHNOLOGY
• like cures like
Since the 1980s, biotechnology has emerged as a major
• activity can be enhanced by dilution
source of new therapeutic agents in the form of antibodies,
The system rapidly drifted into absurdity: for example, enzymes and various regulatory proteins, including hor-
Hahnemann recommended the use of drugs at dilutions mones, growth factors and cytokines (see Clark & Pazdernik,
of 1 : 1060, equivalent to one molecule in a sphere the size 2015). Although such products (known as biopharmaceuticals,
of the orbit of Neptune. biologicals or biologics) are generally produced by genetic
Many other systems of therapeutics have come and gone, engineering rather than by synthetic chemistry, the
and the variety of dogmatic principles that they embodied pharmacological principles are essentially the same as for
have tended to hinder rather than advance scientific pro- conventional drugs, although the details of absorption,
gress. Currently, therapeutic systems that have a basis that
lies outside the domain of science remain popular under
6
the general banner of ‘alternative’ or ‘complementary’ The UK Medicines and Healthcare Regulatory Agency (MHRA)
requires detailed evidence of therapeutic efficacy based on controlled
medicine. Mostly, they reject the ‘medical model’, which clinical trials before a new drug is registered, but no clinical trials data
attributes disease to an underlying derangement of normal for homeopathic products or for the many herbal medicines that were
function that can be defined in physiological or structural on sale before the Medicines Act of 1968. 3
1 SECTION 1 General Principles

distribution and elimination, specificity, harmful effects pharmacokinetics, etc.), which are convenient, if not water-
and clinical effectiveness all differ markedly between high tight, subdivisions. These topics form the main subject
molecular-weight biopharmaceuticals and low molecular- matter of this book. Around the edges are several interface
weight drugs – as does their cost! Looking further ahead, disciplines, not covered in this book, which form important
gene- and cell-based therapies (Ch. 5), although still in two-way bridges between pharmacology and other fields of
their infancy, are beginning to take therapeutics into a new biomedicine. Pharmacology tends to have more of these than
domain. The principles governing gene suppression, the other disciplines. Recent arrivals on the fringe are subjects
design, delivery and control of functioning artificial genes such as pharmacogenomics, pharmacoepidemiology and
introduced into cells, or of engineered cells introduced into pharmacoeconomics.
the body, are very different from those of drug-based Pharmacogenomics. Pharmacogenetics, the study of
therapeutics and will require a different conceptual frame- genetic influences on responses to drugs, initially focused
work, which texts such as this will increasingly need to on familial idiosyncratic drug reactions, where affected
embrace if they are to stay abreast of modern medical individuals show an abnormal – usually adverse – response
treatment. to a class of drug (see Nebert & Weber, 1990). Rebranded
as pharmacogenomics, it now covers broader genetically
PHARMACOLOGY TODAY based variations in drug response, where the genetic basis
As with other biomedical disciplines, the boundaries of is more complex, the aim being to use genetic information
pharmacology are not sharply defined, nor are they constant. to guide the choice of drug therapy on an individual basis
Its exponents are, as befits pragmatists, ever ready to poach – so-called personalised medicine (Ch. 12). The underlying
on the territory and techniques of other disciplines. If it principle is that differences between individuals in their
ever had a conceptual and technical core that it could really response to therapeutic drugs can be predicted from their
call its own, this has now dwindled almost to the point of genetic make-up. Examples that confirm this are steadily
extinction, and the subject is defined by its purpose – to accumulating (see Ch. 12). So far, they mainly involve genetic
understand what drugs do to living organisms, and more polymorphism of drug-metabolising enzymes or receptors.
particularly how their effects can be applied to therapeutics Ultimately, linking specific gene variations with variations
– rather than by its scientific coherence. in therapeutic or unwanted effects of a particular drug
Fig. 1.2 shows the structure of pharmacology as it should enable the tailoring of therapeutic choices on the
appears today. Within the main subject fall a number of basis of an individual’s genotype. Steady improvements
compartments (neuropharmacology, immunopharmacology, in the cost and feasibility of individual genotyping will

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Fig. 1.2 Pharmacology today with its various subdivisions. The grey box contains the general areas of pharmacology covered in this
book. Interface disciplines (brown boxes) link pharmacology to other mainstream biomedical disciplines (green boxes).
4
 What is pharmacology? 1
increase its applicability, potentially with far-reaching Pharmacoeconomics. This branch of health economics
consequences for therapeutics (see Ch. 12). aims to quantify in economic terms the cost and benefit of
Pharmacoepidemiology. This is the study of drug effects drugs used therapeutically. It arose from the concern of
at the population level (see Strom et al., 2013). It is concerned many governments to provide for healthcare from tax
with the variability of drug effects between individuals in revenues, raising questions of what therapeutic procedures
a population, and between populations. It is an increasingly represent the best value for money. This, of course, raises
important topic in the eyes of the regulatory authorities fierce controversy, because it ultimately comes down to
who decide whether or not new drugs can be licensed for putting monetary value on health and longevity. As with
therapeutic use. Variability between individuals or popula- pharmacoepidemiology, regulatory authorities are increas-
tions detracts from the utility of a drug, even though its ingly requiring economic analysis, as well as evidence of
overall effect level may be satisfactory. Pharmacoepide- individual benefit, when making decisions on licensing.
miological studies also take into account patient compliance For more information on this complex subject, see Rascati
and other factors that apply when the drug is used under (2013).
real-life conditions.

REFERENCES AND FURTHER READING

Clark, D.P., Pazderink, N.J., 2015. Biotechnology. Elsevier, New York. Rascati, K.L., 2013. Essentials of Pharmacoeconomics, second ed.
(General account of biotechnology and its potential applications) Lippincott Williams & Wilkins, Philadelphia. (Introduction to a complex
Nebert, D.W., Weber, W.W., 1990. Pharmacogenetics. In: Pratt, W.B., and fraught subject)
Taylor, P. (Eds.), Principles of Drug Action, third ed. Churchill Strom, B.L., Kimmel, S.E., Hennessy, S., 2013. Textbook of
Livingstone, New York. (A detailed account of genetic factors that affect Pharmacoepidemiology, second ed. Wiley, Chichester. (A multiauthor
responses to drugs, with many examples from the pregenomic literature) book covering all aspects of a newly emerged discipline, including aspects of
Porter, R., 1997. The Greatest Benefit to Mankind. Harper-Collins, pharmacoeconomics)
London. (An excellent and readable account of the history of medicine, with
good coverage of the early development of pharmacology and the
pharmaceutical industry)

5
 SECTION 1 GENERAL PRINCIPLES

2 How drugs act: general principles

OVERVIEW These critical binding sites are often referred to as ‘drug

targets’ (an obvious allusion to Ehrlich’s famous phrase
The emergence of pharmacology as a science came ‘magic bullets’, describing the potential of antimicrobial
when the emphasis shifted from describing what drugs drugs). The mechanisms by which the association of a drug
do to explaining how they work. In this chapter we molecule with its target leads to a physiological response
set out some general principles underlying the constitute the major thrust of pharmacological research.
interaction of drugs with living systems (Ch. 3 goes Most drug targets are protein molecules. Even general
into the molecular aspects in more detail). The anaesthetics (see Ch. 42), which were long thought to
interaction between drugs and cells is described, produce their effects by an interaction with membrane lipid,
followed by a more detailed examination of different now appear to interact mainly with membrane proteins
types of drug–receptor interaction. The receptor concept (see Franks, 2008).
has been described as the ‘big idea’ of pharmacology All rules need exceptions, and many antimicrobial and
(Rang, 2006) and will be a recurring theme throughout antitumour drugs (Chs 52 and 57), as well as mutagenic
this book. and carcinogenic agents (Ch. 58), interact directly with DNA
rather than protein; bisphosphonates, used to treat
osteoporosis (Ch. 37), bind to calcium salts in the bone
INTRODUCTION matrix, rendering them toxic to osteoclasts, much like rat
poison. There are also exceptions among the new generation
To begin with, we should gratefully acknowledge Paul of biopharmaceutical drugs that include nucleic acids, proteins
Ehrlich for insisting that drug action must be explicable in and antibodies (see Ch. 5).
terms of conventional chemical interactions between drugs
and tissues, and for dispelling the idea that the remarkable
potency and specificity of action of some drugs put them PROTEIN TARGETS FOR DRUG BINDING
somehow out of reach of chemistry and physics and required
the intervention of magical ‘vital forces’. Although many Four main kinds of regulatory protein are commonly
drugs produce effects in extraordinarily low doses and involved as primary drug targets, namely:
concentrations, low concentrations still involve very large
• receptors
numbers of molecules. One drop of a solution of a drug at
• enzymes
only 10−10 mol/L still contains about 3 × 109 drug molecules,
• carrier molecules (transporters)
so there is no mystery in the fact that it may produce an
• ion channels
obvious pharmacological response. Some bacterial toxins
(e.g. diphtheria toxin) act with such precision that a single Furthermore, many drugs bind (in addition to their primary
molecule taken up by a target cell is sufficient to kill it. targets) to plasma proteins (see Ch. 9) and other tissue
One of the basic tenets of pharmacology is that drug proteins, without producing any obvious physiological
molecules must exert some chemical influence on one or effect. Nevertheless, the generalisation that most drugs act
more cell constituents in order to produce a pharmacological on one or other of the four types of protein listed above
response. In other words, drug molecules must get so close serves as a good starting point.
to these constituent cellular molecules that the two interact Further discussion of the mechanisms by which
chemically in such a way that the function of the latter is such binding leads to cellular responses is given in
altered. Of course, the molecules in the organism vastly Chapters 3–4.
outnumber the drug molecules, and if the drug molecules
were merely distributed at random, the chance of interaction DRUG RECEPTORS
with any particular class of cellular molecule would be
negligible. Therefore pharmacological effects require, in WHAT DO WE MEAN BY RECEPTORS?
general, the non-uniform distribution of the drug molecule ▼ As emphasised in Chapter 1, the concept of receptors is central
within the body or tissue, which is the same as saying that to pharmacology, and the term is most often used to describe the
drug molecules must be ‘bound’ to particular constituents target molecules through which soluble physiological mediators –
hormones, neurotransmitters, inflammatory mediators, etc. – produce
of cells and tissues in order to produce an effect. Ehrlich
their effects. Examples such as acetylcholine receptors, cytokine
summed it up thus: ‘Corpora non agunt nisi fixata’ (in this receptors, steroid receptors and growth hormone receptors abound
context, ‘A drug will not work unless it is bound’).1 in this book, and generally the term receptor indicates a recognition
molecule for a chemical mediator through which a response is
1 transduced.
There are, if one looks hard enough, exceptions to Ehrlich’s dictum –
drugs that act without being bound to any tissue constituent (e.g. osmotic ‘Receptor’ is sometimes used to denote any target molecule with
diuretics, osmotic purgatives, antacids and heavy metal chelating agents). which a drug molecule (i.e. a foreign compound rather than an
6 Nonetheless, the principle remains true for the great majority. endogenous mediator) has to combine in order to elicit its specific
 How drugs act: general principles 2
influence even when no chemical mediator is present
Targets for drug action (see p. 14).
There is an important distinction between agonists, which
• A drug is a chemical applied to a physiological system ‘activate’ the receptors, and antagonists, which combine at
that affects its function in a specific way. the same site without causing activation, and block the
• With some exceptions, drugs act on target proteins, effect of agonists on that receptor. The distinction between
namely: agonists and antagonists only exists for pharmacological
– receptors receptors; we cannot usefully speak of ‘agonists’ for the
– enzymes other classes of drug target described above.
– carriers The characteristics and accepted nomenclature of
– ion channels. pharmacological receptors are described by Neubig et al.
• The term receptor is used in different ways. In (2003). The origins of the receptor concept and its
pharmacology, it describes protein molecules whose pharmacological significance are discussed by Rang (2006).
function is to recognise and respond to endogenous DRUG SPECIFICITY
chemical signals. Other macromolecules with which
drugs interact to produce their effects are known as
For a drug to be useful as either a therapeutic or a scientific
tool, it must act selectively on particular cells and tissues.
drug targets.
In other words, it must show a high degree of binding site
• Specificity is reciprocal: individual classes of drug bind
specificity. Conversely, proteins that function as drug targets
only to certain targets, and individual targets recognise generally show a high degree of ligand specificity; they
only certain classes of drug. bind only molecules of a certain precise type.
• No drugs are completely specific in their actions. In These principles of binding site and ligand specificity
many cases, increasing the dose of a drug will cause it can be clearly recognised in the actions of a mediator such
to affect targets other than the principal one, and this as angiotensin (Ch. 23). This peptide acts strongly on
can lead to side effects. vascular smooth muscle, and on the kidney tubule, but has
very little effect on other kinds of smooth muscle or on the
intestinal epithelium. Other mediators affect a quite different
spectrum of cells and tissues, the pattern in each case
reflecting the specific pattern of expression of the protein
effect. For example, the voltage-sensitive sodium channel is sometimes
referred to as the ‘receptor’ for local anaesthetics (see Ch. 44), or the
receptors for the various mediators. A small chemical
enzyme dihydrofolate reductase as the ‘receptor’ for methotrexate change, such as conversion of one of the amino acids in
(Ch. 51). The term drug target, of which receptors are one type, is angiotensin from L to D form, or removal of one amino
preferable in this context. acid from the chain, can inactivate the molecule altogether,
In the more general context of cell biology, the term receptor is used because the receptor fails to bind the altered form. The
to describe various cell surface molecules (such as T-cell receptors, complementary specificity of ligands and binding sites,
integrins, Toll receptors, etc; see Ch. 7) involved in the cell-to-cell which gives rise to the very exact molecular recognition
interactions that are important in immunology, cell growth, migration properties of proteins, is central to explaining many of the
and differentiation, some of which are also emerging as drug targets. phenomena of pharmacology. It is no exaggeration to say
These receptors differ from conventional pharmacological receptors that the ability of proteins to interact in a highly selective
in that they respond to proteins attached to cell surfaces or extracellular
way with other molecules – including other proteins – is
structures, rather than to soluble mediators.
the basis of living machines. Its relevance to the understand-
Various carrier proteins are often referred to as receptors, such as
ing of drug action will be a recurring theme in this book.
the low-density lipoprotein receptor that plays a key role in lipid
metabolism (Ch. 24) and the transferrin receptor involved in iron
Finally, it must be emphasised that no drug acts with
absorption (Ch. 26). These entities have little in common with complete specificity. Thus tricyclic antidepressant drugs
pharmacological receptors. Though quite distinct from pharmacological (Ch. 48) act by blocking monoamine transporters but are
receptors, these proteins play an important role in the action of drugs notorious for producing side effects (e.g. dry mouth) related
such as statins (Ch. 24). to their ability to block various other receptors. In general,
the lower the potency of a drug and the higher the dose
RECEPTORS IN PHYSIOLOGICAL SYSTEMS needed, the more likely it is that sites of action other than
Receptors form a key part of the system of chemical com- the primary one will assume significance. In clinical terms,
munication that all multicellular organisms use to coordinate this is often associated with the appearance of unwanted
the activities of their cells and organs. Without them, we ‘off-target’ side effects,2 of which no drug is free.
would be unable to function. Since the 1970s, pharmacological research has succeeded
Some fundamental properties of receptors are illus- in identifying the protein targets of many different types
trated by the action of adrenaline (epinephrine) on the of drug. Drugs such as opioid analgesics (Ch. 43), can-
heart. Adrenaline first binds to a receptor protein (the β1 nabinoids (Ch. 20) and benzodiazepine tranquillisers (Ch.
adrenoceptor, see Ch. 15) that serves as a recognition site 45), whose actions had been described in exhaustive detail
for adrenaline and other catecholamines. When it binds for many years, are now known to target well-defined
to the receptor, a train of reactions is initiated (see Ch. 3), receptors, many of which have been fully characterised by
leading to an increase in force and rate of the heartbeat. In
the absence of adrenaline, the receptor is normally function-
ally silent. This is true of most receptors for endogenous 2
‘On-target’ side effects are unwanted effects mediated through the
mediators (hormones, neurotransmitters, cytokines, etc.), same receptor as the clinically desired effect, for example constipation
although there are examples (see Ch. 3) of receptors that and respiratory depression by opioid analgesic drugs (see Ch. 43),
are ‘constitutively active’ – that is, they exert a controlling whereas ‘off target’ side effects are mediated by a different mechanism. 7
2 SECTION 1   General Principles

gene-cloning and protein crystallography techniques (see

Ch. 3). Occupation Activation
governed governed
RECEPTOR CLASSIFICATION by by
▼ Where the action of a drug can be associated with a particular affinity efficacy
receptor, this provides a valuable means for classification and refine-
Drug k+1 β
ment in drug design. For example, pharmacological analysis of the
actions of histamine (see Ch. 18) showed that some of its effects (the A + R AR AR* RESPONSE
(agonist) k-1 α
H1 effects, such as smooth muscle contraction) were strongly antago-
nised by the competitive histamine antagonists then known. Black
and his colleagues suggested in 1970 that the remaining actions of
histamine, which included its stimulant effect on gastric secretion, Drug k+1
might represent a second class of histamine receptor (H2). Testing a B + R BR NO RESPONSE
number of histamine analogues, they found that some were selective (antagonist) k-1
in producing H2 effects, with little H1 activity. By analysing which
parts of the histamine molecule conferred this type of specificity,
Fig. 2.1 The distinction between drug binding and
they were able to develop selective H2 antagonists, which proved to
receptor activation. Ligand A is an agonist, because when it is
be potent in blocking gastric acid secretion, a development of major
bound, the receptor (R) tends to become activated, whereas
therapeutic significance (Ch. 31).3 Two further types of histamine
receptor (H3 and H4) were recognised later. ligand B is an antagonist, because binding does not lead to
activation. It is important to realise that for most drugs, binding
Receptor classification based on pharmacological responses continues
and activation are reversible, dynamic processes. The rate
to be a valuable and widely used approach. Subsequently, newer
experimental approaches produced other criteria on which to base constants k+1, k−1, α and β for the binding, unbinding and
receptor classification. The direct measurement of ligand binding to activation steps vary between drugs. For an antagonist, which
receptors (see later) allowed many new receptor subtypes to be defined does not activate the receptor, β = 0.
that could not easily be distinguished by studies of drug effects.
Molecular sequencing of the amino acid structure (see Ch. 3) provided
a completely new basis for classification at a much finer level of detail
than can be reached through pharmacological analysis. Finally, analysis
of the biochemical pathways that are linked to receptor activation receptors is governed by its affinity, whereas the tendency
(see Ch. 3) provides yet another basis for classification.
for it, once bound, to activate the receptor is denoted by
The result of this data explosion was that receptor classification sud-
its efficacy. These terms are defined more precisely later
denly became much more detailed, with a proliferation of receptor
subtypes for all the main types of ligand. As alternative molecular and
(pp. 9 and 11). Drugs of high potency generally have a
biochemical classifications began to spring up that were incompatible high affinity for the receptors and thus occupy a significant
with the accepted pharmacologically defined receptor classes, the proportion of the receptors even at low concentrations.
International Union of Basic and Clinical Pharmacology (IUPHAR) Agonists also possess significant efficacy, whereas antago-
convened expert working groups to produce agreed receptor classifica- nists, in the simplest case, have zero efficacy. Drugs with
tions for the major types, taking into account the pharmacological, intermediate levels of efficacy, such that even when 100%
molecular and biochemical information available. These wise people of the receptors are occupied the tissue response is sub-
have a hard task; their conclusions will be neither perfect nor final but maximal, are known as partial agonists, to distinguish them
are essential to ensure a consistent terminology. To the student, this from full agonists, the efficacy of which is sufficient that
may seem an arcane exercise in taxonomy, generating much detail
they can elicit a maximal tissue response. These concepts,
but little illumination. There is a danger that the tedious lists of drug
names, actions and side effects that used to burden the subject will be
though clearly an oversimplified description of events at
replaced by exhaustive tables of receptors, ligands and transduction the molecular level (see Ch. 3), provide a useful basis for
pathways. In this book, we have tried to avoid detail for its own sake characterising drug effects.
and include only such information on receptor classification as seems We now discuss certain aspects in more detail, namely
interesting in its own right or is helpful in explaining the actions drug binding, agonist concentration–effect curves, competi-
of important drugs. A comprehensive database of known receptor tive antagonism, partial agonists and the nature of efficacy.
classes is available (see <www.guidetopharmacology.org/>), as well Understanding these concepts at a qualitative level is
as a regularly updated summary (Alexander et al., 2015). sufficient for many purposes, but for more detailed analysis
DRUG–RECEPTOR INTERACTIONS a quantitative formulation is needed (see pp. 19–20).

Occupation of a receptor by a drug molecule may or may THE BINDING OF DRUGS TO RECEPTORS
not result in activation of the receptor. By activation, we ▼ The binding of drugs to receptors can often be measured directly
mean that the receptor is affected by the bound molecule by the use of drug molecules (agonists or antagonists) labelled with
in such a way as to alter the function of the cell and elicit one or more radioactive atoms (usually 3H, 14C or 125I). The usual
a tissue response. The molecular mechanisms associated procedure is to incubate samples of the tissue (or membrane fragments)
with receptor activation are discussed in Chapter 3. Binding with various concentrations of radioactive drug until equilibrium is
and activation represent two distinct steps in the generation reached (i.e. when the rate of association [binding] and dissociation
[unbinding] of the radioactive drug are equal). The bound radioactivity
of the receptor-mediated response by an agonist (Fig. 2.1).
is measured after removal of the supernatant.
If a drug binds to the receptor without causing activation
In such experiments, the radiolabelled drug will exhibit both specific
and thereby prevents the agonist from binding, it is termed
binding (i.e. binding to receptors, which is saturable as there are a
a receptor antagonist. The tendency of a drug to bind to the finite number of receptors in the tissue) and a certain amount of
‘non-specific binding’ (i.e. drug taken up by structures other than
3
For this work, and the development of β-adrenoceptor antagonists by a receptors, which, at the concentrations used in such studies, is normally
similar experimental approach, Sir James Black was awarded the 1984 non-saturable), which obscures the specific component and needs
8 Nobel Prize in Physiology or Medicine. to be kept to a minimum (Fig. 2.2A–B). The amount of non-specific
 How drugs act: general principles 2
A
(i) Radioactive drug binds to specific (ii) Increasing concentration of radioactive (iii) Excess non-radioactive drug displaces
and non-specific sites drug saturates specific sites radioactive drug from specific sites

Specific binding to receptor

R R R

B C D

300 100 100

Bmax Bmax

Specifically bound (fmol/mg)

Specifically bound (fmol/mg)

Total bound (fmol/mg)

Total

K
Non-specific

0 0 0
0 5 10 15 20 0 5 10 15 20 0.001 0.01 0.1 1 10 100
Concentration (nmol/L) Concentration (nmol/L) Concentration (nmol/L, log scale)

Fig. 2.2 Measurement of receptor binding. (A) (i) Cartoon depicting radioligand (shown in red) binding to its receptor (R) in the
membrane as well as to non-specific sites on other proteins and lipid. In (ii) when the concentration of radioligand is increased all the
specific sites become saturated but non-specific binding continues to increase. In (iii) addition of a high concentration of a non-radioactive
drug (shown in green) that also binds to R displaces the radioactive drug from its receptors but not from the non-specific sites. (B–D)
Illustrate actual experimental results for radioligand binding to β adrenoceptors in cardiac cell membranes. The ligand was
[3H]-cyanopindolol, a derivative of pindolol (see Ch. 15). (B) Measurements of total and non-specific binding at equilibrium. Non-specific
binding is measured in the presence of a saturating concentration of a non-radioactive β-adrenoceptor agonist, which prevents the
radioactive ligand from binding to β adrenoceptors. The difference between the two lines represents specific binding. (C) Specific binding
plotted against concentration. The curve is a rectangular hyperbola (Eq. 2.5). (D) Specific binding as in (C) plotted against the concentration
on a log scale. The sigmoid curve is a logistic curve representing the logarithmic scaling of the rectangular hyperbola plotted in panel (C)
from which the binding parameters K (the equilibrium dissociation constant) and Bmax (the binding capacity) can be determined.

binding is estimated by measuring the radioactivity taken up in the Non-invasive imaging techniques, such as positron emission tomography
presence of a saturating concentration of a (non-radioactive) ligand (PET), using drugs labelled with an isotope of short half-life (such
that inhibits completely the binding of the radioactive drug to the as 11C or 18Fl), can also be used to investigate the distribution of
receptors, leaving behind the non-specific component. This is then receptors in structures such as the living human brain. This technique
subtracted from the total binding to give an estimate of specific binding has been used, for example, to measure the degree of dopamine-
(Fig. 2.2C). The binding curve (Fig. 2.2C–D) defines the relationship receptor blockade produced by antipsychotic drugs in the brains of
between concentration and the amount of drug bound (B), and in schizophrenic patients (see Ch. 47).
most cases it fits well to the relationship predicted theoretically (see Binding curves with agonists often reveal an apparent heterogeneity
Fig. 2.14), allowing the affinity of the drug for the receptors to be among receptors. For example, agonist binding to muscarinic receptors
estimated, as well as the binding capacity (Bmax), representing the (Ch. 14) and also to β adrenoceptors (Ch. 15) suggests at least two
density of receptors in the tissue. When combined with functional populations of binding sites with different affinities. This may be
studies, binding measurements have proved very valuable. It has, because the receptors can exist either unattached or coupled within
for example, been confirmed that the spare receptor hypothesis (p. 10) the membrane to another macromolecule, the G protein (see Ch. 3),
for muscarinic receptors in smooth muscle is correct; agonists are which constitutes part of the transduction system through which the
found to bind, in general, with rather low affinity, and a maximal receptor exerts its regulatory effect. Antagonist binding does not show
biological effect occurs at low receptor occupancy. It has also been this complexity, probably because antagonists, by their nature, do
shown, in skeletal muscle and other tissues, that denervation leads not lead to the secondary event of G protein coupling. Because agonist
to an increase in the number of receptors in the target cell, a finding binding results in activation, agonist affinity has proved to be a surpris-
that accounts, at least in part, for the phenomenon of denervation ingly elusive concept, about which aficionados love to argue.
supersensitivity. More generally, it appears that receptors tend to
increase in number, usually over the course of a few days, if the THE RELATION BETWEEN DRUG CONCENTRATION
relevant hormone or transmitter is absent or scarce, and to decrease
AND EFFECT
in number if the receptors are activated for a prolonged period, a
process of adaptation to continued administration of drugs or hormones Although binding can be measured directly, it is usually
(see p. 18). a biological response, such as a rise in blood pressure, 9
2 SECTION 1   General Principles

by the use of recombinant receptors expressed in cells in

100 culture. Thus, even if the concentration–effect curve, as in
Histamine Fig. 2.3, looks just like a facsimile of the binding curve (see
(guinea pig heart) Fig. 2.2D), it cannot be used directly to determine the affinity
of the agonist for the receptors.
Response (% max)

Acetylcholine
(frog rectus muscle) SPARE RECEPTORS
50 ▼ Stephenson (1956), studying the actions of acetylcholine analogues
in isolated tissues, found that many full agonists were capable of
eliciting maximal responses at very low occupancies, often less than
1%. This means that the mechanism linking the response to receptor
occupancy has a substantial reserve capacity. Such systems may be
said to possess spare receptors, or a receptor reserve. The existence of
spare receptors does not imply any functional subdivision of the
0
receptor pool, but merely that the pool is larger than the number
10-7 10-6 10-5 10-4 10-3 10-2 needed to evoke a full response. This surplus of receptors over the
Concentration (mol/L) number actually needed might seem a wasteful biological arrangement.
But in fact it is highly efficient in that a given number of agonist–
Fig. 2.3 Experimentally observed concentration–effect receptor complexes, corresponding to a given level of biological
curves. Although the lines, drawn according to the binding Eq. response, can be reached with a lower concentration of hormone or
2.5, fit the points well, such curves do not give correct estimates neurotransmitter than would be the case if fewer receptors were
of the affinity of drugs for receptors. This is because the provided. Economy of hormone or transmitter secretion is thus
relationship between receptor occupancy and response is achieved at the expense of providing more receptors.
usually non-linear.
COMPETITIVE ANTAGONISM
Though one drug can inhibit the response to another in
several ways (see p. 16), competition at the receptor level
contraction or relaxation of a strip of smooth muscle in an is particularly important, both in the laboratory and in the
organ bath, the activation of an enzyme, or a behavioural clinic, because of the high potency and specificity that can
response, that we are interested in, and this is often plotted be achieved.
as a concentration–effect curve (in vitro) or dose–response curve In the presence of a competitive antagonist, the agonist
(in vivo), as in Fig. 2.3. This allows us to estimate the maximal occupancy (i.e. proportion of receptors to which the agonist
response that the drug can produce (Emax), and the concentra- is bound) at a given agonist concentration is reduced,
tion or dose needed to produce a 50% maximal response because the receptor can accommodate only one molecule
(EC50 or ED50). A logarithmic concentration or dose scale at a time. However, because the two are in competition,
is often used. This transforms the curve from a rectangular raising the agonist concentration can restore the agonist
hyperbola to a sigmoidal curve in which the mid portion occupancy (and hence the tissue response). The antago-
is essentially linear (the importance of the slope of the nism is therefore said to be surmountable, in contrast to
linear portion will become apparent later in this chapter other types of antagonism (see later) where increasing the
when we consider antagonism and partial agonists). The agonist concentration fails to overcome the blocking effect.
Emax, EC50 and slope parameters are useful for comparing A simple theoretical analysis (see p. 20) predicts that in
different drugs that produce qualitatively similar effects the presence of a fixed concentration of the antagonist,
(see Fig. 2.7 and Ch. 8). Although they look similar to the the log concentration–effect curve for the agonist will
binding curve in Fig. 2.2D, concentration–effect curves be shifted to the right, without any change in slope or
cannot be used to measure the affinity of agonist drugs for maximum – the hallmark of competitive antagonism (Fig.
their receptors, because the response produced is not, as 2.4A). The shift is expressed as a dose ratio, r (the ratio by
a rule, directly proportional to receptor occupancy. This which the agonist concentration has to be increased in the
often arises because the maximum response of a tissue presence of the antagonist in order to restore a given level
may be produced by agonists when they occupy less than of response). Theory predicts that the dose ratio increases
100% of the receptors. Under these circumstances the tissue linearly with the concentration of the antagonist (see p.
is said to possess spare receptors (see later). 20). These predictions are often borne out in practice (Fig.
In interpreting concentration–effect curves, it must be 2.5A), providing a relatively simple method for determin-
remembered that the concentration of the drug at the ing the equilibrium dissociation constant of the antagonist
receptors may differ from the known concentration in the (KB; Fig. 2.5B). Examples of competitive antagonism are
bathing solution. Agonists may be subject to rapid enzymic very common in pharmacology. The surmountability of
degradation or uptake by cells as they diffuse from the the block by the antagonist may be important in practice,
surface towards their site of action, and a steady state can because it allows the functional effect of the agonist to
be reached in which the agonist concentration at the recep- be restored by an increase in concentration. With other
tors is very much less than the concentration in the bath. types of antagonism (as detailed below), the block is usually
In the case of acetylcholine, for example, which is hydrolysed insurmountable.
by cholinesterase present in most tissues (see Ch. 14), the The salient features of competitive antagonism are:
concentration reaching the receptors can be less than 1%
of that in the bath, and an even bigger difference has been • shift of the agonist log concentration–effect curve to
found with noradrenaline (norepinephrine), which is avidly the right, without change of slope or maximum (i.e.
taken up by sympathetic nerve terminals in many tissues antagonism can be overcome by increasing the
10 (Ch. 15). The problem is reduced but not entirely eradicated concentration of the agonist)
 How drugs act: general principles 2
A Competitive antagonism
Reversible competitive antagonism
• Reversible competitive antagonism is the commonest
1.0 and most important type of antagonism; it has two
Fractional agonist occupancy

main characteristics.
– In the presence of the antagonist, the agonist log
Antagonist concentration–effect curve is shifted to the right
concentration without change in slope or maximum, the extent of
0.5 0 1 10 100 1000 the shift being a measure of the dose ratio.
– The dose ratio increases linearly with antagonist
concentration.
• Antagonist affinity, measured in this way, is widely
used as a basis for receptor classification.
0
10-2 1 102 104 106
Agonist concentration
IRREVERSIBLE COMPETITIVE ANTAGONISM
B
Irreversible competitive antagonism ▼ Irreversible competitive (or non-equilibrium) antagonism occurs when
the antagonist binds to the same site on the receptor as the agonist
1.0 but dissociates very slowly, or not at all, from the receptors, with the
0
Fractional agonist occupancy

result that no change in the antagonist occupancy takes place when

the agonist is applied.4
The predicted effects of reversible and irreversible antagonists are
Antagonist compared in Fig. 2.4.
concentration
In some cases (Fig. 2.6A), the theoretical effect is accurately reproduced
0.5 1 with the antagonist reducing the maximum response. However, the
distinction between reversible and irreversible competitive antagonism
(or even non-competitive antagonism) is not always so clear. This is
because of the phenomenon of spare receptors (see p. 10); if the agonist
10 occupancy required to produce a maximal biological response is very
0 small (say 1% of the total receptor pool), then it is possible to block
10-2 1 102 104 106 irreversibly nearly 99% of the receptors without reducing the maximal
Agonist concentration response. The effect of a lesser degree of antagonist occupancy will
be to produce a parallel shift of the log concentration–effect curve
Fig. 2.4 Hypothetical agonist concentration–occupancy that is indistinguishable from reversible competitive antagonism (Fig.
curves in the presence of reversible (A) and irreversible (B) 2.6B). Only when the antagonist occupancy exceeds 99% will the
competitive antagonists. The concentrations are normalised maximum response will be reduced.
with respect to the equilibrium dissociation constants, K (i.e. 1.0 Irreversible competitive antagonism occurs with drugs that possess
corresponds to a concentration equal to K and results in 50% reactive groups that form covalent bonds with the receptor. These
occupancy). Note that in (A) increasing the agonist concentration are mainly used as experimental tools for investigating receptor
overcomes the effect of a reversible antagonist (i.e. the block is function, and few are used clinically. Irreversible enzyme inhibitors
surmountable), so that the maximal response is unchanged, that act similarly are clinically used, however, and include drugs
whereas in (B) the effect of an irreversible antagonist is such as aspirin (Ch. 27), omeprazole (Ch. 31) and monoamine oxidase
insurmountable and full agonist occupancy cannot be achieved. inhibitors (Ch. 48).

PARTIAL AGONISTS AND THE CONCEPT

OF EFFICACY
So far, we have considered drugs either as agonists, which
• linear relationship between agonist dose ratio and
in some way activate the receptor when they occupy it, or
antagonist concentration
as antagonists, which cause no activation. However, the
• evidence of competition from binding studies.
ability of a drug molecule to activate the receptor – namely
Competitive antagonism is the most direct mechanism by its efficacy – is actually a graded, rather than an all-or-
which one drug can reduce the effect of another (or of an nothing, property. If a series of chemically related agonist
endogenous mediator). drugs acting on the same receptors is tested on a given
biological system, it is often found that the largest response
▼ The characteristics of reversible competitive antagonism described that can be produced differs from one drug to another.
above reflect the fact that agonist and competitive antagonist molecules Some compounds (known as full agonists) can produce a
do not stay bound to the receptor but dissociate and rebind continu- maximal response (the largest response that the tissue is
ously. The rate of dissociation of the antagonist molecules is sufficiently capable of giving), whereas others (partial agonists) can
high that a new equilibrium is rapidly established on addition of the produce only a submaximal response. Fig. 2.7A shows
agonist. In effect, agonist molecules are able to replace the antagonist
concentration–effect curves for several α-adrenoceptor
molecules on the receptors when the antagonist unbinds, although
they cannot, of course, evict bound antagonist molecules. Displacement
agonists (see Ch. 15), which cause contraction of isolated
occurs because, by occupying a proportion of the vacant receptors,
the agonist effectively reduces the rate of association of the antagonist
molecules; consequently, the rate of dissociation temporarily exceeds 4
This type of antagonism is sometimes called non-competitive, but that
that of association, and the overall antagonist occupancy falls. term is ambiguous and best avoided in this context. 11
2 SECTION 1   General Principles

A B
100 5

Response (% max) 80 4

-9
3 KB = 2.2 x 10 mol/L

Log (r − 1)
60

0 10-8 10-7 10-6

40 2

20 1

0 0
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-9 10-8 10-7 10-6
Isoprenaline concentration (mol/L) Propranolol concentration (mol/L)
Fig. 2.5 Competitive antagonism of isoprenaline by propranolol measured on isolated guinea pig atria. (A) Concentration–effect
curves at various propranolol concentrations (indicated on the curves). Note the progressive shift to the right without a change of slope or
maximum. (B) Schild plot (Eq. 2.10). The equilibrium dissociation constant (KB) for propranolol is given by the abscissal intercept, 2.2 ×
10−9 mol/L. Note that the subscript ‘B’ is now used in ‘KB’ to indicate that the equilibrium dissociation constant is that of the antagonist
(designated drug B) measured in the presence of the agonist (designated drug A). (Results from Potter, L.T., 1967. Uptake of propranolol
by isolated guinea pig atria. J. Pharmacol. Exp. Ther. 55, 91–100.)

A
Antagonist concentration
100 0
Response (% max)

100 nM

800 nM
50

0
10−8 10−7 10−6 10−5 10−4
Normorohine concentration (mol/L)
B
Antagonist concentration
0 2 nM 33 nM
100
Fig. 2.6 Effects of irreversible competitive antagonists
on agonist concentration–effect curves. (A) Rat brain
Response (% max)

neurones responding to the opioid agonist normorphine

before and after being exposed to the irreversible competitive
antagonist β-funaltrexamine for 30 minutes and then washed
50 to remove the antagonist. Note the depression of the
maximum response. (B) Responses of the guinea pig ileum
to histamine before and after treatment with increasing
330 nM concentrations of a receptor alkylating agent (GD121) for 5
minutes and then washed to remove the antagonist. Note
the concentration–response curve is initially shifted to the
0 right with no depression of the maximum response. (Panel
[A] after Williams, J.T., North, R.A., 1984. Mol. Pharmacol.
10−8 10−7 10−6 10−5 10−4 10−3
26, 489–497; panel [B] after Nickerson, M., 1955. Nature
Histamine concentration (mol/L) 178, 696–697.)

12
 How drugs act: general principles 2
strips of rabbit aorta. The full agonist phenylephrine
A produced the maximal response of which the tissue was
capable; the other compounds could only produce sub-
1.00
maximal responses and are partial agonists. The difference
between full and partial agonists lies in the relationship
0.80 between receptor occupancy and response. In the experiment
shown in Fig. 2.7 it was possible to estimate the affinity
Response (E/Emax)

of the various drugs for the receptor, and hence (based on

0.60 the theoretical model described later; p. 19) to calculate the
fraction of receptors occupied (known as occupancy) as a
0.40 function of drug concentration. Plots of response as a
function of occupancy for the different compounds are
shown in Fig. 2.7B, showing that for partial agonists the
0.20 response at a given level of occupancy is less than for full
agonists. The weakest partial agonist, tolazoline, produces
a barely detectable response even at 100% occupancy, and
0.00
is usually classified as a competitive antagonist (see p. 10
0.001 0.01 0.1 1 10 100
and Ch. 15).
Concentration (µmol/L) (log scale)
These differences can be expressed quantitatively in terms
of efficacy (e), a parameter originally defined by Stephenson
B
(1956) that describes the ‘strength’ of the agonist–receptor
1.00 complex in evoking a response of the tissue. In the simple
scheme shown in Fig. 2.1, efficacy describes the tendency
of the drug–receptor complex to adopt the active (AR*),
0.80
rather than the resting (AR), state. A drug with zero efficacy
Response (E/Emax)

(e = 0) has no tendency to cause receptor activation, and

0.60 causes no tissue response. A full agonist is a drug whose
efficacy5 is sufficient that it produces a maximal response
when less than 100% of receptors are occupied. A partial
0.40 agonist has lower efficacy, such that 100% occupancy elicits
only a submaximal response.
0.20 ▼ Subsequently it was appreciated that efficacy is composed of
drug-dependent and tissue-dependent components. The drug-
dependent component is referred to as the intrinsic efficacy, which is
0.00 the ability of the agonist drug molecule, once bound, to activate the
0.00 0.20 0.40 0.60 0.80 1.00 receptor protein (see Kelly, 2013). The tissue-dependent components
of efficacy include the number of receptors that it expresses and the
Fraction of receptors occupied
efficiency of coupling of receptor activation to the measured tissue
response. The number of receptors expressed is especially relevant
Phenylephrine Clonidine to the study of receptors in recombinant expression systems when
Oxymetazoline Tolazoline receptors are often very highly expressed and intermediate efficacy
agonists then appear as full agonists. Across different cell types
Naphazoline expressing the same receptor but at different densities a given drug
of intermediate efficacy may appear as a full agonist in one tissue
Fig. 2.7 Partial agonists. (A) Log concentration–effect curves (high level of receptor expression), a partial agonist in another (lower
for a series of α-adrenoceptor agonists causing contraction of level of receptor expression), and even as an antagonist in another
an isolated strip of rabbit aorta. Phenylephrine is a full agonist. (very low level of receptor expression). The term ‘partial agonist’ is
The others are partial agonists with different efficacies. The lower therefore only applicable when describing the action of a drug on a
the efficacy of the drug the lower the maximum response and specific tissue or cell type.
slope of the log concentration–response curve. (B) The For G protein–coupled receptors the elucidation of their X-ray crystal
relationship between response and receptor occupancy for the structures (described in Ch. 3) and the application of molecular dynamic
series. Note that the full agonist, phenylephrine, produces a simulations of drug binding are beginning to tease out the molecular
near-maximal response when only about half the receptors are basis of receptor activation and why some ligands are agonists and
occupied, whereas partial agonists produce submaximal some are antagonists. For students starting to study pharmacology
responses even when occupying all of the receptors. The the simple theoretical two-state model described below provides a
efficacy of tolazoline is so low that it is classified as an useful starting point.
α-adrenoceptor antagonist (see Ch. 15). In these experiments,
receptor occupancy was not measured directly, but was
PARTIAL AGONISTS AS ANTAGONISTS
calculated from pharmacological estimates of the equilibrium
constants of the drugs. (Data from Ruffolo, R.R. Jr, et al., 1979. In discussing the efficacy of partial agonists above we
J. Pharmacol. Exp. Ther. 209, 429–436.) considered the situation in which the tissue was exposed

5
In Stephenson’s formulation, efficacy is the reciprocal of the occupancy
needed to produce a 50% maximal response, thus e = 25 implies that a
50% maximal response occurs at 4% occupancy. There is no theoretical
upper limit to efficacy. 13
2 SECTION 1   General Principles

100
Full agonist alone

Response due to
the presence of

Response (% max)
the partial agonist
0 10 100 1000 Partial agonist
concentration
50

log10[agonist] (mol/L)

Fig. 2.8 Hypothetical concentration–response curves for a full agonist in the absence and presence of increasing concentrations
of a partial agonist. The partial agonist will have agonist action and hence the initial response increases as the partial agonist
concentration increases, reaching a maximum equal to the maximum response of the partial agonist. However, when the full agonist is
added in the presence of the partial agonist its concentration–response curve is shifted to the right.

psychosis associated with Parkinson’s disease (see Chs 41 and 47).

to only one drug, the partial agonist. What we should also
It turns out that most of the receptor antagonists in clinical use are
consider is how the presence of a partial agonist would actually inverse agonists when tested in systems showing constitutive
alter the response of a tissue to a higher efficacy agonist. receptor activation. However, most receptors – like cats – show a
This is depicted in Fig. 2.8 where it can be seen that the preference for the inactive state, and for these there is no practical
presence of the partial agonist induces some level of difference between a competitive antagonist and an inverse agonist.
response dependent upon the concentration initially applied The following section describes a simple model that explains full,
but in addition because the partial agonist is competing partial and inverse agonism in terms of the relative affinity of different
with the full agonist for the receptors it effectively acts as ligands for the resting and activated states of the receptor.
a competitive antagonist, shifting the concentration–response
curve of the full agonist to the right. This is not just an The two-state receptor model
obscure theoretical point but something which occurs in ▼ As illustrated in Fig. 2.1, agonists and antagonists both bind to
clinical practice. In the treatment of heroin users, buprenor- receptors, but only agonists activate them. How can we express this
phine, a weak partial agonist, not only acts as a weak opioid difference, and account for constitutive activity, in theoretical terms?
substitute but also acts as an antagonist and reduces the The two-state model (Fig. 2.10) provides a simple but useful approach.
likelihood of overdose when users relapse and take heroin As shown in Fig. 2.1, we envisage that the occupied receptor can
again (see Ch. 50). switch from its ‘resting’ (R) state to an activated (R*) state, R* being
favoured by binding of an agonist but not an antagonist molecule.
As described above, receptors may show constitutive activation (i.e.
CONSTITUTIVE RECEPTOR ACTIVATION AND the R* conformation can exist without any ligand being bound), so
INVERSE AGONISTS the added drug encounters an equilibrium mixture of R and R* (see
Fig. 2.10). If it has a higher affinity for R* than for R, the drug will
▼ Although we are accustomed to thinking that receptors are activated
cause a shift of the equilibrium towards R* (i.e. it will promote activa-
only when an agonist molecule is bound, there are examples (see De
tion and be classed as an agonist). If its preference for R* is very
Ligt et al., 2000) where an appreciable level of activation (constitutive
large, nearly all the occupied receptors will adopt the R* conformation
activation) may exist even when no ligand is present. These include
and the drug will be a full agonist; if it shows only a modest degree
receptors for benzodiazepines (see Ch. 45), cannabinoids (Ch. 20),
of selectivity for R* (say 5- to 10-fold), a smaller proportion of occupied
serotonin (Ch. 16) and several other mediators. Furthermore, receptor
receptors will adopt the R* conformation and it will be a partial
mutations occur – either spontaneously, in some disease states (see
agonist; if it shows no preference, the prevailing R : R* equilibrium
Bond & Ijzerman, 2006), or experimentally created (see Ch. 4) – that
will not be disturbed and the drug will be a neutral antagonist (zero
result in appreciable constitutive activation. If a ligand reduces the
efficacy), whereas if it shows selectivity for R it will shift the equilibrium
level of constitutive activation; such drugs are known as inverse agonists
towards R and be an inverse agonist (negative efficacy). We can
(Fig. 2.9; see De Ligt et al., 2000) to distinguish them from neutral
therefore think of efficacy as a property determined by the relative
antagonists, which do not by themselves affect the level of activation.
affinity of a ligand for R and R*, a formulation known as the two-state
Inverse agonists can be regarded as drugs with negative efficacy, to
model, which is useful in that it puts a physical interpretation on the
distinguish them from agonists (positive efficacy) and neutral
otherwise mysterious meaning of efficacy, as well as accounting for
antagonists (zero efficacy). Neutral antagonists, by binding to the
the existence of inverse agonists.
agonist binding site, will antagonise both agonists and inverse agonists.
Inverse agonism was first observed at the benzodiazepine receptor
(Ch. 45) but such drugs are proconvulsive and thus not therapeutically BIASED AGONISM
useful! New examples of constitutively active receptors and inverse
agonists are emerging with increasing frequency (mainly among G A major problem with the two-state model is that, as we
protein–coupled receptors). Pimavanserin, an inverse agonist at the now know, receptors are not actually restricted to two
14 5-HT2A receptor, has recently been developed for the treatment of distinct states but have much greater conformational
 How drugs act: general principles 2
A B

100 100
Change in level of receptor activation (%)

Change in level of receptor activation (%)

Agonist Antagonist in
presence of agonist
Agonist in presence
50 of antagonist 50

Constitutive level of
Antagonist alone
receptor activation
100 100
Inverse agonist
in presence of
antagonist
Inverse agonist Antagonist in presence
of inverse agonist
−50 −50
10-10 10-8 10-6 10-4 10-10 10-8 10-6 10-4
Ligand concentration (M) Antagonist concentration (M)
Fig. 2.9 Inverse agonism. The interaction of a competitive antagonist with normal and inverse agonists in a system that shows receptor
activation in the absence of any added ligands (constitutive activation). (A) The degree of receptor activation (vertical scale) increases in the
presence of an agonist (open squares) and decreases in the presence of an inverse agonist (open circles). Addition of a competitive
antagonist shifts both curves to the right (closed symbols). (B) The antagonist on its own does not alter the level of constitutive activity
(open symbols), because it has equal affinity for the active and inactive states of the receptor. In the presence of an agonist (closed
squares) or an inverse agonist (closed circles), the antagonist restores the system towards the constitutive level of activity. These data
(reproduced with permission from Newman-Tancredi, A., et al., 1997. Br. J. Pharmacol. 120, 737–739) were obtained with cloned human
5-hydroxytryptamine (5-HT) receptors expressed in a cell line. (Agonist, 5-carboxamidotryptamine; inverse agonist, spiperone; antagonist,
WAY 100635; ligand concentration [M = mol/L]; see Ch. 16 for information on 5-HT receptor pharmacology.)

flexibility, so that there is more than one inactive and active

Inverse conformation. The different conformations that they can
Agonist
agonist adopt may be preferentially stabilised by different ligands,
and may produce different functional effects by activating
different signal transduction pathways (see Ch. 3).
Receptors that couple to second messenger systems (see
Ch. 3) can couple to more than one intracellular effector
R R* RESPONSE
pathway, giving rise to two or more simultaneous
Resting Activated responses. One might expect that all agonists that activate
state state the same receptor type would evoke the same array of
responses (Fig. 2.11A). However, it has become apparent
Antagonist that different agonists can exhibit bias for the generation
of one response over another even though they are acting
Fig. 2.10 The two-state model. The receptor is shown in
through the same receptor (Fig. 2.11B), probably because
two conformational states, resting (R) and activated (R*), which
exist in equilibrium. Normally, when no ligand is present, the
they stabilise different activated states of the receptor (see
equilibrium lies far to the left, and few receptors are found in the
Kelly, 2013). Agonist bias has become an important concept
R* state. For constitutively active receptors, an appreciable in pharmacology.
proportion of receptors adopt the R* conformation in the Redefining and attempting to measure agonist efficacy
absence of any ligand. Agonists have higher affinity for R* than for such a multistate model is problematic, however, and
for R, so shift the equilibrium towards R*. The greater the requires a more complicated state transition model than
relative affinity for R* with respect to R, the greater the efficacy the two-state model described above. The errors, pitfalls
of the agonist. An inverse agonist has higher affinity for R than and a possible way forward have been outlined by Kenakin
for R* and so shifts the equilibrium to the left. A neutral & Christopoulos (2013).
antagonist has equal affinity for R and R* so does not by itself
affect the conformational equilibrium but reduces by competition ALLOSTERIC MODULATION
the binding of other ligands.
▼ In addition to the agonist binding site (now referred to as the
orthosteric binding site), to which competitive antagonists also bind,
receptor proteins possess many other (allosteric) binding sites (see
Ch. 3) through which drugs can influence receptor function in
various ways, by increasing or decreasing the affinity of agonists 15
2 SECTION 1   General Principles

Agonists, antagonists and efficacy

A
ag ag • Drugs acting on receptors may be agonists or
R R antagonists.
• Agonists initiate changes in cell function, producing
effects of various types; antagonists bind to receptors
without initiating such changes.
Response 1 Response 2 Response 1 Response 2
• Agonist potency depends on two parameters: affinity
(i.e. tendency to bind to receptors) and efficacy (i.e.
ability, once bound, to initiate changes that lead to
effects).
Conventional agonism
• For antagonists, efficacy is zero.
• Full agonists (which can produce maximal effects) have
high efficacy; partial agonists (which can produce only
B
submaximal effects) have intermediate efficacy.
ag ag • According to the two-state model, efficacy reflects the
relative affinity of the compound for the resting and
R R
activated states of the receptor. Agonists show
selectivity for the activated state; antagonists show no
selectivity. This model, although helpful, fails to
Response 1 Response 2 Response 1 Response 2 account for the complexity of agonist action.
• Inverse agonists show selectivity for the resting state
of the receptor, this being of significance only in
situations where the receptors show constitutive
activity.
Biased agonism • Allosteric modulators bind to sites on the receptor
other than the agonist binding site and can modify
Fig. 2.11 Biased agonism. In (A), the receptor (R) is coupled agonist activity.
to two intracellular responses – response 1 and response 2.
When different agonists indicated in red and green activate the
receptor they evoke both responses in a similar manner. This is
what we can consider as being conventional agonism. In (B),
biased agonism is illustrated in which two agonists bind at the The most important ones are:
same site on the receptor yet the red agonist is better at
evoking response 1 and the green agonist is better at evoking • chemical antagonism
response 2. • pharmacokinetic antagonism
• block of receptor–response linkage
• physiological antagonism
for the agonist binding site, by modifying efficacy or by producing
a response themselves (Fig. 2.12). Depending on the direction of the CHEMICAL ANTAGONISM
effect, the ligands may be allosteric antagonists or allosteric facilitators Chemical antagonism refers to the uncommon situation
of the agonist effect, and the effect may be to alter the slope and where the two substances combine in solution; as a result,
maximum of the agonist log concentration–effect curve (see Fig. 2.12). the effect of the active drug is lost. Examples include the
This type of allosteric modulation of receptor function has attracted
use of chelating agents (e.g. dimercaprol) that bind to
much attention recently and occurs at different types of receptors (see
review by Changeux & Christopoulos, 2016). Well-known examples
heavy metals and thus reduce their toxicity, and the use
of allosteric facilitation include glycine at NMDA receptors (Ch. 39), of the neutralising antibody infliximab, which has an
benzodiazepines at GABAA receptors (Ch. 45) and cinacalcet at the anti-inflammatory action due to its ability to sequester
Ca2+ receptor (Ch. 37). One reason why allosteric modulation may the inflammatory cytokine tumour necrosis factor (TNF;
be important to the pharmacologist and future drug development see Ch. 19).
is that across families of receptors such as the muscarinic receptors
(see Ch. 14) the orthosteric binding sites are very similar and it has PHARMACOKINETIC ANTAGONISM
proven difficult to develop selective agonists and antagonists for Pharmacokinetic antagonism describes the situation in which
individual subtypes. The hope is that there will be greater variation the ‘antagonist’ effectively reduces the concentration of the
in the allosteric sites and that receptor-selective allosteric ligands can
active drug at its site of action. This can happen in various
be developed. Furthermore, positive allosteric modulators will exert
their effects only on receptors that are being activated by endogenous
ways. The rate of metabolic degradation of the active drug
ligands and have no effect on those that are not activated. This might may be increased (e.g. the reduction of the anticoagulant
provide a degree of selectivity (e.g. in potentiating spinal inhibition effect of warfarin when an agent that accelerates its hepatic
mediated by endogenous opioids, see Ch. 43) and a reduction in metabolism, such as phenytoin, is given; see Chs 10 and
side effect profile. 58). Alternatively, the rate of absorption of the active drug
from the gastrointestinal tract may be reduced, or the rate
OTHER FORMS OF DRUG ANTAGONISM of renal excretion may be increased. Interactions of this
Other mechanisms can also account for inhibitory interac- sort, discussed in more detail in Chapter 58, are common
16 tions between drugs. and can be important in clinical practice.
 How drugs act: general principles 2
A

Agonist Allosteric
Affinity drug
modulation

Efficacy
modulation

Agonism Allosteric
(orthosteric) agonism

Response
B

Negative affinity modulation Positive affinity modulation

100 100
% Max. response

% Max. response

50 50

0 0
Log [Agonist] (mol/L) Log [Agonist] (mol/L)

Negative efficacy modulation Positive efficacy modulation

100 100
% Max. response

% Max. response

50 50

0 0
Log [Agonist] (mol/L) Log [Agonist] (mol/L)
Fig. 2.12 Allosteric modulation. (A) Allosteric drugs bind at a separate site on the receptor to ‘traditional’ agonists (now often referred
to as ‘orthosteric’ agonists). They can modify the activity of the receptor by (i) altering agonist affinity, (ii) altering agonist efficacy or (iii)
directly evoking a response themselves. (B) Effects of affinity- and efficacy-modifying allosteric modulators on the concentration–effect curve
of an agonist (blue line). In the presence of the allosteric modulator the agonist concentration–effect curve (now illustrated in red) is shifted
in a manner determined by the type of allosteric modulator until a maximum effect of the modulator is reached. (Panel [A] adapted with
permission from Conn et al., 2009. Nat. Rev. Drug Discov. 8, 41–54; panel [B] courtesy of Christopoulos, A.)
17
2 SECTION 1   General Principles

BLOCK OF RECEPTOR–RESPONSE LINKAGE A 5s

Non-competitive antagonism describes the situation where 10 mV
the antagonist blocks at some point downstream from the
agonist binding site on the receptor, and interrupts the
chain of events that leads to the production of a response
5 mV
by the agonist. For example, ketamine enters the ion channel
pore of the NMDA receptor (see Ch. 39) blocking it, thus
preventing ion flux through the channels. Drugs such as
verapamil and nifedipine prevent the influx of Ca2+ through
the cell membrane (see Ch. 23) and thus non-selectively B
block the contraction of smooth muscle produced by drugs 100
acting at any receptor that couples to these calcium channels.

Percentage of control
As a rule, the effect will be to reduce the slope and maximum 80
β adrenoceptors
of the agonist log concentration–response curve, although
it is quite possible for some degree of rightward shift to
60
occur as well.
PHYSIOLOGICAL ANTAGONISM 40
Physiological antagonism is a term used loosely to describe
the interaction of two drugs whose opposing actions in the 20 Response
body tend to cancel each other. For example, histamine
acts on receptors of the parietal cells of the gastric mucosa 0
to stimulate acid secretion, while omeprazole blocks this 0 4 8 24 56 88
effect by inhibiting the proton pump; the two drugs can Time (h)
be said to act as physiological antagonists. Fig. 2.13 Two kinds of receptor desensitisation.
(A) Acetylcholine (ACh) at the frog motor endplate. Brief
depolarisations (upward deflections) are produced by short
Types of drug antagonism pulses of ACh delivered from a micropipette. A long pulse
(horizontal line) causes the response to decline with a time
Drug antagonism occurs by various mechanisms: course of about 20 seconds, owing to desensitisation, and it
recovers with a similar time course. (B) β adrenoceptors of rat
• chemical antagonism (interaction in solution)
glioma cells in tissue culture. Isoproterenol (1 µmol/L) was
• pharmacokinetic antagonism (one drug affecting the
added at time zero, and the adenylyl cyclase response and
absorption, metabolism or excretion of the other) β-adrenoceptor density measured at intervals. During the early
• competitive antagonism (both drugs binding to the uncoupling phase, the response (blue line) declines with no
same receptors); the antagonism may be reversible or change in receptor density (red line). Later, the response
irreversible declines further concomitantly with disappearance of receptors
• interruption of receptor–response linkage from the membrane by internalisation. The green and orange
• physiological antagonism (two agents producing lines show the recovery of the response and receptor density
opposing physiological effects) after the isoproterenol is washed out during the early or late
phase. (Panel [A] from Katz B., Thesleff S., 1957. J. Physiol.
138, 63; panel [B] from Perkins, J.P., 1981. Trends Pharmacol.
Sci. 2, 326.)

DESENSITISATION AND TOLERANCE

Often, the effect of a drug gradually diminishes when it is • active extrusion of drug from cells (mainly relevant in
given continuously or repeatedly. Desensitisation and cancer chemotherapy; see Ch. 57)
tachyphylaxis are synonymous terms used to describe this
phenomenon, which often develops in the course of a few
minutes. The term tolerance is conventionally used to describe CHANGE IN RECEPTORS
a more gradual decrease in responsiveness to a drug, taking Among receptors directly coupled to ion channels (see Ch.
hours, days or weeks to develop, but the distinction is not 3), desensitisation is often rapid and pronounced. At the
a sharp one. The term refractoriness is also sometimes used, neuromuscular junction (Fig. 2.13A), the desensitised state is
mainly in relation to a loss of therapeutic efficacy. Drug caused by a conformational change in the receptor, resulting
resistance is a term used to describe the loss of effectiveness in tight binding of the agonist molecule without the opening
of antimicrobial or antitumour drugs (see Chs 51 and 57). of the ionic channel. Phosphorylation of intracellular regions
Many different mechanisms can give rise to these phenom- of the receptor protein is a second, slower mechanism by
ena. They include: which ion channels become desensitised.
Most G protein–coupled receptors (see Ch. 3) also show
• change in receptors desensitisation (Fig. 2.13B). Phosphorylation of the receptor
• translocation of receptors interferes with its ability to activate second messenger
• exhaustion of mediators cascades, although it can still bind the agonist molecule.
• increased metabolic degradation of the drug The molecular mechanisms of this ‘uncoupling’ are con-
18 • physiological adaptation sidered further in Chapter 3. This type of desensitisation
 How drugs act: general principles 2
usually takes seconds to minutes to develop, and recovers interaction and which has served well as a framework for interpret-
when the agonist is removed. ing a large body of quantitative experimental data (see Colquhoun,
It will be realised that the two-state model in its simple 2006).
form, discussed earlier, needs to be further elaborated to THE BINDING REACTION
incorporate additional desensitised states of the receptor.
▼ The first step in drug action on specific receptors is the formation
TRANSLOCATION OF RECEPTORS of a reversible drug–receptor complex, the reactions being governed
by the Law of Mass Action. Suppose that a piece of tissue, such as
Prolonged exposure to agonists often results in a gradual heart muscle or smooth muscle, contains a total number of receptors,
decrease in the number of receptors expressed on the cell Ntot, for an agonist such as adrenaline. When the tissue is exposed to
surface, as a result of internalisation of the receptors. This adrenaline at concentration xA and allowed to come to equilibrium,
is shown for β adrenoceptors in Fig. 2.13B and is a slower a certain number, NA, of the receptors will become occupied, and the
process than the uncoupling described above. Similar number of vacant receptors will be reduced to Ntot − NA. Normally,
changes have been described for other types of receptor, the number of adrenaline molecules applied to the tissue in solution
including those for various peptides. The internalised greatly exceeds Ntot, so that the binding reaction does not appreciably
receptors are taken into the cell by endocytosis of patches reduce xA. The magnitude of the response produced by the adrenaline
will be related (even if we do not know exactly how) to the number
of the membrane, a process that normally depends on of receptors occupied, so it is useful to consider what quantitative
receptor phosphorylation and the subsequent binding of relationship is predicted between NA and xA. The reaction can be
arrestin proteins to the phosphorylated receptor (see Ch. represented by:
3, Fig. 3.16). This type of adaptation is common for
hormone receptors and has obvious relevance to the effects A + R
k +1
 
  AR
produced when drugs are given for extended periods. It k −1

is generally an unwanted complication when agonist drugs drug + free receptor complex
are used clinically. (xA ) ( N tot − N A ) (N A )
EXHAUSTION OF MEDIATORS
The Law of Mass Action (which states that the rate of a chemical
In some cases, desensitisation is associated with depletion reaction is proportional to the product of the concentrations of
of an essential intermediate substance. Drugs such as reactants) can be applied to this reaction.
amphetamine, which acts by releasing amines from nerve
Rate of forward reaction = k+1x A ( N tot − N A ) (2.1)
terminals (see Chs 15 and 49), show marked tachyphylaxis
because the amine stores become depleted. Rate of backward reaction = k−1N A (2.2)
ALTERED DRUG METABOLISM At equilibrium, the two rates are equal:

Tolerance to some drugs, for example barbiturates and k+1x A ( N tot − N A ) = k−1N A (2.3)
ethanol (Ch. 49), occurs partly because repeated administra- The affinity constant of binding is given by k+1/k−1 and from Eq. 2.3
tion of the same dose produces a progressively lower plasma equals NA/xA(Ntot –NA). Unfortunately, this has units of reciprocal
concentration, as a result of increased metabolic degradation. concentration (L/mol) which for some of us is a little hard to get our
The degree of tolerance that results is generally modest, heads around. Pharmacologists therefore tend to use the reciprocal
and in both of these examples other mechanisms contribute of the affinity constant, the equilibrium dissociation constant (K), which
to the substantial tolerance that actually occurs. However, has units of concentration (mol/L).
the pronounced tolerance to nitrovasodilators (see Chs 21 For drug A its equilibrium dissociation constant (KA)6 can be repre-
and 23) results mainly from decreased metabolism, which sented as
reduces the release of the active mediator, nitric oxide. K A = k−1 k+1 = x A ( N tot − N A ) N A (2.4)
PHYSIOLOGICAL ADAPTATION The proportion of receptors occupied, or occupancy (PA), is NA/Ntot,
Diminution of a drug’s effect may occur because it is nul- which is independent of Ntot.
lified by a homeostatic response. For example, the blood xA xA
pressure-lowering effect of thiazide diuretics is limited PA = = (2.5)
x A + k−1 k+1 x A + K A
because of a gradual activation of the renin–angiotensin
system (see Ch. 23). Such homeostatic mechanisms are very Thus if the equilibrium dissociation constant of a drug is known we
common, and if they occur slowly the result will be a can calculate the proportion of receptors it will occupy at any
gradually developing tolerance. It is a common experience concentration.
that many side effects of drugs, such as nausea or sleepiness, Eq. 2.5 can be written:
tend to subside even though drug administration is con- xA K A
tinued. We may assume that some kind of physiological PA = (2.6)
adaptation is occurring, presumably associated with altered xA K A + 1
gene expression resulting in changes in the levels of various This important result is known as the Hill–Langmuir equation.7
regulatory molecules, but little is known about the mecha-
nisms involved. 6
Here we now use ‘KA’ rather than just ‘K’ because we will in the next
section be going on to consider the situation when two drugs, A and B,
are present and there we will use ‘KA’ and ‘KB’ to denote the
equilibrium dissociation constants of the two drugs.
QUANTITATIVE ASPECTS OF DRUG– 7
A.V. Hill first published it in 1909, when he was still a medical student.
RECEPTOR INTERACTIONS Langmuir, a physical chemist working on gas adsorption, derived it
independently in 1916. Both subsequently won Nobel Prizes. Until
▼ Here we present some aspects of so-called receptor theory, which recently, it was known to pharmacologists as the Langmuir equation,
is based on applying the Law of Mass Action to the drug–receptor even though Hill deserves the credit. 19
2 SECTION 1   General Principles

BINDING WHEN MORE THAN

A ONE DRUG IS PRESENT
1.0
Fractional occupancy
▼ Suppose that two drugs, A and B, which bind to the same receptor
with equilibrium dissociation constants KA and KB, respectively, are
present at concentrations xA and xB. If the two drugs compete (i.e.
the receptor can accommodate only one at a time), then, by application
0.5
of the same reasoning as for the one-drug situation described above,
KA = 1.0 the occupancy by drug A is given by:

0 xA K A
0 5 10
PA = (2.9)
x A K A + xB K B + 1
Concentration (linear scale)
Comparing this result with Eq. 2.5 shows that adding drug B, as
B expected, reduces the occupancy by drug A. Fig. 2.4A (p. 11) shows
1.0 the predicted binding curves for A in the presence of increasing
Fractional occupancy

concentrations of B, demonstrating the shift without any change of

slope or maximum that characterises the pharmacological effect of a
competitive antagonist (see Fig. 2.5). The extent of the rightward
0.5 shift, on a logarithmic scale, represents the ratio (rA, given by xA′/xA
KA = 1.0 where xA′ is the increased concentration of A) by which the concentra-
tion of A must be increased to overcome the competition by B.
Rearranging Eq. 2.9 shows that
0
0.1 1.0 10.0 rA = ( xB K B ) + 1 (2.10)
Concentration (log scale)
Thus rA depends only on the concentration and equilibrium dissociation
Fig. 2.14 Theoretical relationship between occupancy and constant of the competing drug B, not on the concentration or
ligand concentration. The relationship is plotted according to equilibrium dissociation constant of A.
Eq. 2.5. (A) Plotted with a linear concentration scale, this curve If A is an agonist, and B is a competitive antagonist, and we assume
is a rectangular hyperbola. (B) Plotted with a log concentration that the response of the tissue will be an unknown function of PA,
scale, it is a symmetrical sigmoid curve. KA is defined in the text then the value of rA determined from the shift of the agonist
and footnote 6. concentration–effect curve at different antagonist concentrations can
be used to estimate the equilibrium dissociation constant KB for the
antagonist. Such pharmacological estimates of rA are commonly termed
agonist dose ratios (more properly concentration ratios, although most
pharmacologists use the older term). This simple and very useful Eq.
(2.10) is known as the Schild equation, after the pharmacologist who
first used it to analyse drug antagonism.
The equilibrium dissociation constant, KA, is a characteristic of the drug
and of the receptor; it has the dimensions of concentration and is Eq. 2.10 can be expressed logarithmically in the form:
numerically equal to the concentration of drug required to occupy
log(rA − 1) = log xB − log K B (2.11)
50% of the sites at equilibrium. (Verify from Eq. 2.5 that when xA =
KA then PA = 0.5.) The higher the affinity of the drug for the receptors, Thus a plot of log (rA−1) against log xB, usually called a Schild plot
the lower will be the value of KA. Eq. 2.6 describes the relationship (as in Fig. 2.5, earlier), should give a straight line with unit slope (i.e.
between occupancy and drug concentration, and it generates a its gradient is equal to 1) and an abscissal intercept equal to log KB.
characteristic curve known as a rectangular hyperbola, as shown in Following the pH and pK notation, antagonist potency can be expressed
Fig. 2.14A. It is common in pharmacological work to use a logarithmic as a pA2 value; under conditions of competitive antagonism, pA2 =
scale of concentration; this converts the hyperbola to a symmetrical −log KB. Numerically, pA2 is defined as the negative logarithm of the
sigmoid curve (Fig. 2.14B). molar concentration of antagonist required to produce an agonist
The same approach is used to analyse data from experiments in which dose ratio equal to 2. As with pH notation, its principal advantage
drug binding is measured directly (see pp. 8–9, Fig. 2.2). In this case, is that it produces simple numbers, a pA2 of 6.5 being equivalent to
the relationship between the amount bound (B) and ligand concentra- a KB of 3.2 × 10−7 mol/L.
tion (xA) should be: For competitive antagonism, r shows the following characteristics:
• It depends only on the concentration and equilibrium
B = Bmax x A ( x A + K A ) (2.7) dissociation constant of the antagonist, and not on the size of
response that is chosen as a reference point for the
where Bmax is the total number of binding sites in the preparation
measurements (so long as it is submaximal).
(often expressed as pmol/mg of protein). To display the results in
linear form, Eq. 2.6 may be rearranged to: • It does not depend on the equilibrium dissociation constant of
the agonist.
B x A = Bmax K A − B K A (2.8) • It increases linearly with xB, and the slope of a plot of (rA−1)
against xB is equal to 1/KB; this relationship, being independent
A plot of B/xA against B (known as a Scatchard plot) gives a straight of the characteristics of the agonist, should be the same for an
line from which both Bmax and KA can be estimated. Statistically, this antagonist against all agonists that act on the same population
procedure is not without problems, and it is now usual to estimate of receptors.
these parameters from the untransformed binding values by an iterative These predictions have been verified for many examples of competitive
non-linear curve-fitting procedure. antagonism (see Fig. 2.5).
To this point, our analysis has considered the binding of one ligand
to a homogeneous population of receptors. To get closer to real-life In this section, we have avoided going into great detail and have
pharmacology, we must consider (a) what happens when more than oversimplified the theory considerably. As we learn more about the
one ligand is present, and (b) how the tissue response is related to actual molecular details of how receptors work to produce their
20 receptor occupancy. biological effects (see Ch. 3), the shortcomings of this theoretical
 How drugs act: general principles 2
treatment become more obvious. The two-state model can be incor-
porated without difficulty, but complications arise when we include
Drug + target
the involvement of G proteins (see Ch. 3) in the reaction scheme (as
they shift the equilibrium between R and R*), and when we allow
for the fact that receptor activation is not a simple on–off switch, as
the two-state model assumes, but may take different forms. Despite Rapid Rapid
strenuous efforts by theoreticians to allow for such possibilities, the
molecules always seem to remain one step ahead. Nevertheless, this
type of basic theory applied to the two-state model remains a useful
basis for developing quantitative models of drug action. The book Rapid
Altered gene
by Kenakin (1997) is recommended as an introduction, and the later physiological Slow
expression
review (Kenakin & Christopoulos, 2011) presents a detailed account responses
of the value of quantification in the study of drug action.

Slow

Delayed
Binding of drugs to receptors responses
• Binding of drugs to receptors necessarily obeys the
Fig. 2.15 Early and late responses to drugs. Many drugs
Law of Mass Action.
act directly on their targets (left-hand arrow) to produce a rapid
• At equilibrium, receptor occupancy is related to drug
physiological response. If this is maintained, it is likely to cause
concentration by the Hill–Langmuir equation (Eq. 2.6). changes in gene expression that give rise to delayed effects.
• The higher the affinity of the drug for the receptor, the Some drugs (right-hand arrow) have their primary action on
lower the concentration at which it produces a given gene expression, producing delayed physiological responses.
level of occupancy. Drugs can also work by both pathways. Note the bidirectional
• The same principles apply when two or more drugs interaction between gene expression and response.
compete for the same receptors; each has the effect
of reducing the apparent affinity for the other.
as acute drug effects is becoming increasingly important.
Pharmacologists have traditionally tended to focus on
short-term physiological responses, which are much easier
to study, rather than on delayed effects. The focus is now
THE NATURE OF DRUG EFFECTS clearly shifting.

In discussing how drugs act in this chapter, we have focused

mainly on the rapid consequences of receptor activation.
Details of the receptors and their linkage to effects at the Drug effects
cellular level are described in Chapter 3. We now have a
fairly good understanding at this level. It is important, • Drugs act mainly on cellular targets, producing effects
however, particularly when considering drugs in a thera- at different functional levels (e.g. biochemical, cellular,
peutic context, that their direct effects on cellular function physiological and structural).
generally lead to secondary, delayed effects, which are often
• The direct effect of the drug on its target produces
highly relevant in a clinical situation in relation to both
acute responses at the biochemical, cellular or
therapeutic efficacy and harmful effects (Fig. 2.15). For
example, activation of cardiac β adrenoceptors (see Chs 3 physiological levels.
and 22) causes rapid changes in the functioning of the heart • Prolonged receptor activation generally leads to
muscle, but also slower (minutes to hours) changes in the delayed long-term effects, such as desensitisation or
functional state of the receptors (e.g. desensitisation), and down-regulation of receptors, hypertrophy, atrophy or
even slower (hours to days) changes in gene expression remodelling of tissues, tolerance, dependence and
that produce long-term changes (e.g. hypertrophy) in cardiac addiction.
structure and function. Opioids (see Ch. 43) produce an • Long-term delayed responses result from changes in
immediate analgesic effect, but after a time, tolerance and gene expression, although the mechanisms by which
dependence ensue, and in some cases long-term addiction. the acute effects bring this about are often uncertain.
In these and many other examples, the nature of the • Therapeutic effects may be based on acute responses
intervening mechanism is unclear, although as a general (e.g. the use of bronchodilator drugs to treat asthma;
rule any long-term phenotypic change necessarily involves Ch. 29) or delayed responses (e.g. antidepressants;
alterations of gene expression. Drugs are often used to treat Ch. 48).
chronic conditions, and understanding long-term as well

21
2 SECTION 1   General Principles

REFERENCES AND FURTHER READING

General Receptor mechanisms: agonists and efficacy
Alexander, S.P.H., Kelly, E., Marrion, N., et al., 2015. The Concise Guide Bond, R.A., Ijzerman, A.P., 2006. Recent developments in constitutive
to Pharmacology. Br. J. Pharmacol. 172, 5729–6202. (Summary data on a receptor activity and inverse agonism, and their potential for GPCR
vast array of receptors, ion channels, transporters and enzymes and of the drug discovery. Trends Pharmacol. Sci. 27, 92–96. (Discussion of
drugs that interact with them – valuable for reference) pathophysiological consequences of constitutive receptor activation and
Colquhoun, D., 2006. The quantitative analysis of drug–receptor therapeutic potential of inverse agonists)
interactions: a short history. Trends Pharmacol. Sci. 27, 149–157. (An Changeux, J.P., Christopoulos, A., 2016. Allosteric modulation as a
illuminating account for those interested in the origins of one of the central unifying mechanism for receptor function and regulation. Cell 166,
ideas in pharmacology) 1084–1102. (Extensive review describing allosteric modulation at different
Franks, N.P., 2008. General anaesthesia: from molecular targets to types of receptor)
neuronal pathways of sleep and arousal. Nat. Rev. Neurosci. 9, De Ligt, R.A.F., Kourounakis, A.P., Ijzerman, A.P., 2000. Inverse
370–386. (Describes how we now understand that general anaesthetics agonism at G protein-coupled receptors: (patho)physiological
interact with specific membrane proteins rather than by accumulating in relevance and implications for drug discovery. Br. J. Pharmacol. 130,
membrane lipids) 1–12. (Useful review article giving many examples of constitutively active
Kenakin, T., 1997. Pharmacologic Analysis of Drug–Receptor receptors and inverse agonists, and discussing the relevance of these concepts
Interactions, third ed. Lippincott-Raven, New York. (Useful and for disease mechanisms and drug discovery)
detailed textbook covering most of the material in this chapter in greater Kelly, E., 2013. Efficacy and ligand bias at the µ-opioid receptor. Br. J.
depth) Pharmacol. 169, 1430–1446. (A readable account of the problems of
Kenakin, T., Christopoulos, A., 2013. Signalling bias in new drug measuring efficacy as well as a discussion of agonist bias at an important
discovery: detection, quantification and therapeutic impact. Nat. Rev. receptor)
Drug Discov. 12, 205–216. (Detailed discussion of the difficulties in Kenakin, T., Christopoulos, A., 2011. Analytical pharmacology: the
measuring agonist efficacy and bias) impact of numbers on pharmacology. Trends Pharmacol. Sci. 32,
Neubig, R., Spedding, M., Kenakin, T., Christopoulos, A., 2003. 189–196. (A theoretical treatment that attempts to take into account recent
International Union of Pharmacology Committee on receptor knowledge of receptor function at the molecular level)
nomenclature and drug classification: XXXVIII. Update on terms and May, L.T., Leach, K., Sexton, P.M., Christopoulos, A., 2007. Allosteric
symbols in quantitative pharmacology. Pharmacol. Rev. 55, 597–606. modulation of G protein-coupled receptors. Annu. Rev. Pharmacol.
(Summary of IUPHAR-approved terms and symbols relating to Toxicol. 47, 1–51. (Comprehensive review describing the characteristics,
pharmacological receptors – useful for reference purposes) mechanisms and pharmacological implications of allosteric interactions at
Rang, H.P., 2006. The receptor concept: pharmacology’s big idea. Br. J. GPCRs)
Pharmacol. 147 (Suppl. 1), 9–16. (Short review of the origin and status of
the receptor concept)
Stephenson, R.P., 1956. A modification of receptor theory. Br. J.
Pharmacol. 11, 379–393. (Classic analysis of receptor action introducing the
concept of efficacy)

22
 GENERAL PRINCIPLES SECTION 1

How drugs act: molecular aspects 3

function and responses of all the different cells in the body,
OVERVIEW the chemical messengers being the various hormones,
transmitters and other mediators discussed in Section 2 of
In this chapter, we move from the general principles this book. Many therapeutically useful drugs act, either as
of drug action outlined in Chapter 2 to the molecules agonists or antagonists, on receptors for known endogenous
that are involved in recognising chemical signals and mediators. In most cases, the endogenous mediator was
translating them into cellular responses. Molecular discovered before – often many years before – the receptor
pharmacology is advancing rapidly, and the new was characterised pharmacologically and biochemically.
knowledge is changing our understanding of drug In some cases, such as the cannabinoid and opioid receptors
action and opening up many new therapeutic possibili- (see Chs 20 and 43), the endogenous mediators were identi-
ties, further discussed in other chapters. fied later; in others, known as orphan receptors (see later)
First, we consider the types of target proteins on the mediator, if it exists, still remains unknown. The host
which drugs act. Next, we describe the main families defence system also utilises a set of receptors (e.g. the ‘Toll’
of receptors and ion channels. Finally, we discuss the receptors) that are adept at recognising fragments of ‘foreign’
various forms of receptor–effector linkage (signal bacterial and other invading organisms. These are considered
transduction mechanisms) through which receptors separately in Chapter 7.
are coupled to the regulation of cell function. The
relationship between the molecular structure of a ION CHANNELS
receptor and its functional linkage to a particular Ion channels1 are essentially gateways in cell membranes
type of effector system is a principal theme. In the that selectively allow the passage of particular ions, and
next two chapters, we see how these molecular events that are induced to open or close by a variety of mechanisms.
alter important aspects of cell function – a useful basis Two important types are ligand-gated channels and voltage-
for understanding the effects of drugs on intact living gated channels. The former open only when one or more
organisms. We are confident that tomorrow’s phar- agonist molecules are bound, and are properly classified
macology will rest solidly on the advances in cellular as receptors, since agonist binding is needed to activate
and molecular biology that are discussed here. them. Voltage-gated channels are gated by changes in the
transmembrane potential rather than by agonist binding.
In general, drugs can affect ion channel function in several
PROTEIN TARGETS FOR DRUG ACTION ways:
1. By binding to the channel protein itself, either to the
The protein targets for drug action on mammalian cells
ligand-binding (orthosteric) site of ligand-gated
(Fig. 3.1) that are described in this chapter can be broadly
channels, or to other (allosteric) sites, or, in the
divided into:
simplest case, exemplified by the action of local
• receptors anaesthetics on the voltage-gated sodium channel (see
• ion channels Ch. 44), the drug molecule plugs the channel
• enzymes physically (see Fig. 3.1B), blocking ion permeation.
• transporters (carrier molecules) Examples of drugs that bind to allosteric sites on the
channel protein and thereby affect channel gating
The great majority of important drugs act on one or other
include:
of these types of protein, but there are exceptions. For
• benzodiazepines (see Ch. 45). These drugs bind to a
example, colchicine used to treat arthritic gout attacks (Ch.
region of the GABAA receptor–chloride channel
27) interacts with the structural protein tubulin, while several
complex (a ligand-gated channel) that is distinct
immunosuppressive drugs (e.g. ciclosporin, Ch. 27) bind
from the GABA binding site and facilitate the
to cytosolic proteins known as immunophilins. Therapeutic
opening of the channel by the inhibitory
antibodies that act by sequestering cytokines (protein
neurotransmitter GABA (see Ch. 39)
mediators involved in inflammation; see Chs 5 and 27) are
• vasodilator drugs of the dihydropyridine type (see
also used. Targets for chemotherapeutic drugs (Chs 51–57),
Ch. 23), which inhibit the opening of L-type calcium
where the aim is to suppress invading microorganisms or
channels (see Ch. 4).
cancer cells, include DNA and cell wall constituents as
well as other proteins.
RECEPTORS 1
‘Ion channels and the electrical properties they confer on cells are
involved in every human characteristic that distinguishes us from the
Receptors (see Fig. 3.1A) are the sensing elements in the stones in a field’ (Armstrong, C.M., 2003. Voltage-gated K channels. Sci.
system of chemical communications that coordinates the STKE 188, re10). 23
3 SECTION 1 General Principles

angiotensin-converting enzyme; Ch. 23); in other cases,

A RECEPTORS the binding is irreversible and non-competitive (e.g.
Ion channel
Direct opening/closing aspirin, acting on cyclo-oxygenase; Ch. 27). Drugs may
Enzyme also act as false substrates, where the drug molecule
activation/inhibition undergoes chemical transformation to form an abnormal
Transduction
Agonist/
mechanisms
Ion channel product that subverts the normal metabolic pathway.
inverse modulation
An example is the anticancer drug fluorouracil, which
agonist DNA
transcription replaces uracil as an intermediate in purine biosyn-
thesis but cannot be converted into thymidylate, thus
No effect blocking DNA synthesis and preventing cell division
Antagonist
Endogenous mediators blocked
(Ch. 57).
It should also be mentioned that drugs may require
enzymic degradation to convert them from an inactive form,
B ION CHANNELS the prodrug (see Ch. 10), to an active form (e.g. enalapril
Blockers Permeation is converted by esterases to enalaprilat, which inhibits
blocked angiotensin-converting enzyme). Furthermore, as discussed
Increased or in Chapter 58, drug toxicity often results from the enzymic
Modulators decreased conversion of the drug molecule to a reactive metabolite.
opening probability Paracetamol (see Ch. 27) causes liver damage in this way.
As far as the primary action of the drug is concerned, this
is an unwanted side reaction, but it is of major practical
C ENZYMES
importance.
Normal reaction
Inhibitor
inhibited
TRANSPORTERS
False Abnormal The movement of ions and small polar organic molecules
substrate metabolite produced across cell membranes generally occurs either through
channels, or through the agency of a transport protein
(see Fig. 3.1D), because the permeating molecules are
Prodrug Active drug produced often insufficiently lipid-soluble to penetrate lipid mem-
branes on their own. Many such transporters are known;
examples of particular pharmacological importance include
D TRANSPORTERS those responsible for the transport of ions and many
organic molecules across the renal tubule, the intestinal
Normal epithelium and the blood–brain barrier, the transport of
transport Na+ and Ca2+ out of cells, the uptake of neurotransmit-
ter precursors (such as choline) or of neurotransmitters
themselves (such as amines and amino acids) by nerve
Inhibitor or Transport terminals, and the transport of drug molecules and their
blocked
metabolites across cell membranes and epithelial barri-
False Abnormal compound ers. We shall encounter transporters frequently in later
substrate accumulated chapters.
In many cases, hydrolysis of ATP provides the energy
for transport of substances against their electrochemical
Agonist/substrate Abnormal product gradient. Such transport proteins include a distinct ATP-
Antagonist/inhibitor Prodrug binding site, and are termed ABC (ATP-Binding Cassette)
transporters. Important examples include the sodium
Fig. 3.1 Types of target for drug action. pump (Na+-K+-ATPase; see Ch. 4) and multidrug resist-
ance (MDR) transporters that eject cytotoxic drugs from
cancer and microbial cells, conferring resistance to these
therapeutic agents (see Ch. 57). In other cases, including
2. By an indirect interaction, involving an activated G the neurotransmitter transporters, the transport of organic
protein subunit or other intermediary (see p. 34). molecules is coupled to the transport of ions (usually Na+),
3. By altering the level of expression of ion channels on either in the same direction (symport) or in the opposite
the cell surface. For example, gabapentin reduces the direction (antiport), and therefore relies on the electrochemi-
insertion of neuronal calcium channels into the cal gradient for Na+ generated by the ATP-driven sodium
plasma membrane (Ch. 46). pump. The carrier proteins embody a recognition site that
makes them specific for a particular permeating species, and
A summary of the different ion channel families and their these recognition sites can also be targets for drugs whose
functions is given later. effect is to block the transport system (e.g. cocaine blocks
monoamine neurotransmitter uptake into nerve terminals;
ENZYMES see Ch. 49).
Many drugs target enzymes (see Fig. 3.1C). Often, the The importance of transporters as a source of individual
drug molecule is a substrate analogue that acts as a com- variation in the pharmacokinetic characteristics of various
24 petitive inhibitor of the enzyme (e.g. captopril, acting on drugs is increasingly recognised (see Ch. 11).
 How drugs act: molecular aspects 3
agonist-induced receptor conformational changes and how
RECEPTOR PROTEINS signalling is initiated.
Now that the genes have been clearly identified, the
CLONING OF RECEPTORS
emphasis has shifted to characterising the receptors phar-
In the 1970s, pharmacology entered a new phase when macologically and determining their molecular character-
receptors, which had until then been theoretical entities, istics and physiological functions.
began to emerge as biochemical realities following the
development of receptor-labelling techniques (see Ch. 2), TYPES OF RECEPTOR
which made it possible to extract and purify the receptor Receptors elicit many different types of cellular effect. Some
material. of them are very rapid, such as those involved in fast
Once receptor proteins were isolated and purified, it was synaptic transmission, operating within milliseconds,
possible to analyse the amino acid sequence of a short whereas other receptor-mediated effects, such as many of
stretch, allowing the corresponding base sequence of the those produced by thyroid hormone or various steroid
mRNA to be deduced and full-length DNA to be isolated hormones, occur over hours or days. There are many
by conventional cloning methods, starting from a cDNA examples of intermediate timescales – catecholamines, for
library obtained from a tissue source rich in the receptor example, usually act in a matter of seconds, whereas many
of interest. The first receptor clones were obtained in this peptides take rather longer to produce their effects. Not
way, but subsequently expression cloning and, with the surprisingly, very different types of linkage between receptor
sequencing of the entire genome of various species, including occupation and the ensuing response are involved. Based
human, cloning strategies based on sequence homologies, on molecular structure and the nature of this linkage (the
which do not require prior isolation and purification of the transduction mechanism), we can distinguish four receptor
receptor protein, were widely used, and now several types, or superfamilies (Figs 3.2 and 3.3; Table 3.1).
hundred receptors of all four structural families (see Fig.
• Type 1: ligand-gated ion channels (also known as
3.3) have been cloned. Sequence data so obtained has
ionotropic receptors3). The chain of discoveries
revealed many molecular variants (subtypes) of known
culminating in the molecular characterisation of these
receptors that had not been evident from pharmacological
receptors is described by Halliwell (2007). Typically,
studies (see IUPHAR/BPS, Guide to Pharmacology). Much
these are the receptors on which fast neurotransmitters
remains to be discovered about the pharmacological,
act (see Table 3.1).
functional and clinical significance of this abundant molecu-
• Type 2: G protein–coupled receptors (GPCRs). These
lar polymorphism. It is expected, however, that such vari-
are also known as metabotropic receptors or
ations will account for part of the variability between
7-transmembrane (7-TM, serpentine or heptahelical)
individuals in response to therapeutic agents (see Ch. 12)
receptors. They are membrane receptors that are
Endogenous ligands for many of the novel receptors
coupled to intracellular effector systems primarily via
identified by gene cloning are so far unknown, and they
a G protein (see p. 32). They constitute the largest
are described as ‘orphan receptors’.2 Identifying ligands
family,4 and include receptors for many hormones and
for these presumed receptors is often difficult. Increasingly,
slow transmitters (Table 3.1).
there are examples (e.g. free fatty acid receptors) where
• Type 3: kinase-linked and related receptors. This is a
important endogenous ligands have been linked to hitherto
large and heterogeneous group of membrane receptors
orphan receptors. There is optimism that novel therapeutic
responding mainly to protein mediators. They
agents will emerge by targeting this pool of unclaimed
comprise an extracellular ligand-binding domain
receptors.
linked to an intracellular domain by a single
Much information has been gained by introducing the
transmembrane helix. In many cases, the intracellular
cloned DNA encoding individual receptors into cell lines,
domain is enzymic in nature (with protein kinase or
producing cells that express the foreign receptors in a
guanylyl cyclase activity). Some lack enzymic activity
functional form. Such engineered cells allow much more
themselves but link to intracellular effector enzymes
precise control of the expressed receptors than is possible
through their binding of adaptor proteins. Examples
with natural cells or intact tissues, and the technique is widely
of these latter receptor types include cytokine
used to study the binding and pharmacological characteristics
receptors (e.g. tumour necrosis factor [TNF] receptors)
of cloned receptors. Expressed human receptors, which often
and pattern recognition receptors (PRRs) that
differ in their sequence and pharmacological properties from
recognise pathogen-associated molecular patterns
their animal counterparts, can be studied in this way.
(PAMPs) or danger-associated molecular patterns
Obtaining crystals of a protein allows its structure to be
(DAMPs) found in pathogens, which stimulate the
analysed at very high resolution by X-ray diffraction
innate immune system host defence network (see Ch.
techniques, but unfortunately, since many receptors are
7). PRR receptors include the cell surface Toll-like
normally embedded in membrane lipid, they have, until
receptors (TLRs), and the cytoplasmic receptors such
relatively recently, proven difficult to crystallise. Much of
the information obtained relates to how ligands bind to
receptors, but we are now beginning to learn more about 3
Here, focusing on receptors, we include ligand-gated ion channels as
an example of a receptor family. Other types of ion channels are
described later (p. 46); many are also drug targets, although not
2
An oddly Dickensian term that seems inappropriately condescending. receptors in the strict sense.
4
Because we can assume that these receptors play defined roles in There are 865 human GPCRs comprising 1.6% of the genome
physiological signalling, their ‘orphanhood’ reflects our ignorance, not (Fredriksson & Schiöth, 2005). Nearly 500 of these are believed to be
their status. More information on orphan receptors can be found at odorant receptors involved in smell and taste sensations, the remainder
<www.guidetopharmacology.org/GRAC/FamilyDisplayForward?famil being receptors for known or unknown endogenous mediators
yId=115#16>. – enough to keep pharmacologists busy for some time yet. 25
3 SECTION 1 General Principles

1. Ligand-gated ion 2. G protein−coupled 3. Kinase-linked 4. Nuclear receptors

channels receptors receptors
(ionotropic receptors) (metabotropic)

Ions Ions

R R R E R/E
G G
or or NUCLEUS
Hyperpolarisation Second messengers
Change Protein
or R
in excitability phosphorylation
depolarisation
Gene
transcription
Gene transcription
Ca2+ release Protein Other
phosphorylation
Protein synthesis Protein synthesis

Cellular effects Cellular effects Cellular effects Cellular effects

Time scale
Milliseconds Seconds Hours Hours
Examples
Nicotinic Muscarinic Cytokine receptors Oestrogen
ACh receptor ACh receptor receptor

Fig. 3.2 Types of receptor–effector linkage. ACh, acetylcholine; E, enzyme; G, G protein; R, receptor.

Table 3.1 The four main types of receptor

Type 1: Ligand-gated Type 2: G protein– Type 3: Receptor Type 4: Nuclear

ion channels coupled receptors kinases receptors

Location Membrane Membrane Membrane Intracellular

Effector Ion channel Channel or enzyme Protein kinases Gene transcription
Coupling Direct G protein or arrestin Direct Via DNA
Examples Nicotinic acetylcholine Muscarinic acetylcholine Insulin, growth factors, Steroid receptors
receptor, GABAA receptor receptor, adrenoceptors cytokine receptors
Structure Oligomeric assembly of Monomeric or oligomeric Single transmembrane Monomeric structure with
subunits surrounding assembly of subunits helix linking extracellular receptor- and DNA-binding
central pore comprising seven receptor domain to domains
transmembrane helices intracellular kinase
with intracellular domain
G protein–coupling domain

as RIG-I-like receptors (RLRs) and NOD-like receptors also recognise many foreign molecules, inducing the
(NLRs). All these immune receptors signal their expression of enzymes that metabolise them.
intracellular effects through adaptor proteins and
kinases to alter the cell’s transcription to elicit the
correct immune response needed to fight against any MOLECULAR STRUCTURE OF RECEPTORS
pathogenic invaders. The molecular organisation of typical members of each of
• Type 4: nuclear receptors. These are receptors that these four receptor superfamilies is shown in Fig. 3.3.
regulate gene transcription.5 Receptors of this type Although individual receptors show considerable sequence
variation in particular regions, and the lengths of the main
5
The term nuclear receptor is something of a misnomer, because some are
intracellular and extracellular domains also vary from one
actually located in the cytosol and migrate to the nuclear compartment to another within the same family, the overall structural
26 when a ligand is present. patterns and associated signal transduction pathways are
 How drugs act: molecular aspects 3
very consistent. The realisation that just four main receptor
superfamilies provide a solid framework for interpreting
A N the complex welter of information about the effects of a
Binding
Type 1
domain large proportion of the drugs that have been studied has
Ligand-gated been one of the most refreshing developments in modern
ion channels C pharmacology.
(ionotropic
receptors) x 4 or 5
RECEPTOR HETEROGENEITY AND SUBTYPES
Channel Receptors within a given family generally occur in several
lining molecular varieties, or subtypes, with similar architecture
but significant differences in their sequences, and often in
their pharmacological properties.6 Nicotinic acetylcholine
B receptors are typical in this respect; distinct subtypes occur
Type 2 N Binding
in different brain regions (see Table 40.2), and these differ
G protein− domains
coupled from the muscle receptor. Some of the known pharm-
receptors acological differences (e.g. sensitivity to blocking agents)
(metabotropic between muscle and brain acetylcholine receptors correlate
receptors)
G protein− with specific sequence differences; however, as far as we
coupling know, all nicotinic acetylcholine receptors respond to the
domain same physiological mediator and produce the same kind
C of synaptic response, so why many variants should have
evolved is still a puzzle.
C N
Binding ▼ Much of the sequence variation that accounts for receptor diversity
Type 3 domain arises at the genomic level, that is, different genes give rise to distinct
Kinase-linked receptor subtypes. Additional variation arises from alternative mRNA
receptors splicing, which means that a single gene can give rise to more than
one receptor isoform. After translation from genomic DNA, the mRNA
normally contains non-coding regions (introns) that are excised by
Catalytic mRNA splicing before the message is translated into protein. Depend-
domain ing on the location of the splice sites, splicing can result in inclusion
C or deletion of one or more of the mRNA coding regions, giving rise
to long or short forms of the protein. This is an important source of
variation, particularly for GPCRs, producing receptors with different
D C binding characteristics and different signal transduction mechanisms,
Binding
Type 4 although its pharmacological relevance remains to be clarified. Another
domain
Nuclear process that can produce different receptors from the same gene is
receptors mRNA editing, which involves the mischievous substitution of one
DNA-binding base in the mRNA for another, and hence potentially a small variation
domain
in the amino acid sequence of the expressed receptor.
(‘zinc
fingers’) Molecular heterogeneity of this kind is a feature of all kinds
N of receptors – indeed of functional proteins in general. New
receptor subtypes and isoforms continue to be discovered,
and regular updates of the catalogue are available
Fig. 3.3 General structure of four receptor families. The
(www.guidetopharmacology.org/). The problems of clas-
rectangular segments represent hydrophobic α-helical regions of
sification, nomenclature and taxonomy resulting from this
the protein comprising approximately 20 amino acids, which
flood of data have been mentioned earlier.
form the membrane-spanning domains of the receptors. The
pink shaded areas illustrate the region of the orthosteric
We will now describe the characteristics of each of the
ligand-binding domains. (A) Type 1: ligand-gated ion channels. four receptor superfamilies.
The example illustrated here shows the subunit structure of the
TYPE 1: LIGAND-GATED ION CHANNELS
nicotinic acetylcholine receptor. The subunit structure of other
ligand-gated ion channels is shown in Fig. 3.5. Many ligand- The nicotinic acetylcholine receptor, which we find at the
gated ion channels comprise four or five subunits of the type skeletal neuromuscular junction (Ch. 14), in autonomic
shown, the whole complex containing 16–20 membrane- ganglia (Ch. 14) and in the brain (Ch. 40), is a typical example
spanning segments surrounding a central ion channel. (B) Type of a ligand-gated ion channel, known as the cys-loop recep-
2: G protein–coupled receptors (GPCRs). The two ligand-binding tors (so called because they have in their structure a large
domains shown illustrate the position of the orthosteric intracellular domain between transmembrane domains 3
ligand-binding domains on different types of GPCRs, there and 4 containing multiple cysteine residues [see Fig. 3.3A]).
would be only one on each GPCR. (C) Type 3: kinase-linked Others of this type include the GABAA and glycine receptors
receptors. Most growth factor receptors incorporate the (Ch. 39) as well as the 5-hydroxytryptamine type 3 (5-HT3;
ligand-binding and enzymatic (kinase) domains in the same Chs 16 and 40) receptor. Other types of ligand-gated ion
molecule, as shown, whereas cytokine receptors lack an
intracellular kinase domain but link to cytosolic kinase molecules.
Other structural variants also exist. (D) Type 4: nuclear receptors
6
that control gene transcription. Receptors for 5-hydroxytryptamine (see Ch. 16) are currently the
champions with respect to diversity, with 13 subtypes of GPCR and 1
ligand-gated ion channel all responding to the same endogenous ligand. 27
3 SECTION 1 General Principles

channel exist – namely ionotropic glutamate receptors (Ch.

39) and purinergic P2X receptors (Chs 17 and 40) that differ A
in several respects from the nicotinic acetylcholine receptor β δ
(see Fig. 3.5). In addition to the ligand-gated ion channels α α
ACh ACh
found on the cell membrane that mediate fast synaptic 6 nm
transmission, there are also intracellular ligand-gated ion
channels – namely the inositol trisphosphate (IP3) and
ryanodine receptors (see Ch. 4) that release Ca2+ from Exterior
intracellular stores.
MOLECULAR STRUCTURE Membrane 3 nm

Ligand-gated ion channels have structural features in

common with other ion channels, described on p. 46. The Cytosol
2 nm
nicotinic acetylcholine receptor cloned from the Torpedo
electric ray (Fig. 3.4),7 consists of a pentameric assembly
of different subunits, of which there are four types, termed α-Helices forming gate
α, β, γ and δ, each of molecular weight (Mr) 40–58 kDa.
The subunits show marked sequence homology, and each
contains four membrane-spanning α-helices, inserted into β δ
the membrane as shown in Fig. 3.4B. The pentameric
structure (α2, β, γ, δ) possesses two acetylcholine binding ACh
sites, each lying at the interface between one of the two α Pore ~0.7 nm
subunits and its neighbour. Both must bind acetylcholine diameter α α
molecules for the receptor to be activated. Fig. 3.4B shows
ACh
the receptor structure. Each subunit spans the membrane γ
four times, so the channel comprises no fewer than 20
membrane-spanning helices surrounding a central pore.
▼ One of the transmembrane helices (M2) from each of the five subunits
B
forms the lining of the ion channel (see Fig. 3.4). The five M2 helices
that form the pore are sharply kinked inwards halfway through the
membrane, forming a constriction. When acetylcholine molecules
bind, a conformation change occurs in the extracellular part of the
receptor, which twists the α subunits, causing the kinked M2 segments
to swivel out of the way, thus opening the channel. The channel
lining contains a series of anionic residues, making the channel
selectively permeable to cations (primarily Na+ and K+, although some
types of nicotinic receptor are permeable to Ca2+ as well).
The use of site-directed mutagenesis, which enables short regions,
or single residues, of the amino acid sequence to be altered, has
shown that a mutation of a critical residue in the M2 helix changes
the channel from being cation permeable (hence excitatory in the
context of synaptic function) to being anion permeable (typical of
receptors for inhibitory transmitters such as GABA and glycine). Other Fig. 3.4 Structure of the nicotinic acetylcholine receptor
mutations affect properties such as gating and desensitisation of (a typical ligand-gated ion channel). (A) Schematic diagram in
ligand-gated channels. side view (upper) and plan view (lower). The five receptor
Other ligand-gated ion channels, such as glutamate receptors (see subunits (α2, β, γ, δ) form a cluster surrounding a central
Ch. 39) and P2X receptors (see Chs 17 and 40), whose structures are transmembrane pore, the lining of which is formed by the M2
shown in Fig. 3.5, have a different architecture. Ionotropic glutamate helical segments of each subunit. These contain a
receptors are tetrameric and the pore is built from loops rather than preponderance of negatively charged amino acids, which makes
transmembrane helices, in common with many other (non-ligand-gated) the pore cation selective. There are two acetylcholine binding
ion channels (see Fig. 3.20). P2X receptors are trimeric and each subunit sites in the extracellular portion of the receptor, at the interface
has only two transmembrane domains (North, 2002). The nicotinic between the α and the adjoining subunits. When acetylcholine
receptor and other cys-loop receptors are pentamers with two agonist binds, the kinked α-helices either straighten out or swing out of
binding sites on each receptor. Binding of one agonist molecule to the way, thus opening the channel pore. (B) High-resolution
one site increases the affinity of binding at the other site (positive image showing revised arrangement of intracellular domains.
cooperativity) and both sites need to be occupied for the receptor to (Panel [A] based on Unwin, N., 1993. Nicotinic acetylcholine
be activated and the channel to open. Some ionotropic glutamate receptor at 9Å resolution. J. Mol. Biol. 229, 1101–1124, and
receptors have as many as four agonist binding sites and P2X receptors
Unwin, N., 1995. Acetylcholine receptor channel imaged in the
have three, but they appear to open when two agonist molecules are
open state. Nature 373, 37–43; panel [B] reproduced with
bound. Once again we realise that the simple model of receptor
permission from Unwin, N., 2005. Refined structure of the
nicotinic acetylcholine receptor at 4Å resolution. J. Mol. Biol.
346(4), 967–989.)
7
In early studies the Torpedo electric ray was used to isolate and purify
the nicotinic receptor as it expresses a very high density of nicotinic
receptors on its electroplaques. We now realise that the subunit
compositions of the mammalian neuromuscular (Ch. 14) and neuronal
(Chs 14 and 40) nicotinic receptors are different from that of the
28 Torpedo but here we focus on the Torpedo receptor to keep it simple.
 How drugs act: molecular aspects 3
Ionotropic Calcium release type
Cys-loop type P2X type
glutamate type
N N N

C C

C
Examples: nAChR, GABAA, Examples: NMDA Example: P2XR Example: IP3R, RyR
5-HT3

(pentameric assembly) (tetrameric assembly) (trimeric assembly) (tetrameric assembly)

Fig. 3.5 Molecular architecture of ligand-gated ion channels. Red and blue rectangles represent membrane-spanning α-helices and
blue hairpins represent the P loop pore-forming regions. 5-HT3, 5-hydroxytryptamine type 3 receptor; GABAA, GABA type A receptor; IP3R,
inositol trisphosphate receptor; nAChR, nicotinic acetylcholine receptor; NMDA, N-methyl-D-aspartatic acid receptor; P2XR, purine P2X
receptor; RyR, ryanodine receptor.

activation shown in Fig. 2.1 is an oversimplification as it only con- be characterised by β/α ≫ 1, whereas for a drug of low efficacy β/α
sidered one agonist molecule binding to produce a response. For two has a lower value.
or more agonist molecules binding, more complex mathematical At some ligand-gated ion channels the situation is more complicated
models are needed (see Colquhoun, 2006). because different agonists may cause individual channels to open to
one or more of several distinct conductance levels (see Fig. 3.6B).
THE GATING MECHANISM This implies that there is more than one R* conformation. Furthermore,
Receptors of this type control the fastest synaptic events desensitisation of ligand-gated ion channels (see Ch. 2) also involves
in the nervous system, in which a neurotransmitter acts one or more additional agonist-induced conformational states. These
findings necessitate some elaboration of the simple scheme in which
on the postsynaptic membrane of a nerve or muscle cell only a single open state, R*, is represented, and are an example of
and transiently increases its permeability to particular ions. the way in which the actual behaviour of receptors makes our theoreti-
Most excitatory neurotransmitters, such as acetylcholine cal models look a little threadbare.
at the neuromuscular junction (Ch. 14) or glutamate in the
central nervous system (Ch. 39), cause an increase in Na+
and K+ permeability and in some instances Ca2+ permeability.
At negative membrane potentials this results in a net inward Ligand-gated ion channels
current carried mainly by Na+, which depolarises the cell
and increases the probability that it will generate an action • These are sometimes called ionotropic receptors.
potential. The action of the transmitter reaches a peak in • They are involved mainly in fast synaptic transmission.
a fraction of a millisecond, and usually decays within a • There are several structural families, the commonest
few milliseconds. The sheer speed of this response implies being heteromeric assemblies of four or five subunits,
that the coupling between the receptor and the ion channel with transmembrane helices arranged around a central
is a direct one, and the molecular structure of the receptor– aqueous channel.
channel complex (see earlier) agrees with this. In contrast • Ligand binding and channel opening occur on a
to other receptor families, no intermediate biochemical steps millisecond timescale.
are involved in the transduction process. • Examples include the nicotinic acetylcholine, GABA
type A (GABAA), glutamate (e.g. N-methyl-D-aspartatic
▼ The patch clamp recording technique, devised by Neher and Sakmann, acid receptor [NMDA]) and ATP (P2X) receptors.
allows the very small current flowing through a single ion channel
to be measured directly (Fig. 3.6). The patch clamp technique provides
a view, rare in biology, of the physiological behaviour of individual
protein molecules in real time, and has given many new insights into TYPE 2: G PROTEIN–COUPLED RECEPTORS
the gating reactions and permeability characteristics of both ligand-
gated channels and voltage-gated channels. The magnitude of the GPCRs constitute the commonest single class of targets for
single channel conductance confirms that permeation occurs through therapeutic drugs. The GPCR family comprises many of
a physical pore through the membrane, because the ion flow is too the receptors that are familiar to pharmacologists, such as
large (about 107 ions per second) to be compatible with a carrier muscarinic AChRs, adrenoceptors, dopamine receptors,
mechanism. The channel conductance produced by different agonists 5-HT (serotonin) receptors, receptors for many peptides,
is the same, whereas the mean channel lifetime varies. The ligand– purine receptors and many others, including the chemo-
receptor interaction scheme shown in Chapter 2 is a useful model
receptors involved in olfaction and pheromone detection,
for ion-channel gating. The conformation R*, representing the open
state of the ion channel, is thought to be the same for all agonists,
and also many ‘orphans’ (see Fredriksson & Schiöth, 2005).
accounting for the finding that the channel conductance does not For most of these, pharmacological and molecular studies
vary. Kinetically, the mean open time is determined mainly by the have revealed a variety of subtypes. All have the charac-
closing rate constant, α, and this varies from one drug to another. teristic heptahelical structure (see Fig. 3.3B).
As explained in Chapter 2 (see Fig. 2.1), an agonist of high efficacy Many neurotransmitters, apart from peptides, can interact
that activates a large proportion of the receptors that it occupies will with both GPCRs and ligand-gated channels, allowing the 29
3 SECTION 1 General Principles

Positive allosteric
A Nicotinic acetylcholine channel openings
modulator

Number of open channels

0
Cell exterior

Membrane

3 pA 2 Agonist

10 ms

B NMDA channel openings

Cell interior
Conductance states

Fig. 3.7 Structure of the M2 muscarinic receptor. High-

18 pS resolution image showing the conformation of the M2 muscarinic
38 pS receptor bound with both an agonist (orthosteric) and a positive
allosteric modulator. The brown cylinders represent the
transmembrane domains. The full extent of the N- and
2 pA C-terminal domains and the third intracellular loop are not
shown. (Courtesy A. Christopoulos.)
20 ms

Fig. 3.6 Single channel openings recorded by the patch biology caught up very rapidly with pharmacology, and
clamp technique. (A) Acetylcholine-operated ion channels at with the sequencing of the human genome the amino acid
the frog motor endplate. The pipette, which was applied tightly sequence of all the GPCRs hitherto identified by their
to the surface of the membrane, contained 10 µmol/L ACh. The pharmacological properties was revealed, as was the struc-
downward deflections show the currents flowing through single
ture of many novel GPCRs. More recently the difficulties
ion channels in the small patch of membrane under the pipette
of crystallising GPCRs have been overcome, allowing the
tip. Towards the end of the record, two channels can be seen
to open with a discrete step from the first to the second. (B)
use of the powerful technique of X-ray crystallography to
Single-channel N-methyl-D-aspartic acid receptor (NMDA)
study the three-dimensional molecular structure of these
receptor currents recorded from cerebellar neurons in the receptors in detail (Fig. 3.7) (Zhang et al., 2015). Also,
outside-out patch conformation. NMDA was added to the computational molecular docking and nuclear magnetic
outside of the patch to activate the channel. The channel opens resonance (NMR) methods have been developed to study
to multiple conductance levels. In (B) the openings to the higher ligand binding and subsequent conformational changes
conductance level and the subsequent closings are smooth, associated with activation (see Sounier et al., 2015). This
indicating that one channel is opening (two channels would not is starting to provide important information on agonist-
be expected to open and close simultaneously) whereas in (A) and antagonist-bound receptor conformations as well as
there are discrete steps indicating two channels. (Panel [A] receptor–G protein interactions. From such studies we are
courtesy D. Colquhoun and D.C. Ogden; panel [B] reproduced gaining a clearer picture of the mechanism of activation of
with permission from Cull-Candy, S.G. & Usowicz, M.M., 1987. GPCRs and the factors determining agonist efficacy, as well
Nature 325, 525–528.) as having a better basis for designing new GPCR ligands.
GPCRs consist of a single polypeptide chain, usually of
350–400 amino acid residues, but in some cases up to 1100
same molecule to produce fast (through ligand-gated ion residues. The general anatomy is shown in Fig. 3.3B. Their
channels) and relatively slow (through GPCRs) effects. characteristic structure comprises seven transmembrane
Individual peptide hormones, however, generally act either α-helices, similar to those of the ion channels discussed
on GPCRs or on kinase-linked receptors (see later), but previously, with an extracellular N-terminal domain of
rarely on both, and a similar choosiness applies to the many varying length, and an intracellular C-terminal domain.
ligands that act on nuclear receptors.8 GPCRs are divided into three main classes – A, B and
C (Table 3.2). There is considerable sequence homology
MOLECULAR STRUCTURE between the members of one class, but little between dif-
In 1986 the first pharmacologically relevant GPCR, the β2 ferent classes. They share the same seven transmembrane
adrenoceptor (Ch. 15), was cloned. Thereafter molecular helix (heptahelical) structure, but differ in other respects,
principally in the length of the extracellular N-terminus
and the location of the agonist binding domain. Class A is
8
Examples of promiscuity are increasing, however. Steroid hormones, by far the largest, comprising most monoamine, neuropep-
normally faithful to nuclear receptors, make the occasional pass at ion
channels and GPCRs, and some eicosanoids act on nuclear receptors as
tide and chemokine receptors. Class B includes receptors
well as GPCRs. Nature is quite open-minded, although such examples for some other peptides, such as calcitonin and glucagon.
30 are liable to make pharmacologists frown and students despair. Class C is the smallest, its main members being the
 How drugs act: molecular aspects 3
Table 3.2 Main G protein–coupled receptor classesa,b

Class Receptorsb Structural features

A: rhodopsin family The largest group. Receptors for most amine Short extracellular (N-terminal) tail.
neurotransmitters, many neuropeptides, purines, Ligand binds to transmembrane helices
prostanoids, cannabinoids, etc. (amines) or to extracellular loops (peptides)
B: secretin/glucagon receptor Receptors for peptide hormones, including Intermediate extracellular tail incorporating
family secretin, glucagon, calcitonin ligand-binding domain
C: metabotropic glutamate Small group. Metabotropic glutamate receptors, Long extracellular tail incorporating
receptor/calcium sensor family GABAB receptors, Ca2+-sensing receptors ligand-binding domain
a
Other classes include frizzled G protein–coupled receptors (GPCRs), adhesion GPCRs and receptors for pheromones.
b
For full lists, see <www.guidetopharmacology.org>.

metabotropic glutamate and GABA receptors, and the PROTEINASE-ACTIVATED RECEPTORS12

Ca2+-sensing receptors.9 ▼ Although activation of GPCRs is normally the consequence of a dif-
fusible agonist, it can be the result of proteinase activation. Four types
▼ The understanding of the function of receptors of this type owes
of protease-activated receptors (PARs) have been identified (see review
much to studies of a closely related protein, rhodopsin, which is
by Ramachandran et al., 2012). Many proteinases, such as thrombin (a
responsible for transduction in retinal rods. This protein is abundant
proteinase involved in the blood-clotting cascade; see Ch. 25), activate
in the retina, and much easier to study than receptor proteins (which
PARs by snipping off the end of the extracellular N-terminal tail of
are anything but abundant); it is built on an identical plan to that
the receptor (Fig. 3.8) to expose five or six N-terminal residues that
shown in Fig. 3.3B and also produces a response in the rod (hyperpo-
bind to receptor domains in the extracellular loops, functioning as a
larisation, associated with inhibition of Na+ conductance) through a
‘tethered agonist’. Receptors of this type occur in many tissues and
mechanism involving a G protein (see p. 32, Fig. 3.9). The most obvious
they appear to play a role in inflammation and other responses to
difference is that a photon, rather than an agonist molecule, produces
tissue damage where tissue proteinases are released. A PAR molecule
the response. In effect, rhodopsin can be regarded as incorporating its
can be activated only once, because the cleavage cannot be reversed,
own inbuilt agonist molecule, namely retinal, which isomerises from
and thus continuous resynthesis of the receptor protein is necessary.
the trans (inactive) to the cis (active) form when it absorbs a photon.
Inactivation occurs by a further proteolytic cleavage that frees the
For small molecules, such as noradrenaline (norepinephrine) tethered ligand, or by desensitisation, involving phosphorylation (see
Fig. 3.8), after which the receptor is internalised and degraded, to be
and acetylcholine, the ligand-binding domain of class A
replaced by newly synthesised protein.
receptors is buried in the cleft between the α-helical segments
within the membrane (see Figs 3.3B and 3.7), similar to the
slot occupied by retinal in the rhodopsin molecule.10 Peptide
ligands, such as substance P (Ch. 19), bind more superficially G protein–coupled receptors
to the extracellular loops, as shown in Fig. 3.3B. From crystal
structures and single-site mutagenesis experiments, it is • These are sometimes called metabotropic or seven-
possible to map the ligand-binding domain of these receptors. transmembrane-domain (7-TDM) receptors.
Recent advances in computational molecular docking of • Structures comprise seven membrane-spanning
ligands into the ligand–receptor-binding domain have made α-helices.
it possible to design novel synthetic ligands based primarily • The G protein is a membrane protein comprising three
on knowledge of the receptor structure (see Manglik et al., subunits (α, β, γ), the α subunit possessing GTPase
2016) – an important milestone in drug development, which activity.
has relied up to now mainly on the structure of endogenous • The G protein interacts with a binding pocket on the
mediators (such as histamine) or plant alkaloids (such as intracellular surface of the receptor.
morphine) for its chemical inspiration.11
• When the G protein binds to an agonist-occupied
receptor, the α subunit binds GTP, dissociates and is
9
then free to activate an effector (e.g. a membrane
The Ca2+-sensing receptor (see Conigrave et al., 2000) is an unusual
enzyme). In some cases, the βγ subunit is the activator
GPCR that is activated not by conventional mediators, but by
extracellular Ca2+ in the range of 1–10 mmol/L – an extremely low species.
affinity in comparison with other GPCR agonists. It is expressed by cells • Activation of the effector is terminated when the bound
of the parathyroid gland, and serves to regulate the extracellular Ca2+ GTP molecule is hydrolysed, which allows the α
concentration by controlling parathyroid hormone secretion (Ch. 37).
This homeostatic mechanism is quite distinct from the mechanisms for subunit to recombine with βγ.
regulating intracellular Ca2+, discussed in Chapter 4. • There are several types of G protein, which interact
10
Hydrophilic small molecules access their ligand-binding domain from with different receptors and control different effectors.
the extracellular space down the water-filled cleft, however for highly
lipophilic molecules such as those activating the cannabinoid CB1 and
• Examples include muscarinic acetylcholine receptors,
lysophospholipid S1P1 receptors access appears to be through a adrenoceptors, neuropeptide and chemokine
membrane-embedded access channel in the side of the receptor. receptors, and proteinase-activated receptors.
11
In the past many lead compounds have come from screening huge
chemical libraries (see Ch. 60). No inspiration was required, just robust
assays, large computers and efficient robotics. Now with the generation
of crystal structures we have moved to a more sophisticated age in
drug discovery. 12
These receptors were formerly called protease-activated receptors. 31
3 SECTION 1 General Principles

N
Tethered agonist
Cleavage by thrombin
N
N Released N
fragment

Phosphorylation

P
INACTIVE ACTIVE DESENSITISED

Fig. 3.8 Activation of a proteinase-activated receptor by cleavage of the N-terminal extracellular domain. Inactivation occurs by
phosphorylation. Recovery requires resynthesis of the receptor.

Resting state
Receptor Receptor occupied by agonist

Target Target Target Target

1 2 1 2
α βγ α βγ
Inactive GDP Inactive Inactive GDP Inactive

GTP

Target proteins
GTP hydrolysed
activated

Target Target Target Target

1 α 2 1 α 2
βγ βγ
Active GDP Active Active GTP Active
+
P

Fig. 3.9 The function of the G protein. The G protein consists of three subunits (α, β, γ), which are anchored to the membrane through
attached lipid residues. Coupling of the α subunit to an agonist-occupied receptor causes the bound GDP to exchange with intracellular
GTP; the α–GTP complex then dissociates from the receptor and from the βγ complex, and interacts with a target protein (target 1, which
may be an enzyme, such as adenylyl cyclase or phospholipase C). The βγ complex also activates a target protein (target 2, which may be
an ion channel or a kinase). The GTPase activity of the α subunit is increased when the target protein is bound, leading to hydrolysis of the
bound GTP to GDP, whereupon the α subunit reunites with βγ.

proteins, but were actually called G proteins because of

G PROTEINS AND THEIR ROLE their interaction with the guanine nucleotides, GTP and
G proteins comprise a family of membrane-resident proteins GDP. For more detailed information on the structure and
whose function is to respond to GPCR activation and pass functions of G proteins, see reviews by Milligan and Kostenis
on the message inside the cell to the effector systems that (2006), and Oldham and Hamm (2008). G proteins consist
generate a cellular response. They represent the level of of three subunits: α, β and γ (Fig. 3.9). Guanine nucleotides
middle management in the organisational hierarchy, bind to the α subunit, which has enzymic (GTPase) activity,
intervening between the receptors – choosy mandarins, catalysing the conversion of GTP to GDP. The β and γ
alert to the faintest whiff of their preferred chemical – and subunits remain together as a βγ complex. The ‘γ’ subunit
the effector enzymes or ion channels – the blue-collar brigade is anchored to the membrane through a fatty acid chain,
that gets the job done without needing to know which coupled to the G protein through a reaction known as
32 hormone authorised the process. They are the go-between prenylation. In the ‘resting’ state (see Fig. 3.9), the G protein
 How drugs act: molecular aspects 3
Table 3.3 The main G protein subtypes and their functionsa

Subtypes Main effectors Notes

b
Gα subunits
Gαs Stimulates adenylyl cyclase, causing increased cAMP formation Activated by cholera toxin, which
blocks GTPase activity, thus
preventing inactivation
Gαi Inhibits adenylyl cyclase, decreasing cAMP formation Blocked by pertussis toxin, which
prevents dissociation of αβγ complex
Gαo ? Limited effects of α subunit (effects mainly due to βγ subunits) Blocked by pertussis toxin. Occurs
mainly in nervous system
Gαq Activates phospholipase C, increasing production of second messengers
inositol trisphosphate and diacylglycerol (see pp. 36–38) thus releasing
Ca2++ from intracellular stores and activating protein kinase C (PKC)
Gα12/13 Activates Rho and thus Rho kinase
Gβγ subunits
Activate potassium channels Many βγ isoforms identified, but
Inhibit voltage-gated calcium channels specific functions are not yet known
Activate GPCR kinases (GRKs, pp. 38–39)
Activate mitogen-activated protein kinase cascade
Interact with some forms of adenylyl cyclase and with phospholipase Cβ
a
This table lists only those isoforms of major pharmacological significance. Many more have been identified, some of which play roles in
olfaction, taste, visual transduction and other physiological functions (see Offermanns, 2003).
b
Initially the subscripts ‘s’ and ‘i’ were used to denote stimulatory and inhibitory actions on adenylyl cyclase but, subsequently, the terms
used, ‘q’ and ‘12/13’, have little logic behind their use.
GPCR, G protein–coupled receptor.

exists as an αβγ trimer, which may or may not be precoupled Signalling is terminated when the hydrolysis of GTP to
to the receptor, with GDP occupying the site on the α GDP occurs through the inherent GTPase activity of the α
subunit. When a GPCR is activated by an agonist this subunit. The resulting α–GDP then dissociates from the
induces small changes in residues around the ligand-binding effector, and reunites with βγ, completing the cycle.
pocket that translate to larger rearrangements of the ▼ Attachment of the α subunit to an effector molecule actually
intracellular regions of the receptor that open a cavity on increases its GTPase activity, the magnitude of this increase being
the intracellular side of the receptor into which the G protein different for different types of effector. Because GTP hydrolysis is
can bind, resulting in a high-affinity interaction of αβγ and the step that terminates the ability of the α subunit to produce its
the receptor. This agonist-induced interaction of αβγ with effect, regulation of its GTPase activity by the effector protein means
the receptor occurs within about 50 ms, causing the bound that the activation of the effector tends to be self-limiting. In addition,
GDP to dissociate and to be replaced with GTP (GDP–GTP there is a family of about 20 cellular proteins, regulators of G protein
exchange), which in turn causes dissociation of the G protein signalling (RGS) proteins (see review by Sjögren, 2017), that possess
a conserved sequence that binds specifically to α subunits to increase
trimer, releasing α–GTP from the βγ subunits; these are
greatly their GTPase activity, so hastening the hydrolysis of GTP and
the ‘active’ forms of the G protein, which diffuse in the inactivating the complex. RGS proteins thus exert an inhibitory effect
membrane and can associate with various enzymes and on G protein signalling, a mechanism that is thought to have a regula-
ion channels, causing activation of the target (see Fig. 3.9). tory function in many situations.
It was originally thought that only the α subunit had a
signalling function, the βγ complex serving merely as a Different GPCRs couple to different G proteins and thus
chaperone to keep the flighty α subunits out of range of produce distinct cellular responses. For example, M2 mus-
the various effector proteins that they might otherwise carinic acetylcholine receptors (mAChRs) and β1 adrenocep-
excite. However, the βγ complexes actually make assigna- tors, both of which occur in cardiac muscle cells, produce
tions of their own, and control effectors in much the same opposite functional effects (Chs 14 and 15). Four main classes
way as the α subunits. Association of α or βγ subunits with of G protein (Gs, Gi, Go and Gq) are of pharmacological
target enzymes or channels can cause either activation or importance (Table 3.3). These differ primarily in the α
inhibition, depending on which G protein is involved (see subunit they contain.13 G proteins show selectivity with
Table 3.3). G protein activation results in amplification,
because a single agonist–receptor complex can activate
several G protein molecules in turn, and each of these can 13
In humans there are 21 known subtypes of Gα, 6 of Gβ and 12 of Gγ,
remain associated with their effector enzyme for long enough providing, in theory, about 1500 variants of the trimer. We know little
about the role of different α, β and γ subtypes, but it would be rash to
to produce many molecules of product. The product (see assume that the variations are functionally irrelevant. By now, you will
later) is often a ‘second messenger’, and further amplification be unsurprised (even if somewhat bemused) by such a display of
occurs before the final cellular response is produced. molecular heterogeneity, for it is the way of evolution. 33
3 SECTION 1 General Principles

Inhibitory Target Stimulatory

receptor enzyme receptor
Gi Gs

Ri Rs
βγ αi αs βγ

Fig. 3.10 Bidirectional control of a target enzyme, such as adenylyl cyclase by Gs and Gi. Heterogeneity of G proteins allows
different receptors to exert opposite effects on a target enzyme.

respect to both the receptors and the effectors with which introduced the concept of second messengers in signal
they couple, having specific recognition domains in their transduction. cAMP is a nucleotide synthesised within the
structure complementary to specific G protein-binding cell from ATP by the action of a membrane-bound enzyme,
domains in the receptor and effector molecules. For example, adenylyl cyclase. It is produced continuously and inactivated
Gs and Gi produce, respectively, stimulation and inhibition by hydrolysis to 5′-AMP by the action of a family of enzymes
of the enzyme adenylyl cyclase (Fig. 3.10). known as phosphodiesterases (PDEs). Many different drugs,
One functional difference that has been useful as an hormones and neurotransmitters act on GPCRs and increase
experimental tool to distinguish which type of G protein or decrease the catalytic activity of adenylyl cyclase (see
is involved in different situations concerns the action of Fig. 3.10), thus raising or lowering the concentration of
two bacterial toxins, cholera toxin and pertussis toxin (see cAMP within the cell. In mammalian cells there are 10
Table 3.3). These toxins, which are enzymes, catalyse a different molecular isoforms of the enzyme, some of which
conjugation reaction (ADP ribosylation) on the α subunit respond selectively to Gαs or Gαi.
of G proteins. Cholera toxin acts only on Gs, and it causes Cyclic AMP regulates many aspects of cellular function
persistent activation. Many of the symptoms of cholera, including, for example, enzymes involved in energy
such as the excessive secretion of fluid from the gastro- metabolism, cell division and cell differentiation, ion
intestinal epithelium (leading to ‘rice-water stools’), are transport, ion channels and the contractile proteins in smooth
due to the uncontrolled activation of adenylyl cyclase that muscle. These varied effects are, however, all brought about
occurs. Pertussis toxin specifically blocks Gi and Go by by a common mechanism, namely the activation of protein
preventing dissociation of the G protein trimer. Pertussis kinases by cAMP (known as cyclic AMP-dependent protein
toxin is released from Bordetella pertussis bacteria, which kinases) in eukaryotic cells. One important cyclic AMP-
cause whooping cough. As with cholera toxin, the symptoms dependent protein kinase is protein kinase A (PKA). Protein
caused by pertussis toxin are related to its effects on G kinases regulate the function of many different cellular
proteins, but in this case by inhibiting Gi and Go rather proteins by controlling protein phosphorylation. Fig. 3.11
than activating Gs and leading to changes in respiratory shows how increased cAMP production in response to
tract secretion and a distinctive cough rather than the β-adrenoceptor activation affects enzymes involved in
copious diarrhoea of cholera. glycogen and fat metabolism in liver, fat and muscle cells.
The result is a coordinated response in which stored energy
TARGETS FOR G PROTEINS in the form of glycogen and fat is made available as glucose
The main targets for G proteins, through which GPCRs to fuel muscle contraction.
control different aspects of cell function (see Table 3.3), are: Other examples of regulation by PKA include the
increased activity of voltage-gated calcium channels in heart
• adenylyl cyclase, the enzyme responsible for cAMP
muscle cells (see Ch. 22). Phosphorylation of these channels
formation;
increases the amount of Ca2+ entering the cell during the
• phospholipase C, the enzyme responsible for inositol
action potential, and thus increases the force of contraction
phosphate and diacylglycerol (DAG) formation;
of the heart.
• ion channels, particularly calcium and potassium
In smooth muscle, PKA phosphorylates (thereby inactivat-
channels;
ing) another enzyme, myosin light-chain kinase, which is
• Rho A/Rho kinase, a system that regulates the activity
required for contraction. This accounts for the smooth muscle
of many signalling pathways controlling cell
relaxation produced by many drugs that increase cAMP
growth, proliferation and motility, smooth muscle
production in smooth muscle (see Ch. 4).
contraction, etc.;
As mentioned earlier, receptors linked to Gi rather than
• mitogen-activated protein kinase (MAP kinase), a system
Gs inhibit adenylyl cyclase, and thus reduce cAMP formation
that controls many cell functions, including cell
to elicit opposing responses to those receptors which activate
division and is also a target of several kinase-linked
Gs. Examples include certain types of mAChR (e.g. the M2
receptors.
receptor of cardiac muscle; see Ch. 14), α2 adrenoceptors
in smooth muscle (Ch. 15) and opioid receptors (see Ch.
The adenylyl cyclase/cAMP system 43). Adenylyl cyclase can be activated directly by drugs
The discovery by Sutherland and his colleagues of the role such as forskolin, which is used experimentally to study
of cAMP (cyclic 3′,5′-adenosine monophosphate) as an the role of the cAMP system.
intracellular mediator demolished at a stroke the barriers Cyclic AMP is hydrolysed within cells by PDEs, an
34 that existed between biochemistry and pharmacology, and important and ubiquitous family of enzymes. Twenty-four
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