SECOND MESSENGERs

CAMP
cGMP
Faraza Javed
PhD Pharmacology
 second MESSENGERS

Second messengers are intracellular signaling

molecules released by the cell to trigger physiological
changes such as proliferation, differentiation, migration,
survival, and apoptosis.

 Second messengers are therefore one of the initiating

components of intracellular signal
transduction cascades.
 Examples of second messengers include cyclic AMP,
cyclic GMP, Inositol trisphosphate, Diacylglycerol
and Calcium.

 Releases in response to exposure to extracellular

signaling molecules/ligands the first messenger, such
as neurotransmitters, hormones (epinephrine,
growth hormone and serotonin).
 The first messengers such as peptide hormones,
neurotransmitters usually do not physically cross the
phospholipid bilayers.

 First messengers need to be transduced into

secondary messengers, so that the extracellular signal
may be propagated intracellularly.
 Second messengers greatly amplify the strength of the
signal.

 Activate or inhibit the target enzymes of the pathway.

History
 Earl Wilbur Sutherland Jr. discovered secondary
messengers.

 He saw that epinephrine stimulate glycogenolysis in liver

cells, but epinephrine alone would not convert glycogen to
glucose

 He found that epinephrine had to trigger a secondary

messenger, cyclic AMP for the liver to convert glycogen to
glucose. He won the 1971 Nobel Prize in Medicine.
Common mechanisms of second messenger
systems

There are several different secondary messenger systems

(cAMP system, phosphoinositol system), but they all are
quite similar in overall mechanism, although the
substances involved and overall effects can vary.
 Types Of Second Messenger Molecules

 Three basic types of second messenger

molecules:

 Hydrophobic molecules:
membrane-associated e.g.
diacylglycerol, phosphatidylinositol

 Hydrophilic molecules: water-

soluble molecules, such as cAMP,
cGMP, IP3, and Ca2+, located
within the cytosol.

 Gases: nitric oxide (NO), carbon

monoxide (CO) and hydrogen
sulfide (H2S) which can diffuse both
through cytosol and across cellular
membranes.
 1. cAMP
 cAMP is a second
messenger, synthesized
from ATP by enzyme
adenylyl cyclase.
 Adenylate cyclase is
activated by stimulatory
G (Gs)-protein-coupled
receptors.
 Inhibited by adenylate
cyclase inhibitory G
(Gi)-protein-coupled
receptors.
 Cyclic AMP generated by adenylyl cyclases has three
major targets in most cells,
 The cyclic AMP dependent protein kinase (PKA),
 cAMP-regulated guanine nucleotide exchange factors
termed EPACs (exchange protein directly activated by
cAMP), and
 via PKA phosphorylation, a transcription factor termed
CREB (cAMP response element binding protein).
 PKA REGULATION by cAMP
 The most common downstream
effector of cAMP is Protein kinase
A(PKA).
 PKA is normally inactive as
tetrameric holoenzyme(two catalytic
and two regulatory units).
 The regulatory unit always block the
catalytic center of catalytic unit.
 Two cAMP molecules bind to each
PKA regulatory subunit.
 The regulatory subunit dissociate from
the catalytic subunit.
 The free catalytic subunits interact
with proteins to phosphorylate Ser or
Thr residues, thus producing cellular
response.
 PKA can phosphorylate a diverse array of
physiological targets such as metabolic enzymes and
transport proteins, and numerous regulatory proteins
including other protein kinases, regulation
of glycogen, sugar, and lipid metabolism, ion
channels, and transcription factors.
cAMP Response Element-Binding (CREB) Protein

Cellular transcription factor e.g. the neurotrophin

BDNF, tyrosine hydroxylase & many neuropeptides
binds to certain DNA sequences called cAMP response
elements (CRE) thereby increasing or decreasing the
transcription of the downstream genes.
 Protein synthesis-PKA
directly activate CREB,
which bind the cAMP
response element
(CRE). The activated
CREB protein then binds
to a CRE region, and is
then bound
to CBP (CREB binding
protein), which co-
activates it, allowing it to
switch certain genes on or
off and altering the
transcription.
 CREB has many functions in many different organs,
and some of its functions have been studied in relation
to the brain. CREB proteins in neurons are thought to
be involved in the formation of long-term memories.
CREB is also important for the survival of neurons, as
studied in genetically engineered mice, where CREB
and CREM were deleted in the brain. If CREB is lost
in the whole developing mouse embryo, the mice die
immediately after birth, again highlighting the critical
role of CREB in promoting survival.
 Cyclic AMP–Regulated Guanine Nucleotide Exchange
Factors

The small GTP-binding proteins are monomeric GTPases

and key regulators of cell function. The small GTPases
operate as binary switches that exist in GTP- or GDP-
liganded conformations. They integrate extracellular signals
from membrane receptors with cytoskeletal changes and
activation of diverse signaling pathways, regulating such
processes as phagocytosis, progression through the cell
cycle, cell adhesion, gene expression, and apoptosis.
 For example, many small GTPases are regulated by GEFs
(Guanine Nucleotide Exchange Factors). GEFs act by
binding to the GDP-liganded GTPase and catalyzing the
exchange of GDP for GTP.
 The two GEFs regulated by cAMP are able to activate
members of the Ras small GTPase family, Rap1 and Rap2;
these GEFs are termed exchange proteins activated by
cyclic AMP (EPAC-1 and EPAC-2). The EPAC pathway
provides an additional effector system for cAMP signaling
and drug action.
 These pathways play major role in Cell
differentiation/proliferation, cytoskeletal organization,
vesicular trafficking & nuclear transport.

 Potential target for cancer therapy.

Therapeutic Applications
Selected Drugs that target cAMP signalling

Drug cAMP Therapeutic Application

Salmeterol ↑ Asthma, COPD

Haloperidol ↑ Schizophrenia

Metoclopramide ↑ Nausea, Vomiting

Desmopressin ↑ Diabetes Insipidus

Metoprolol ↓ Angina, Hypertension,
CHF

Morphine ↓ Pain
Sumatriptan ↓ Migraine

Ibuprofen ↓ Inflammation, Pain

Ranitidine ↓ Peptic ulcer, GERD

Misoprostol ↓ Prevention of NSAID

ulcers
Cabergoline ↓ Parkinson’s disease
 Cyclic nucleotide phosphodiesterases (PDEs)

 Cyclic nucleotide phosphodiesterases form another family

of important signaling proteins whose activities are
regulated via the rate of gene transcription as well as by
second messengers (cyclic nucleotides or Ca2+) and
interactions with other signaling proteins such as β arrestin
and protein kinases. PDEs hydrolyze the cyclic 3′,5′-
phosphodiester bond in cAMP and cGMP, thereby
terminating their action.
 Phosphodiesterase 4 (PDE4) is the predominant cAMP-
degrading enzyme expressed in inflammatory cells.

 cAMP helps regulate T cell function.

 cAMP helps maintain immune homeostasis by suppressing

the release of proinflammatory mediators (eg, TNF-α, IL-
17, and IFN-γ). cAMP promote the release of anti-
inflammatory mediators (eg, IL-10) by immune cells.
Decrease in PDE4 increases cAMP, leads to increased
transcription of genes that have CRE sites, including the
gene for IL-10, which is an anti-inflammatory mediator.
 PDEs are drug targets for treatment of diseases such as
asthma, COPD, cardiovascular diseases such as heart
failure, atherosclerotic coronary and peripheral arterial
disease, and neurological disorders.
 Recent Development

A new Second Messenger, c-di-AMP was

discovered in Staphylococcus aureus with a Role in
Controlling Cell Size and Envelope Stress. This work was
published in the September 2011 Issue of PLoS Pathogens.
 Most of work on c-di-AMP signaling has been done in
Gram-positive bacteria and actinobacteria, where c-di-AMP
signaling pathways affect potassium transport, cell wall
structure, and antibiotic resistance.
 These findings greatly expand the c-di-AMP signaling role
and reveal a central metabolic regulatory role for a cyclic
dinucleotide.
 However, the molecular mechanisms of c-di-AMP signaling
are still poorly understood.
cGMP

The signaling pathways that regulate the synthesis of

cyclic GMP in cells include hormonal regulation of
transmembrane guanylate cyclases such as the atrial
natriuretic peptide receptor (ANP) and the activation
of soluble forms of guanylate cyclase by nitric oxide
(NO).
Unlike cAMP, cGMP has established signaling roles in
only few cell types

Signaling pathways that regulate synthesis include:

 Nitric Oxide

 Hormonal Regulation (ANP/BNP/CNP)

Nitric Oxide

 Nitric oxide (NO) is a

gas, diffuse through the
plasma membrane and
affect nearby cells.
 Synthesized from arginine
and oxygen by the NO
synthase.

 NO then activate soluble

guanylyl cyclase, to
produce cGMP.
 The function of NO is the
dilation of blood vessels.
 The acetylcholine
(neurotransmitter) acts on
endothelial cells to
stimulate NO synthesis.
 NO, diffuses to
neighboring smooth
muscle cells where it
interacts with the guanylyl
cyclase.
 This increase enzymatic
activity resulting in the
synthesis of cGMP.
 The cGMP then induces
muscle relaxation and
blood vessel dilation.
Nitric Oxide Synthase (NOS) exists as 4 isoforms:

 Neuronal type I isoform (nNOS)

 Inducible type II isoform (iNOS)
 Endothelial type III isoform (eNOS)
 Mitochondrial isoform (mtNOS)
NOS constitutively expressed in:

eNOS
Endothelium, cardiac myocytes, renal mesangial cells,
osteoblasts, platelets and is involved with regulating
vascular function (Vasodilation).

nNOS
CNS nerves and skeletal muscles, performs a role in cell
communication and is associated with plasma membranes
iNOS
Macrophages, Kupffer cells, neutrophils, fibroblasts,
vascular smooth muscle cells & endothelium and involved
in immune defence against pathogens.

mtNOS
NO in the mitochondrial matrix may regulate ATP
synthesis.
 Natriuretic Peptide

3 small peptide ligands:

 Atrial Natriuretic Peptide (ANP)

 Brain Natriuretic Peptide (BNP)
 C-type Natriuretic Peptide (CNP)

These peptides possess potent natriuretic, diuretic,

and vasodilating activities and are implicated in body fluid
homeostasis and blood pressure control.
The major physiological effects of these hormones are to
decrease blood pressure (ANP, BNP), to reduce cardiac
hypertrophy and fibrosis (BNP), and to stimulate long bone
growth (CNP).
The transmembrane receptors for ANP, BNP, and CNP
are ligand-activated guanylate cyclases. The ANP and
BNP receptors contain 450 amino acid extracellular
domain that binds the peptide, a short 20 amino acid
transmembrane domain, and large intracellular
domains that contain a kinase homology region, and a
C-terminal guanylate cyclase domain. Phosphorylation
of serine residues in the kinase domain is important for
activity; dephosphorylation of these residues leads to
desensitization of the receptor.
 Ligand brings
juxtamembrane regions
together

Phosphorylation of
serine residues→
Stimulation of guanyl
cyclase
Downstream reactions of cGMP:

 Activation of Protein Kinase G

 cGMP gated ion channels
 cGMP-modulated Phosphodiesterase

Pharmacologically important effects of elevated cyclic GMP

include modulation of platelet activation and relaxation of
smooth muscle. PDEs hydrolyze the cyclic 3′,5′-phosphodiester
bond in cAMP and cGMP, thereby terminating their action.
 Therapeutic Applications
Selected Drugs that target cGMP signalling

Drug cGMP Therapeutic Application

Nitroglycerine NO Donor Angina

Sodium Nitroprusside NO Donor Hypertensive

Emergencies
Nesiritide Synthetic Acutely decompensated
BNP CHF
Ecadotril ↑ BNP Congestive Heart Failure
BAY Series of Specific Asthma
Compounds activators & Graft survival after liver
BAY 41-2272 inhibitors of & lung transplantation.
8-pCPT-cGMP PKG Pulmonary Hypertension
Riociguat (Adempas)
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