THE JOURNAL OF BIOLOGICAL CHEMISTRY

1999 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 274, No. 14, Issue of April 2, pp. 96739676, 1999

Printed in U.S.A.

Urate Synthesis in the Blood-sucking Insect Rhodnius prolixus

STIMULATION BY HEMIN IS MEDIATED BY PROTEIN KINASE C*
(Received for publication, November 5, 1998, and in revised form, January 18, 1999)

Aurelio V. Graca-Souza, Mario A. C. Silva-Neto, and Pedro L. Oliveira

From the Departamento de Bioqumica Medica, Instituto de Ciencias Biomedicas, Centro de Ciencias da Saude,
Universidade Federal do Rio de Janeiro, Rio de Janeiro-RJ, Brasil, CEP 21910-590

Hemin is a catalyst of the formation of reactive oxygen

species. We proposed that hematophagous insects are
exposed to intense oxidative stress because of hemoglobin hydrolysis in their midgut (Petretsky, M. D., Ribeiro,
J. M. C., Atella, G. C., Masuda, H., and Oliveira, P. L.
(1995) J. Biol. Chem. 270, 1089310896). We have shown
that hemin stimulates urate synthesis in the blood-sucking insect Rhodnius prolixus (Graca-Souza, A. V., Petretsky, J. H., Demasi, M., Bechara, E. J. H., and Oliveira,
P. L. (1997) Free Radical Biol. Med. 22, 209 214). Once
released by fat body cells, urate accumulates in the hemolymph, where this radical scavenger constitutes an
important defense against blood-feeding derived oxidative stress.
Incubation of Rhodnius fat bodies with okadaic acid
raises the level of urate synthesis, suggesting that urate
production can be controlled by protein phosphorylation/dephosphorylation. Urate synthesis is stimulated
by dibutyryl cAMP and inhibited by N(2((p-bromocinnamil)amino)ethyl)-5-isoquinolinesulfonamide (H-89),
an inhibitor of protein kinase A, as well as activated by
the protein kinase C activator phorbol 12-myristate 13acetate. In the presence of hemin, however, inhibition of
urate synthesis by H-89 does not occur, suggesting that
the hemin stimulatory effect is not mediated by protein
kinase A. Calphostin C completely inhibits the hemininduced urate production, suggesting that the triggering of urate antioxidant response depends on protein
kinase C activation. This conclusion is reinforced by the
observation that in fat bodies exposed to hemin, both
protein kinase C activity and phosphorylation of specific
endogenous polypeptides are significantly increased.

Oxygen is toxic because of its ability to generate reactive

species that can damage cellular components such as nucleic
acids, proteins and lipids (1 4). To survive in an oxygen-rich
environment, aerobic organisms have developed an array of
antioxidant mechanisms to prevent or repair oxidative injury
(5). Uric acid has been proposed to be an important free radical
scavenger (6 8). High levels of allantoin, the product of urate
oxidation, were found in patients under oxidative stress (9).
According to the free radical theory of aging, high concentrations of urate in plasma may be correlated with lengthening of
* This work was supported by grants from the Conselho Nacional de
Desenvolvimento Cientfico e Tecnologico (CNPq), Fundacao de Coordenacao de Aperfeicoamento do Pessol de Nvel Superior (CAPES),
Financiadora de Estudos e Projetos (Finep), Programa de Nucleos de
Excelencia (PRONEX), and Programa de Apoio ao Desenvolvimento
Cientfico e Tecnologico (PADCT). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked advertisement in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 55-21-270-8647;
E-mail: [email protected].
This paper is available on line at http://www.jbc.org

the life span (10). Uric acid is the main end-product of nitrogen
metabolism in insects (11). It is synthesized in the fat body cells
(12) and secreted to the hemolymph for posterior absorption at
the Malpighian tubules during formation of insect urine (13,
14). Increased susceptibility to oxidative stress was reported in
mutants of Drosophila melanogaster (15) that are not able to
synthesize uric acid, pointing to an antioxidative role of urate
in insects.
Blood digestion by hematophagous insects creates an especially intense source of oxidative stress, because hemin, iron,
and hemoglobin itself are promoters of free radical formation,
mainly through Fenton-type reactions (16 21). We have recently shown that in the blood-sucking insect Rhodnius prolixus, urate is the most important low molecular weight antioxidant present in the hemolymph (22). The maintenance of
high urate titer in its extracellular fluids (up to 5 mM) protects
Rhodnius against oxidative damage caused by the intake of
large amounts of hemin in a blood meal.
Reactive oxygen species have been shown to modulate the
activity of protein kinases and phosphatases (23). However, the
triggering of a signal transduction cascade by an oxidant challenge, resulting in the activation of a low molecular weight
antioxidant defense has not been reported. Here we present
evidence that the stimulation of urate synthesis by hemin in R.
prolixus is exerted through protein kinase C activation.
EXPERIMENTAL PROCEDURES

ChemicalsAllopurinol, hemin, okadaic acid, Bt2cAMP, PMA,1 soybean trypsin inhibitor, leupeptin, benzamidine, and b-mercaptoethanol
were purchased from Sigma. Calphostin C, H-89, and H-8 were purchased from Calbiochem. Other reagents were of analytical grade.
InsectsR. prolixus were kept at 28 C and 80% relative humidity.
Experimental animals were adult, mated females, fed directly on rabbits in their first cycle after the imaginal moult.
Organ Culture and Urate DeterminationFat bodies were dissected
from adult females on the 4th or 5th day after a blood meal, rinsed for
2 min in 50 ml of Rhodnius physiological saline (24) and transferred to
a 96-well microplate (3 fat bodies/well) containing 200 ml of the same
solution per well. After a 30 min pre-incubation, the saline was discarded and fat bodies were incubated in Rhodnius saline with the
additions indicated in the figure legends. Because Me2SO was used to
solubilize most compounds employed as effectors, all incubation media
(including controls) contained 1% Me2SO. Urate secreted to the medium
was determined enzymatically (25) in a U-1100 Hitachi spectrophotometer using a medical diagnosis kit supplied by Doles (Goiania, GO).
RadioisotopesCarrier-free 32Pi was purchased from Comissao Nacional de Energia Nuclear (Sao Paulo, SP), purified by ion-exchange
chromatography on a Dowex 1X-10 column (26), and used in metabolic
labeling experiments and in the enzymatic synthesis of [g-32P]ATP (27).
PKC AssayTypically 30 40 fat bodies were dissected and homogenized in a Potter-Elvehjem tissue grinder in the presence of 150 mM
NaCl, 250 mM sucrose, 2.5 mM MgCl2, 2.5 mM EGTA, 50 mM b-mercap1
The abbreviations used are: PMA, phorbol 12-myristate 13-acetate;
H-89, N-(2-((p-bromocinnamil)amino)ethyl)-5-isoquinolinesulfonamide;
H-8, N(-2-(methylamino)ethyl)-5-isoquinolinesulfonamide; Me2SO, dimethyl sulfoxide; PKC, protein kinase C; PS, phosphatidylserine.

9673

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Protein Phosphorylation and Urate Synthesis

toethanol, 0.05 mg/ml soybean trypsin inhibitor, 0.05 mg/ml leupeptin,

and 1 mM benzamidine in 10 mM Tris-Cl, pH 7.4. After centrifugation at
4,000 3 g for 10 min to remove tissue debris, the supernatant was
centrifuged at 100,000 3 g for 1 h at 4 C. The pellet was discarded, and
the supernatant was used as a crude extract to measure PKC activity.
PKC activity against endogenous substrates was determined as described by Kikkawa et al. (28). Briefly, reaction medium (0.2 ml) contained 10 mM Tris-Cl, pH 7.4, 2.5 mM MgCl2, 1 mM CaCl2, and 100 mg
of crude fat body extract. Reactions were started by addition of
[g-32P]ATP to a final concentration of 10 mM (1,000 cpm/pmol). After 10
min of incubation at 37 C, 25-ml aliquots of the reaction medium were
transferred to a phosphocellulose sheet and washed three times for 15
min each with 10 ml of 25% ice-cold trichloroacetic acid. Incorporated
radioactivity was determined by liquid scintillation. Phosphatidylserine, PMA, calphostin C, and hemin were added as described in the
figure legends. Protein concentration was estimated accordingly Lowry
et al. (29).
Protein Phosphorylation in Intact CellsFat bodies from adult females on the 5th day after a blood meal were dissected and incubated
under a Zeiss stereomicroscope, rinsed for 2 min in 50 ml of phosphatefree Rhodnius saline, and transferred to a 96-well microplate (3 fat
bodies/well) containing 200 ml of Rhodnius saline in the presence of 100
mCi 32Pi. After 30 min of pre-incubation to allow endogenous formation
of [g-32P]ATP, the medium was discarded and replaced by Rhodnius
saline with the additions indicated in the figure legends. All incubations
were carried out at 28 C. 1 h later, organs were homogenized in SDS
sample buffer, immediately heated at 100 C for 3 min, and samples
were separated by 10% SDS-polyacrylamide gel electrophoresis (30).
After Coomassie Blue staining, gels were dried and exposed to a Kodak
X-Omat AR-5 film for 3 days at 70 C. Densitometry of the autoradiographs was carried out using a computer scanner (4,800 dpi) and the gel
analysis software QuantiScan (Biosoft, Cambridge, UK). Molecular
masses were determined using the following protein standards: myosin
(205 kDa), b-galactosidase (116 kDa), phosphorylase b (98 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and
cytochrome c (12.3 kDa).

FIG. 1. Urate synthesis is regulated by protein phosphorylation in R. prolixus fat body. Fat bodies were dissected and preincubated in a 96-multiwell microplate with Rhodnius physiological
saline during 30 min. After this time the medium was removed and
replaced by physiological saline alone (control), saline 1 400 mM allopurinol (allopurinol), 0.1 or 1 mM okadaic acid, or 400 mM allopurinol 1
1 mM okadaic acid. After incubation for 10 90 min, the media were
collected, and the amount of urate produced was determined as described under Experimental Procedures. Data are presented as
mean 6 S.D. for four determinations.

RESULTS AND DISCUSSION

In a previous report we observed that hemin injection or

exposure to 70% O2, two conditions that cause oxidative stress,
lead to increased urate levels in R. prolixus hemolymph (22).
Brief (20 min) exposure of isolated fat bodies to hemin were
enough to induce higher rates of urate synthesis (data not
shown), suggesting the existence of short-term regulatory
mechanisms. Here we show that treatment of Rhodnius fat
bodies with okadaic acid, an inhibitor of protein phosphatases
(31, 32), greatly enhances uric acid release (Fig. 1). This result
suggests that purine metabolism depends on protein phosphorylation in the fat body cell. The effect of okadaic acid was
caused by increased de novo synthesis of uric acid, and not
merely increased uric acid secretion by the organ, because
addition of allopurinol, a specific inhibitor of xanthine dehydrogenase, prevented the okadaic acid stimulation of urate
production (Fig. 1).
When the specific protein kinase A inhibitors H-8 (33) and
H-89 (34), were added to the incubation medium, a significant
(p , 0.05) inhibition (30 and 38%, respectively) was observed in
both cases (Fig. 2). Incubation of fat bodies with Bt2cAMP
resulted in a two-fold stimulation of urate synthesis (Fig. 2), an
effect that was counteracted by H-8 and H-89, confirming that
the stimulatory effect of Bt2cAMP should be attributed to protein kinase A activation. An analogous experiment was performed to test the role of PKC in the control of urate synthesis.
The phorbol ester analogue PMA (35, 36) was a very effective
stimulator of urate synthesis (Fig. 3), and the activation of
urate production by this compound was blocked by calphostin C
(37) and sphingosine (38) at the concentration used (2.5 and 50
mM, respectively). When we incubated the fat bodies with calphostin C and sphingosine in the absence of PMA (Fig. 3), we
did not observe inhibition of urate formation, indicating that
this signaling cascade was not actively modulating the urate
formation pathway.

FIG. 2. Protein kinase A and urate synthesis. Fat bodies were

dissected and pre-incubated as described in the legend to Fig. 1, and
then incubated for 90 min with the following additions: Rhodnius saline
(ctrl), 50 mM H-8, 1 mM H-89, 20 mM Bt2cAMP (cAMP), 50 mM H-8 1 20
mM Bt2cAMP, 1 mM H-89 1 20 mM Bt2cAMP, 400 mM allopurinol (allo),
and 400 mM allopurinol 1 20 mM Bt2cAMP. After incubation, the media
were collected and the amount of urate produced was determined as
described under Experimental Procedures. Data are presented as
mean 6 S.D. for four determinations.

When hemin was added to the incubation medium, high

rates of urate synthesis were observed (Fig. 4). This augmented
urate production seems to involve PKC, because calphostin C
reduced urate synthesis to levels close to those of the control
without hemin. On the other hand, addition of H-89 together
with hemin had no effect on urate secretion by the fat body,
indicating that protein kinase A does not participate in the
stimulation of urate production by hemin. However, the urate
overproduction induced by hemin seems to be a PKC-dependent phenomenon, a conclusion that led us to investigate the
effect of hemin on protein phosphorylation. When 32Pi-labeled
fat bodies were incubated with hemin, phosphorylation of specific polypeptides (151 and 73 kDa) increased by 190 and 100%,
respectively (Fig. 5, lanes 2 and 3) when compared with control
(Fig. 5, lane 1). This phosphorylation was blocked by calphostin
C (Fig. 5, lane 4), suggesting once more that the action of hemin
on fat body cells involves PKC activation. Although regulation
of PKC activity by superoxide (39) and other pro-oxidant species (40, 41) has already been reported, this is the first study

Protein Phosphorylation and Urate Synthesis

9675

FIG. 3. Protein kinase C and urate synthesis. Fat bodies were

dissected and pre-incubated as described in the legend to Fig. 1 and
then incubated for 90 min with the following additions: Rhodnius saline
(ctrl), 20 mM sphingosine (sphg), 2.5 mM calphostin C (cph C), 50 mM PMA
(pma), 20 mM sphingosine 1 50 mM PMA, 2.5 mM calphostin C 1 50 mM
PMA, 400 mM allopurinol (allo), and 400 mM allopurinol 1 50 mM PMA.
After incubation, the media were collected, and the amount of urate
produced was determined as described under Experimental Procedures. Data are presented as mean 6 S.D. for four determinations.

FIG. 5. Hemin stimulates the phosphorylation of 151- and 73kDa bands through PKC activation. Fat bodies were dissected and
pre-incubated with 100 mCi 32Pi for 30 min. After this period, the
medium was discarded and replaced by Rhodnius physiological saline
alone or with the additions described below. After 1 h of incubation, the
medium was discarded, and the organs were homogenized in SDSpolyacrylamide gel electrophoresis sample buffer and analyzed by 10%
SDS-polyacrylamide gel electrophoresis. Panel A, autoradiogram of the
gel: 1, control; 2, 0.1 mM hemin; 3, 0.5 mM hemin; 4, 0.5 mM hemin 1 2.5
mM calphostin C. Panel B, densitometric analysis of the 151- and 73-kDa
bands. Further conditions as described under Experimental Procedures.
FIG. 4. Hemin stimulation of urate synthesis is mediated by
protein kinase C. Fat bodies were dissected and pre-incubated as
described in Fig. 1 and then incubated for 90 min with the following
additions: Rhodnius saline alone (none), 500 mM hemin (ctrl), 500 mM
hemin 1 400 mM allopurinol (allo), 500 mM hemin 1 20 mM sphingosine
(sphg), 500 mM hemin 1 2.5 mM calphostin C (cph C), or 500 mM hemin
1 1 mM H-89. After incubation, the media were collected and urate
levels determined (see Experimental Procedures). Data are presented
as mean 6 S.D. for four determinations.

providing evidence that the synthesis of a low molecular weight

antioxidant can be regulated by an oxidant agent through PKC
activation.
Because of the amphiphilic nature of the hemin molecule, its
partition into cellular membranes is expected, and therefore
these membranes could be the site where hemin exerts its
stimulatory effect. However, PKC activation induced by hemin
may not be dependent on the presence of the plasma membrane. Incubation of fat body cytosolic (100,000 3 g) fraction
with different concentrations of hemin resulted in a 120% increase in PMA-stimulated/calphostin-inhibited phosphorylation of endogenous substrates (Fig. 6). This could be explained
either by activation of some other upstream soluble component
of the cascade or by a direct action of hemin (or hemin-derived
reactive species) on PKC itself.
PKC has been demonstrated to have its activity directly
stimulated by superoxide anion, redox cycling quinones and
micromolar levels of periodate (42, 43). Several mechanisms
have been proposed to explain PKC modulation by these effectors, including oxidation of regulatory and binding domains of
the kinase itself (44). Another possible mechanism for PKC
activity modulation is a thiol-dependent inactivation of protein
phosphatases 1 and 2A (45). Threonine/tyrosine phosphoryla-

FIG. 6. Hemin stimulates PKC activity in R. prolixus fat body

cytosolic fraction. Fat bodies were homogenized and PKC activity
was assayed in a cytosolic fraction obtained as described under Experimental Procedure. Incubation media were as follows: Rhodnius saline
(control); 20 mM PMA 1 20 mg PS/tube; 20 mM PMA 1 20 mg PS/tube in
the presence of 50 nM calphostin C; 0.01, 0.1, or 0.5 mM hemin or the
same concentrations of hemin in the presence of 50 nM calphostin C.

tion catalyzed by mitogen-activated protein kinase has been

shown to be stimulated by H2O2, ionizing radiations, and phorbol esters in NIH-3T3 cells, but a PKC-independent pathway
was involved (46). Nevertheless, the hemin-stimulated urate
synthesis reported here could involve a cross-talk between mitogen-activated protein kinase and PKC, because it has been
demonstrated that mitogen-activated protein kinase can be
activated by PKC (47, 48).
As pointed out before, hemin is produced in large amounts in
the digestive tract of Rhodnius, and it constitutes an intense

9676

Protein Phosphorylation and Urate Synthesis

physiological source of oxidative stress for this animal (49, 50).

The regulation of urate production according to hemin availability seems to be an important adaptation of this insect to
blood feeding. Urate has been identified as an important low
molecular weight antioxidant in extracellular fluids of vertebrates (9). It would be interesting to test whether the control of
urate formation by oxidative stress also occurs in mammalian
systems.
AcknowledgmentsWe express our gratitude to Dr. Martha M.
Sorenson for a critical reading of the manuscript, to Rosane O. M. M. da
Costa, Helosa S. L. Coelho, Llian S. C. Gomes, Jose de Souza L. Junior,
and Jose F. de Souza Neto for excellent technical assistance.
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