Urate Synthesis in the Blood-sucking Insect Rhodnius prolixus
STIMULATION BY HEMIN IS MEDIATED BY PROTEIN KINASE C*

Aurélio V. Graça-SouzaDagger , Mário A. C. Silva-Neto, and Pedro L. Oliveira

From the Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro-RJ, Brasil, CEP 21910-590

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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, 10893-10896). We have shown that hemin stimulates urate synthesis in the blood-sucking insect Rhodnius prolixus (Graça-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 13-acetate. 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 hemin-induced 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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 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
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Chemicals-- Allopurinol, hemin, okadaic acid, Bt2cAMP, PMA,1 soybean trypsin inhibitor, leupeptin, benzamidine, and beta -mercaptoethanol were purchased from Sigma. Calphostin C, H-89, and H-8 were purchased from Calbiochem. Other reagents were of analytical grade.

Insects-- R. 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 Determination-- Fat 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 µl 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 (Goiânia, GO).

Radioisotopes-- Carrier-free 32Pi was purchased from Comissão Nacional de Energia Nuclear (São 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 [gamma -32P]ATP (27).

PKC Assay-- Typically 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 beta -mercaptoethanol, 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 × g for 10 min to remove tissue debris, the supernatant was centrifuged at 100,000 × 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 µg of crude fat body extract. Reactions were started by addition of [gamma -32P]ATP to a final concentration of 10 µM (1,000 cpm/pmol). After 10 min of incubation at 37 °C, 25-µl 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 Cells-- Fat 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 phosphate-free Rhodnius saline, and transferred to a 96-well microplate (3 fat bodies/well) containing 200 µl of Rhodnius saline in the presence of 100 µCi 32Pi. After 30 min of pre-incubation to allow endogenous formation of [gamma -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), beta -galactosidase (116 kDa), phosphorylase b (98 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.3 kDa).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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).


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Fig. 1.   Urate synthesis is regulated by protein phosphorylation in R. prolixus fat body. Fat bodies were dissected and pre-incubated 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 + 400 µM allopurinol (allopurinol), 0.1 or 1 µM okadaic acid, or 400 µM allopurinol + 1 µM 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 ± S.D. for four determinations.

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 µM, 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.


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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 µM H-8, 1 µM H-89, 20 µM Bt2cAMP (cAMP), 50 µM H-8 + 20 µM Bt2cAMP, 1 µM H-89 + 20 µM Bt2cAMP, 400 µM allopurinol (allo), and 400 µM allopurinol + 20 µM 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 ± S.D. for four determinations.


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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 µM sphingosine (sphg), 2.5 µM calphostin C (cph C), 50 µM PMA (pma), 20 µM sphingosine + 50 µM PMA, 2.5 µM calphostin C + 50 µM PMA, 400 µM allopurinol (allo), and 400 µM allopurinol + 50 µM 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 ± 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 providing evidence that the synthesis of a low molecular weight antioxidant can be regulated by an oxidant agent through PKC activation.


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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 µM hemin (ctrl), 500 µM hemin + 400 µM allopurinol (allo), 500 µM hemin + 20 µM sphingosine (sphg), 500 µM hemin + 2.5 µM calphostin C (cph C), or 500 µM hemin + 1 µM H-89. After incubation, the media were collected and urate levels determined (see "Experimental Procedures"). Data are presented as mean ± S.D. for four determinations.


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Fig. 5.   Hemin stimulates the phosphorylation of 151- and 73-kDa bands through PKC activation. Fat bodies were dissected and pre-incubated with 100 µCi 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 SDS-polyacrylamide 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 + 2.5 µM calphostin C. Panel B, densitometric analysis of the 151- and 73-kDa bands. Further conditions as described under "Experimental Procedures."

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 × 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.


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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 µM PMA + 20 µg PS/tube; 20 µM PMA + 20 µg 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.

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 phosphorylation 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 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.

    ACKNOWLEDGEMENTS

We express our gratitude to Dr. Martha M. Sorenson for a critical reading of the manuscript, to Rosane O. M. M. da Costa, Heloísa S. L. Coelho, Lílian S. C. Gomes, José de Souza L. Júnior, and José F. de Souza Neto for excellent technical assistance.

    FOOTNOTES

* This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Coordenação de Aperfeiçoamento do Pessol de Nível Superior (CAPES), Financiadora de Estudos e Projetos (Finep), Programa de Núcleos de Excelência (PRONEX), and Programa de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Fax: 55-21-270-8647; E-mail: avsouza{at}server.bioqmed.ufrj.br.

    ABBREVIATIONS

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.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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