- Email: [email protected]
Nonadrenergic, noncholinergic relaxation of human isolated corpus cavernosum induced by scorpion venom
BASIC SCIENCE
NONADRENERGIC, NONCHOLINERGIC RELAXATION OF HUMAN ISOLATED CORPUS CAVERNOSUM INDUCED BY SCORPION VENOM CLEBER E. TEIXEIRA, RENATO FARO, RONILSON A. MORENO, NELSON RODRIGUES NETTO, JR, ADRIANO FREGONESI, EDSON ANTUNES, AND GILBERTO DE NUCCI
ABSTRACT Objectives. To examine the effects of Tityus serrulatus scorpion venom (TSV) on human corpus cavernosum (HCC) using a bioassay cascade. Priapism is occasionally observed in scorpion envenomation, mostly in children. Methods. HCC strips were suspended in a cascade system and superfused with aerated and warmed Krebs’ solution at 5 mL/min. Noradrenaline (3 mol/L) was infused to induce a submaximal contraction of the HCC strips. The release of cyclooxygenase products was prevented by infusing indomethacin (6 mol/L). Results. N-nitro-L-arginine methyl ester (10 mol/L; n ⫽ 10) increased the tone of the preparations and significantly reduced (P ⬍0.01) the acetylcholine (ACh) and TSV-induced relaxations. Subsequent infusion of L-arginine (300 mol/L) partially reversed the increased tone and significantly restored the relaxations induced by TSV and ACh (P ⬍0.01). The soluble guanylyl cyclase inhibitor ODQ (10 mol/L; n ⫽ 8) markedly reduced (P ⬍0.01) the relaxations induced by TSV, ACh, glyceryl trinitrate, and bradykinin. 7-Nitroindazole (10 mol/L; n ⫽ 8) inhibited the relaxations induced by TSV by 84% (P ⬍0.01) and also caused small, but significant, reductions in the ACh and bradykinin-induced HCC relaxations (P ⬍0.05). Atropine (1 mol/L; n ⫽ 6) abolished the relaxations evoked by ACh (P ⬍0.01), but had no effect on those elicited by TSV. Tetrodotoxin (1 mol/L; n ⫽ 6) abolished the relaxations induced by TSV (P ⬍0.01) and also reversed the established TSV-induced relaxation (n ⫽ 4). Conclusions. Our results indicate that TSV relaxes HCC through the release of nitric oxide from nonadrenergic, noncholinergic (NANC) nerves. The elucidation of the mechanism responsible for the TSV-induced relaxations might be useful for a better understanding of the development of priapism in cases of scorpion envenomation. UROLOGY 57: 816–820, 2001. © 2001, Elsevier Science Inc.
T
he mechanisms involved in the regulation of either contraction or relaxation of the corpus cavernosum and penile vasculature have been intensely investigated during the past decades.1 Erection follows the relaxation of penile corpus cavernosum smooth muscle, which is initiated by a change from vasoconstriction to vasodilation.2 Nitric oxide (NO) has been suggested as the main mediator of nonandrenergic, noncholinergic (NANC) nerve-induced Cleber E. Teixeira is supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP). From the Department of Pharmacology and Discipline of Urology, Faculty of Medical Sciences, Campinas, Sa˜o Paulo, Brazil Reprint requests: Cleber E. Teixeira, Department of Pharmacology, Faculty of Medical Sciences, UNICAMP P.O. Box 6111, 13081-970, Campinas, Sa˜o Paulo, Brazil Submitted: March 13, 2000, accepted (with revisions): November 2, 2000
816
© 2001, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
relaxation of the corpora cavernosa in vitro, as well as of increased intracavernous pressure in vivo in a variety of mammals, including rats,3,4 rabbits,5,6 dogs,7 monkeys,8 and humans.9 Scorpion envenomation usually results in generalized depolarization of the peripheral nerves, causing a massive neurotransmitter release10,11 that can account for the development of a variety of symptoms such as abdominal distress (pain, nausea, and vomiting), acute toxicity of the central nervous system (rapid and uncoordinated movements of the eyes and limbs), and cardiac effects (tachycardia followed by bradycardia and blood pressure elevation). The occurrence of priapism in humans stung by scorpions has also been reported.12–14 Tityus serrulatus scorpion venom (TSV) stimulates the release of either acetylcholine (ACh) or catecholamines in a wide range of tissues 0090-4295/01/$20.00 PII S0090-4295(00)01047-5
and organs,15 and relaxes rabbit corpus cavernosum by selectively activating nitrergic NANC nerve fibers, causing the release of NO.6 We investigated the effects of TSV on human corpus cavernosum (HCC) in vitro by using a bioassay cascade. MATERIAL AND METHODS HCC from 11 patients (age range 22 to 43 years) who underwent multiple organ donation were used after informed consent was obtained from the appropriate person. The protocol was approved by the University Hospital Ethics Committee.
BIOASSAY CASCADE The excised tissues were immediately placed in Krebs’ solution and kept at 4°C until use, which never exceeded 24 hours after removal. HCC tissues were cut in strips approximately 2 cm long (four strips from each patient). The tissues were then suspended in a cascade system16 and continuously superfused with oxygenated (95% oxygen plus 5% carbon dioxide) and warmed (37°C) Krebs’ solution at a flow rate of 5 mL/min. The responses of the HCC strips were detected with auxotonic levers attached to Harvard heart/smooth muscle transducers and displayed on a Watanabe multichannel pen recorder (model WTR 381). After an equilibration period of approximately 90 minutes, noradrenaline (3 mol/L) was infused (0.1 mL/min) to induce a submaximal contraction of HCC strips. The release of cyclooxygenase products was prevented by infusing at 0.1 mL/min indomethacin (6 mol/L). The HCC strips were calibrated by injecting a single bolus of the nitrovasodilator glyceryl trinitrate (GTN), and the sensitivity of the tissues was adjusted electronically to be roughly equal. TSV and other substances (GTN, bradykinin, histamine, and ACh) were always injected as a single bolus (up to 100 L). N-nitro-L-arginine methyl ester (L-NAME), L-arginine, atropine, 1H-[1,2,4]oxadiazolo[4,3,-alquinoxalin-1-one] (ODQ), 7-nitroindazole (7-NI), and tetrodotoxin (TTX) were infused over the HCC tissues 20 minutes before and during bolus injection of the appropriate agonists.
DRUGS AND TSV TSV was provided by the Butantan Institute (Sa˜o Paulo). The crude venom (lot No. 041088) was obtained by electrostimulation of the telsons of scorpions in captivity and was lyophilized and stored at ⫺20°C. ACh, atropine, bradykinin, histamine, indomethacin, L-arginine, L-NAME, 7-NI, noradrenaline, ODQ, and TTX were obtained from Sigma (St. Louis, Mo). GTN (ampoules containing 1 mg/mL isotonic solution) was acquired from Lipha Pharmaceuticals (London, UK). The Krebs’ solution (pH 7.4) had the following composition: sodium chloride 118 mmol/L, potassium chloride 4.7 mmol/L, KH2PO4 1.2 mmol/L, MgSO4 䡠 7H2O 1.17 mmol/L, CaCl2 䡠 2H2O 2.5 mmol/L, NaHCO3 25 mmol/L, and glucose 5.6 mmol/L.
STATISTICAL ANALYSIS The relaxations induced by TSV and the agonists (ACh, histamine, and bradykinin) were measured considering the maximal relaxation induced by GTN (dose in moles) as 100%. Results were expressed as the mean ⫾ the standard error of the mean of n experiments. Analysis of variance and Student’s unpaired two-tailed t test were used to evaluate the data. P ⬍0.05 was taken as significant. UROLOGY 57 (4), 2001
FIGURE 1. Effects of L-NAME (10 mol/L) and L-arginine (300 mol/L) on HCC strips. Infusion of L-NAME increased the HCC tone and markedly reduced the relaxations induced by ACh (60 nmol) and TSV (30 g). The relaxations induced by GTN (4 nmol) were not significantly affected by L-NAME infusion. Subsequent infusion of L-arginine partially reversed the increased HCC tone and also greatly restored the relaxations induced by ACh and TSV. Representative tracing of 10 HCC strips obtained from 3 donors.
RESULTS INVOLVEMENT OF NO ON THE TSV-INDUCED HCC RELAXATIONS TSV (10 to 100 g) caused dose-dependent and non-tachyphylactic HCC relaxations (45% ⫾ 6%, 73% ⫾ 8%, and 102% ⫾ 11% for 10, 30, and 100 g, respectively; n ⫽ 4). Bolus injections of GTN (4 nmol), ACh (60 nmol), histamine (100 nmol), and bradykinin (10 nmol) also significantly relaxed the HCC tissues. Figure 1 shows that infusion of the NO synthase inhibitor L-NAME (10 mol/L) increased the tone of the HCC tissues and markedly reduced (P ⬍0.01) the relaxations induced by ACh (60 nmol) and TSV (30 g) (Table I). The GTN (4 nmol)induced HCC relaxations were not affected by LNAME. The subsequent infusion of L-arginine (300 mol/L) partially reversed the increased tone (Fig. 1) and significantly (P ⬍0.01) restored the relaxations induced by ACh (5% ⫾ 3% during LNAME infusion and 85% ⫾ 7% during L-arginine infusion) and TSV (11% ⫾ 6% during L-NAME infusion and 79% ⫾ 9% during L-arginine infusion). 7-NI (10 mol/L) increased the tone of the preparations and significantly (P ⬍0.01) inhibited the HCC relaxations induced by TSV (30 g) (Table I and Fig. 2). 7-NI also reduced the relaxations induced by both ACh (60 nmol; P ⬍0.05) and bradykinin (10 nmol; P ⬍0.05) without affecting those evoked by GTN (4 nmol; Table I). At the end of the 7-NI infusion, the tone of the tissues decreased and the relaxations induced by TSV were partially restored (11% ⫾ 3% during and 44% ⫾ 6% after the 7-NI infusion; P ⬍0.01) as were those induced by ACh (29% ⫾ 4% during and 46% ⫾ 9% after the 7-NI infusion; P ⬍0.05) and bradykinin 817
TABLE I. Effect of nitric oxide synthase and soluble guanylyl cyclase inhibition on human corpus cavernosum relaxations induced by acetylcholine, bradykinin, and Tityus serrulatus venom L-NAME
7-NI
ODQ
Agents
Control
Treated
Control
Treated
Control
Treated
ACh Bk TSV
98 ⫾ 9 ND 91 ⫾ 10
5 ⫾ 3* ND 11 ⫾ 6*
41 ⫾ 7 54 ⫾ 8 68 ⫾ 10
29 ⫾ 4† 39 ⫾ 7† 11 ⫾ 3*
62 ⫾ 7 70 ⫾ 11 81 ⫾ 8
3 ⫾ 1* 5 ⫾ 1* 7 ⫾ 5*
KEY: L-NAME ⫽ N-nitro-L-arginine methyl ester; 7-NI ⫽ 7-nitroindazole; ODQ ⫽ 1H-[1,2,4] oxadiazolo [4,3-alquinoxalin-1-one]; ACh ⫽ acetylcholine; Bk ⫽ bradykinin; ND ⫽ not determined; TSV ⫽ Tityus serrulatus venom; HCC ⫽ human corpus cavernosum. L-NAME (10 mol/L; n ⫽ 10), 7-NI (10 mol/L; n ⫽ 8), and ODQ (10 mol/L; n ⫽ 8) were infused over HCC tissues at a flow rate of 0.1 mL/min for at least 20 minutes before injection of the substances; HCC relaxations induced by ACh, Bk, and TSV expressed (as the mean ⫾ SEM) relative to the submaximal relaxation induced by glyceryl trinitrate, which was taken to be 100%. * P ⬍0.01 compared with the respective control. † P ⬍0.05 compared with the respective control.
FIGURE 2. Effects of 7-NI (10 mol/L) on HCC tissues. Infusion of 7-NI increased the HCC tone and markedly reduced the relaxations induced by TSV (30 g). Relaxations induced by ACh (60 nmol) and bradykinin (Bk, 10 nmol) were also inhibited by 7-NI. Relaxations induced by GTN (4 nmol) were not significantly affected by 7-NI. After stopping the 7-NI infusion, the HCC relaxations induced by ACh, Bk, and TSV were partially restored. Representative tracing of eight HCC strips obtained from 2 donors.
(39% ⫾ 7% during and 58% ⫾ 7% after the 7-NI infusion; P ⬍0.05). The infusion of the soluble guanylyl cyclase inhibitor ODQ (10 mol/L) significantly increased the tone of the HCC tissues and markedly reduced (P ⬍0.01) the HCC relaxations induced by ACh (60 nmol), bradykinin (10 nmol), and TSV (30 g) (Fig. 3 and Table I). The GTN (4 nmol)-induced HCC relaxations were also significantly reduced by ODQ (97% ⫾ 2% inhibition; P ⬍0.01). The relaxations evoked by these agents were partially restored 30 minutes after stopping the ODQ infusion (Fig. 3). EFFECT OF ATROPINE The muscarinic receptor antagonist atropine (1 mol/L) virtually abolished the ACh (60 nmol)induced HCC relaxations (66% ⫾ 9% before and 2% ⫾ 1% during the atropine infusion; P ⬍0.01) but had no effect on those induced by either histamine (100 nmol; 86% ⫾ 11% before and 94% ⫾ 10% during the atropine infusion) or TSV (30 g; 818
FIGURE 3. Inhibition by the selective soluble guanylate cyclase inhibitor ODQ (10 mol/L) of the HCC relaxations induced by GTN (4 nmol), ACh (60 nmol), bradykinin (Bk, 10 nmol), and TSV (30 g). Infusion of ODQ increased the tone of the tissues and virtually abolished the relaxations induced by the above-mentioned agonists. After stopping the ODQ infusion, the relaxations were significantly restored. Representative tracing of eight HCC strips obtained from 2 donors.
72% ⫾ 7% before and 80% ⫾ 7% during the atropine infusion). The GTN (4 nmol)-induced HCC relaxations were not affected by atropine. EFFECT OF TTX The infusion of TTX (1 mol/L) virtually abolished the HCC relaxations induced by TSV (30 g; 102% ⫾ 13% before and 2% ⫾ 2% during the TTX infusion; P ⬍0.01). TTX had no effect on the relaxant responses elicited by ACh (60 nmol; 92% ⫾ 10% before and 85% ⫾ 8% during the TTX infusion). The HCC relaxations evoked by GTN (4 nmol) also remained unaffected in the presence of TTX (Fig. 4a). Interestingly, TTX promptly reversed the response to the venom (100 g) when infused during the relaxation phase (Fig. 4b). COMMENT We have demonstrated in this study that TSV relaxes HCC preparations in vitro due to NO release. Nonspecific NO synthesis inhibitors, such as N-nitro-L-arginine, N-monomethyl-L-arginine, UROLOGY 57 (4), 2001
FIGURE 4. Effects of the sodium channel blocker TTX (1 mol/L) on HCC preparations. (a) Infusion of TTX abolished HCC relaxations elicited by TSV (30 g), without affecting those induced by ACh (60 nmol) or GTN (4 nmol). Representative tracing of six strips obtained from 2 donors. (b) TTX promptly reversed the response to TSV (100 g) when infused during the relaxation phase. Representative tracing of four strips obtained from 1 donor.
and L-NAME, reduce the relaxations of cavernosal tissue caused by electrical field stimulation and muscarinic receptor activation.17–21 The findings that L-NAME markedly reduced the relaxations elicited by TSV and L-arginine significantly reversed this inhibition indicate that TSV relaxed HCC tissues through the release of NO. The involvement of NO in the TSV-induced HCC relaxations was further confirmed by the finding that ODQ, a potent and selective inhibitor of NO-stimulated soluble guanylyl cyclase,22 virtually abolished the relaxations. Similar to the findings in rabbits,6 these results provide evidence that NO and cyclic guanosine monophosphate mediate the relaxant response elicited by TSV in human cavernosal tissue. The erectile tissues have been shown to be densely innervated by adrenergic excitatory, cholinergic inhibitory and NANC inhibitory nerve fibers.1 The biochemical18 and immunohistochemical23 demonstration of NO synthase within cavernosal nerves, together with the findings that nerve-evoked relaxation is preserved after the removal of endothelium,24 indicates that the most important source of NO in penile tissue is neuronal, appointing a pivotal role for NO in erectile function. It remains possible, however, that the endothelium provides an important additional source of NO for the initiation and maintenance of penile erection. The muscarinic receptor antagonist atropine failed to affect the TSV-induced HCC relaxations, indicating that the venom does not relax this preparation by activating cholinergic fibers. The classic sodium channel blocker TTX ties together in a highly efficient manner various parts of the sodium channel. As a result, the passage of sodium ions through the channel is physically prevented and a conduction blockade results.25 TTX virtually abolished the TSV-induced HCC relaxations, strongly indicating that NO is generated within, and released from, NANC nerve fibers. InUROLOGY 57 (4), 2001
terestingly, TTX also promptly reversed the response to TSV when infused during the established relaxation phase, suggesting a persistent activation of the sodium channels elicited by TSV. 7-NI has been shown to be a potent inhibitor of neuronal NO synthase because of its potent antinociceptive effect26 and its ability to inhibit brain NO synthase activity.26,27 The infusion of 7-NI markedly reduced the HCC relaxations induced by TSV, indicating activation of brain NO synthase in the cavernosal tissue. However, 7-NI also caused small reductions in the relaxations elicited by both ACh and bradykinin because of nonspecific action on endothelium NO synthase. Indeed, 7-NI inhibits NO synthase activity in the homogenates of endothelial cells,26,28 although it has no effect on arterial blood pressure.26 Priapism is most commonly seen in children as a complication of systemic scorpion envenomation. Since our findings demonstrated a potent local effect of the scorpion toxin, it is possible that the amount of venom injected by the Tityus serrulatus scorpion (approximately 0.7 mg)29 may not be sufficient to achieve effective corpus cavernosum concentrations in an adult. Indeed, child envenomation is often accompanied by other systemic symptoms, and adult envenomation is characterized mainly by a restricted local response. CONCLUSIONS We have demonstrated that TSV relaxes the HCC through the release of NO from NANC nerves as a consequence of sodium channel activation. The purification of the responsible toxins should provide a valuable tool in the further understanding of the mechanisms underlying NANC nerve activation in human penile erectile tissue. The finding that sodium channel blockers and NO synthase inhibitors abolish both toxin-induced and electrical field stimulation-induced relaxation of HCC19 indicates a therapeutic potential for these drugs in 819
the treatment of priapism. Although the former is extremely toxic to be used clinically, this limitation does not apply to specific inhibitors of neuronal NO synthase. Indeed, nonspecific inhibitors of NO synthase have already been used in different clinical settings.30 REFERENCES 1. Andersson K-E, and Wagner G: Physiology of penile erection. Physiol Rev 75: 191–236, 1995. 2. Lerner SE, Melman A, and Christ GJ: A review of erection dysfunction: new insights and more questions. J Urol 149: 1246 –1255, 1993. 3. Burnett AL, Lowenstein CJ, Bredt DS, et al: Nitric oxide: a physiologic mediator of penile erection. Science 257: 401– 403, 1992. 4. Finberg JPM, Levy S, and Vardi Y: Inhibition of nerve stimulation-induced vasodilation in corpora cavernosa of the pithed rat by blockade of nitric oxide synthase. Br J Pharmacol 108: 1038 –1042, 1993. 5. Knispel HH, Goessl C, and Beckmann R: Nitric oxide mediates neurogenic relaxation induced in rabbit cavernous smooth muscle by electric field stimulation. Urology 40: 471– 476, 1996. 6. Teixeira CE, Bento AC, Lopes-Martins RAB, et al: Tityus serrulatus scorpion venom relaxes the isolated rabbit corpus cavernosum by activating NANC nitrergic nerve fibers. Br J Pharmacol 123: 435– 442, 1998. 7. Hayashida H, Okamura T, Tomoyoshi T, et al: Neurogenic nitric oxide mediates relaxation of canine corpus cavernosum. J Urol 155: 1122–1127, 1996. 8. Okamura T, Ayajiki K, and Toda N: Monkey corpus cavernosum relaxation mediated by NO and other relaxing factor derived from nerves. Am J Physiol 274: H1075–H1081, 1998. 9. Leone AM, Wiklund NP, Hokfelt T, et al: Release of nitric oxide by nerve stimulation in the human urogenital tract. Neuroreport 24: 733–736, 1994. 10. Couraud F, and Jover E: Mechanism of action of scorpion toxins, in Tu AT (Ed): Handbook of Natural Toxins. New York, Marcel Dekker, 1984, vol 2, pp 659 – 678. 11. Simard J, and Watt DD: Venoms and toxins, in Polis GA (Ed): The Biology of Scorpions. Standford, Standford University Press, 1990, pp 414 – 444. 12. Bawaskar HS: Diagnostic cardiac premonitory signs and symptoms of red scorpion sting. Lancet 1: 552–554, 1982. 13. Amitai Y, Mines Y, Aker M, et al: Scorpion sting in children: a review of 51 cases. Clin Pediatr 24: 136 –140, 1985. 14. Hershkovich Y, Elitsur Y, Margolis CZ, et al: Criteria map audit of scorpion envenomation in Negev, Israel. Toxicon 23: 845– 854, 1985. 15. Freire-Maia L: Peripheral effects of Tityus serrulatus scorpion venom. J Toxicol 14: 423– 435, 1995. 16. Vane JR: The use of isolated organs for detecting active
820
substances in the circulating blood. Br J Pharmacol Chemother 23: 360 –373, 1964. 17. Ignarro LJ, Bush PA, Buga GM, et al: Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle. Biochem Biophys Res Commun 170: 843– 850, 1990. 18. Bush PA, Gonzalez NE, and Ignarro LJ: Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum. Biochem Biophys Res Commun 186: 308 –314, 1992. 19. Holmquist F, Andersson K-E, and Hedlund H: Characterization of inhibitory neurotransmission in the isolated corpus cavernosum from rabbit and man. J Physiol (Lond) 449: 295–311, 1992. 20. Pickard RS, Powell PH, and Zar MA: The effect of inhibitors of nitric oxide biosynthesis and cyclic GMP formation on nerve-evoked relaxation of human cavernosal smooth muscle. Br J Pharmacol 104: 755–759, 1991. 21. Rajfer J, Aronson WJ, Bush PA, et al: Nitric oxide as mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med 326: 90 –94, 1992. 22. Garthwaite J, Southam E, Boulton CL, et al: Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-alquinoxalin-1-one]. Mol Pharmacol 48: 184 –188, 1995. 23. Burnett AL, Tillman SL, Chang TSK, et al: Immunohistochemical localization of nitric oxide synthase in the autonomic innervation of the human penis. J Urol 150: 73–76, 1993. 24. Trigo-Rocha F, Hsu G-L, Donatucci CF, et al: The role of cyclic adenosine monophosphate, cyclic guanosine monophosphate, endothelium and nonadrenergic, noncholinergic neurotransmission in canine penile erection. J Urol 149: 872– 877, 1993. 25. Smythies JR, Benington F, and Morin RD: Model for the action of tetrodotoxin and batrachotoxin. Nature 231: 188 – 190, 1971. 26. Moore PK, Wallace P, Gaffen ZA, et al: Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects. Br J Pharmacol 110: 219 –224, 1993. 27. Mayer B, Klatt P, Werner ER, et al: Molecular mechanisms of inhibition of porcine brain nitric oxide synthase by the antinociceptive drug 7-nitro-indazole. Neuropharmacology 33: 1253–1259, 1994. 28. Wolff DJ, Lubeskie A, and Umansky C: The inhibition of the constitutive bovine endothelial nitric oxide synthase by imidazole and indazole agents. Arch Biochem Biophys 314: 360 –366, 1994. 29. Bu¨cherl W: Classification, biology, and venom extraction of scorpions, in Bu¨cherl W, and Buckley EE (Eds): Venomous Animals and Their Venoms. New York, Academic Press, 1971, vol 3, pp 317–347. 30. Cotter G, Kaluski E, Blatt A, et al: L-NMMA (a nitric oxide synthase inhibitor) is effective in the treatment of cardiogenic shock. Circulation 101: 1358 –1361, 2000.
UROLOGY 57 (4), 2001
NONADRENERGIC, NONCHOLINERGIC RELAXATION OF HUMAN ISOLATED CORPUS CAVERNOSUM INDUCED BY SCORPION VENOM CLEBER E. TEIXEIRA, RENATO FARO, RONILSON A. MORENO, NELSON RODRIGUES NETTO, JR, ADRIANO FREGONESI, EDSON ANTUNES, AND GILBERTO DE NUCCI
ABSTRACT Objectives. To examine the effects of Tityus serrulatus scorpion venom (TSV) on human corpus cavernosum (HCC) using a bioassay cascade. Priapism is occasionally observed in scorpion envenomation, mostly in children. Methods. HCC strips were suspended in a cascade system and superfused with aerated and warmed Krebs’ solution at 5 mL/min. Noradrenaline (3 mol/L) was infused to induce a submaximal contraction of the HCC strips. The release of cyclooxygenase products was prevented by infusing indomethacin (6 mol/L). Results. N-nitro-L-arginine methyl ester (10 mol/L; n ⫽ 10) increased the tone of the preparations and significantly reduced (P ⬍0.01) the acetylcholine (ACh) and TSV-induced relaxations. Subsequent infusion of L-arginine (300 mol/L) partially reversed the increased tone and significantly restored the relaxations induced by TSV and ACh (P ⬍0.01). The soluble guanylyl cyclase inhibitor ODQ (10 mol/L; n ⫽ 8) markedly reduced (P ⬍0.01) the relaxations induced by TSV, ACh, glyceryl trinitrate, and bradykinin. 7-Nitroindazole (10 mol/L; n ⫽ 8) inhibited the relaxations induced by TSV by 84% (P ⬍0.01) and also caused small, but significant, reductions in the ACh and bradykinin-induced HCC relaxations (P ⬍0.05). Atropine (1 mol/L; n ⫽ 6) abolished the relaxations evoked by ACh (P ⬍0.01), but had no effect on those elicited by TSV. Tetrodotoxin (1 mol/L; n ⫽ 6) abolished the relaxations induced by TSV (P ⬍0.01) and also reversed the established TSV-induced relaxation (n ⫽ 4). Conclusions. Our results indicate that TSV relaxes HCC through the release of nitric oxide from nonadrenergic, noncholinergic (NANC) nerves. The elucidation of the mechanism responsible for the TSV-induced relaxations might be useful for a better understanding of the development of priapism in cases of scorpion envenomation. UROLOGY 57: 816–820, 2001. © 2001, Elsevier Science Inc.
T
he mechanisms involved in the regulation of either contraction or relaxation of the corpus cavernosum and penile vasculature have been intensely investigated during the past decades.1 Erection follows the relaxation of penile corpus cavernosum smooth muscle, which is initiated by a change from vasoconstriction to vasodilation.2 Nitric oxide (NO) has been suggested as the main mediator of nonandrenergic, noncholinergic (NANC) nerve-induced Cleber E. Teixeira is supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP). From the Department of Pharmacology and Discipline of Urology, Faculty of Medical Sciences, Campinas, Sa˜o Paulo, Brazil Reprint requests: Cleber E. Teixeira, Department of Pharmacology, Faculty of Medical Sciences, UNICAMP P.O. Box 6111, 13081-970, Campinas, Sa˜o Paulo, Brazil Submitted: March 13, 2000, accepted (with revisions): November 2, 2000
816
© 2001, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
relaxation of the corpora cavernosa in vitro, as well as of increased intracavernous pressure in vivo in a variety of mammals, including rats,3,4 rabbits,5,6 dogs,7 monkeys,8 and humans.9 Scorpion envenomation usually results in generalized depolarization of the peripheral nerves, causing a massive neurotransmitter release10,11 that can account for the development of a variety of symptoms such as abdominal distress (pain, nausea, and vomiting), acute toxicity of the central nervous system (rapid and uncoordinated movements of the eyes and limbs), and cardiac effects (tachycardia followed by bradycardia and blood pressure elevation). The occurrence of priapism in humans stung by scorpions has also been reported.12–14 Tityus serrulatus scorpion venom (TSV) stimulates the release of either acetylcholine (ACh) or catecholamines in a wide range of tissues 0090-4295/01/$20.00 PII S0090-4295(00)01047-5
and organs,15 and relaxes rabbit corpus cavernosum by selectively activating nitrergic NANC nerve fibers, causing the release of NO.6 We investigated the effects of TSV on human corpus cavernosum (HCC) in vitro by using a bioassay cascade. MATERIAL AND METHODS HCC from 11 patients (age range 22 to 43 years) who underwent multiple organ donation were used after informed consent was obtained from the appropriate person. The protocol was approved by the University Hospital Ethics Committee.
BIOASSAY CASCADE The excised tissues were immediately placed in Krebs’ solution and kept at 4°C until use, which never exceeded 24 hours after removal. HCC tissues were cut in strips approximately 2 cm long (four strips from each patient). The tissues were then suspended in a cascade system16 and continuously superfused with oxygenated (95% oxygen plus 5% carbon dioxide) and warmed (37°C) Krebs’ solution at a flow rate of 5 mL/min. The responses of the HCC strips were detected with auxotonic levers attached to Harvard heart/smooth muscle transducers and displayed on a Watanabe multichannel pen recorder (model WTR 381). After an equilibration period of approximately 90 minutes, noradrenaline (3 mol/L) was infused (0.1 mL/min) to induce a submaximal contraction of HCC strips. The release of cyclooxygenase products was prevented by infusing at 0.1 mL/min indomethacin (6 mol/L). The HCC strips were calibrated by injecting a single bolus of the nitrovasodilator glyceryl trinitrate (GTN), and the sensitivity of the tissues was adjusted electronically to be roughly equal. TSV and other substances (GTN, bradykinin, histamine, and ACh) were always injected as a single bolus (up to 100 L). N-nitro-L-arginine methyl ester (L-NAME), L-arginine, atropine, 1H-[1,2,4]oxadiazolo[4,3,-alquinoxalin-1-one] (ODQ), 7-nitroindazole (7-NI), and tetrodotoxin (TTX) were infused over the HCC tissues 20 minutes before and during bolus injection of the appropriate agonists.
DRUGS AND TSV TSV was provided by the Butantan Institute (Sa˜o Paulo). The crude venom (lot No. 041088) was obtained by electrostimulation of the telsons of scorpions in captivity and was lyophilized and stored at ⫺20°C. ACh, atropine, bradykinin, histamine, indomethacin, L-arginine, L-NAME, 7-NI, noradrenaline, ODQ, and TTX were obtained from Sigma (St. Louis, Mo). GTN (ampoules containing 1 mg/mL isotonic solution) was acquired from Lipha Pharmaceuticals (London, UK). The Krebs’ solution (pH 7.4) had the following composition: sodium chloride 118 mmol/L, potassium chloride 4.7 mmol/L, KH2PO4 1.2 mmol/L, MgSO4 䡠 7H2O 1.17 mmol/L, CaCl2 䡠 2H2O 2.5 mmol/L, NaHCO3 25 mmol/L, and glucose 5.6 mmol/L.
STATISTICAL ANALYSIS The relaxations induced by TSV and the agonists (ACh, histamine, and bradykinin) were measured considering the maximal relaxation induced by GTN (dose in moles) as 100%. Results were expressed as the mean ⫾ the standard error of the mean of n experiments. Analysis of variance and Student’s unpaired two-tailed t test were used to evaluate the data. P ⬍0.05 was taken as significant. UROLOGY 57 (4), 2001
FIGURE 1. Effects of L-NAME (10 mol/L) and L-arginine (300 mol/L) on HCC strips. Infusion of L-NAME increased the HCC tone and markedly reduced the relaxations induced by ACh (60 nmol) and TSV (30 g). The relaxations induced by GTN (4 nmol) were not significantly affected by L-NAME infusion. Subsequent infusion of L-arginine partially reversed the increased HCC tone and also greatly restored the relaxations induced by ACh and TSV. Representative tracing of 10 HCC strips obtained from 3 donors.
RESULTS INVOLVEMENT OF NO ON THE TSV-INDUCED HCC RELAXATIONS TSV (10 to 100 g) caused dose-dependent and non-tachyphylactic HCC relaxations (45% ⫾ 6%, 73% ⫾ 8%, and 102% ⫾ 11% for 10, 30, and 100 g, respectively; n ⫽ 4). Bolus injections of GTN (4 nmol), ACh (60 nmol), histamine (100 nmol), and bradykinin (10 nmol) also significantly relaxed the HCC tissues. Figure 1 shows that infusion of the NO synthase inhibitor L-NAME (10 mol/L) increased the tone of the HCC tissues and markedly reduced (P ⬍0.01) the relaxations induced by ACh (60 nmol) and TSV (30 g) (Table I). The GTN (4 nmol)induced HCC relaxations were not affected by LNAME. The subsequent infusion of L-arginine (300 mol/L) partially reversed the increased tone (Fig. 1) and significantly (P ⬍0.01) restored the relaxations induced by ACh (5% ⫾ 3% during LNAME infusion and 85% ⫾ 7% during L-arginine infusion) and TSV (11% ⫾ 6% during L-NAME infusion and 79% ⫾ 9% during L-arginine infusion). 7-NI (10 mol/L) increased the tone of the preparations and significantly (P ⬍0.01) inhibited the HCC relaxations induced by TSV (30 g) (Table I and Fig. 2). 7-NI also reduced the relaxations induced by both ACh (60 nmol; P ⬍0.05) and bradykinin (10 nmol; P ⬍0.05) without affecting those evoked by GTN (4 nmol; Table I). At the end of the 7-NI infusion, the tone of the tissues decreased and the relaxations induced by TSV were partially restored (11% ⫾ 3% during and 44% ⫾ 6% after the 7-NI infusion; P ⬍0.01) as were those induced by ACh (29% ⫾ 4% during and 46% ⫾ 9% after the 7-NI infusion; P ⬍0.05) and bradykinin 817
TABLE I. Effect of nitric oxide synthase and soluble guanylyl cyclase inhibition on human corpus cavernosum relaxations induced by acetylcholine, bradykinin, and Tityus serrulatus venom L-NAME
7-NI
ODQ
Agents
Control
Treated
Control
Treated
Control
Treated
ACh Bk TSV
98 ⫾ 9 ND 91 ⫾ 10
5 ⫾ 3* ND 11 ⫾ 6*
41 ⫾ 7 54 ⫾ 8 68 ⫾ 10
29 ⫾ 4† 39 ⫾ 7† 11 ⫾ 3*
62 ⫾ 7 70 ⫾ 11 81 ⫾ 8
3 ⫾ 1* 5 ⫾ 1* 7 ⫾ 5*
KEY: L-NAME ⫽ N-nitro-L-arginine methyl ester; 7-NI ⫽ 7-nitroindazole; ODQ ⫽ 1H-[1,2,4] oxadiazolo [4,3-alquinoxalin-1-one]; ACh ⫽ acetylcholine; Bk ⫽ bradykinin; ND ⫽ not determined; TSV ⫽ Tityus serrulatus venom; HCC ⫽ human corpus cavernosum. L-NAME (10 mol/L; n ⫽ 10), 7-NI (10 mol/L; n ⫽ 8), and ODQ (10 mol/L; n ⫽ 8) were infused over HCC tissues at a flow rate of 0.1 mL/min for at least 20 minutes before injection of the substances; HCC relaxations induced by ACh, Bk, and TSV expressed (as the mean ⫾ SEM) relative to the submaximal relaxation induced by glyceryl trinitrate, which was taken to be 100%. * P ⬍0.01 compared with the respective control. † P ⬍0.05 compared with the respective control.
FIGURE 2. Effects of 7-NI (10 mol/L) on HCC tissues. Infusion of 7-NI increased the HCC tone and markedly reduced the relaxations induced by TSV (30 g). Relaxations induced by ACh (60 nmol) and bradykinin (Bk, 10 nmol) were also inhibited by 7-NI. Relaxations induced by GTN (4 nmol) were not significantly affected by 7-NI. After stopping the 7-NI infusion, the HCC relaxations induced by ACh, Bk, and TSV were partially restored. Representative tracing of eight HCC strips obtained from 2 donors.
(39% ⫾ 7% during and 58% ⫾ 7% after the 7-NI infusion; P ⬍0.05). The infusion of the soluble guanylyl cyclase inhibitor ODQ (10 mol/L) significantly increased the tone of the HCC tissues and markedly reduced (P ⬍0.01) the HCC relaxations induced by ACh (60 nmol), bradykinin (10 nmol), and TSV (30 g) (Fig. 3 and Table I). The GTN (4 nmol)-induced HCC relaxations were also significantly reduced by ODQ (97% ⫾ 2% inhibition; P ⬍0.01). The relaxations evoked by these agents were partially restored 30 minutes after stopping the ODQ infusion (Fig. 3). EFFECT OF ATROPINE The muscarinic receptor antagonist atropine (1 mol/L) virtually abolished the ACh (60 nmol)induced HCC relaxations (66% ⫾ 9% before and 2% ⫾ 1% during the atropine infusion; P ⬍0.01) but had no effect on those induced by either histamine (100 nmol; 86% ⫾ 11% before and 94% ⫾ 10% during the atropine infusion) or TSV (30 g; 818
FIGURE 3. Inhibition by the selective soluble guanylate cyclase inhibitor ODQ (10 mol/L) of the HCC relaxations induced by GTN (4 nmol), ACh (60 nmol), bradykinin (Bk, 10 nmol), and TSV (30 g). Infusion of ODQ increased the tone of the tissues and virtually abolished the relaxations induced by the above-mentioned agonists. After stopping the ODQ infusion, the relaxations were significantly restored. Representative tracing of eight HCC strips obtained from 2 donors.
72% ⫾ 7% before and 80% ⫾ 7% during the atropine infusion). The GTN (4 nmol)-induced HCC relaxations were not affected by atropine. EFFECT OF TTX The infusion of TTX (1 mol/L) virtually abolished the HCC relaxations induced by TSV (30 g; 102% ⫾ 13% before and 2% ⫾ 2% during the TTX infusion; P ⬍0.01). TTX had no effect on the relaxant responses elicited by ACh (60 nmol; 92% ⫾ 10% before and 85% ⫾ 8% during the TTX infusion). The HCC relaxations evoked by GTN (4 nmol) also remained unaffected in the presence of TTX (Fig. 4a). Interestingly, TTX promptly reversed the response to the venom (100 g) when infused during the relaxation phase (Fig. 4b). COMMENT We have demonstrated in this study that TSV relaxes HCC preparations in vitro due to NO release. Nonspecific NO synthesis inhibitors, such as N-nitro-L-arginine, N-monomethyl-L-arginine, UROLOGY 57 (4), 2001
FIGURE 4. Effects of the sodium channel blocker TTX (1 mol/L) on HCC preparations. (a) Infusion of TTX abolished HCC relaxations elicited by TSV (30 g), without affecting those induced by ACh (60 nmol) or GTN (4 nmol). Representative tracing of six strips obtained from 2 donors. (b) TTX promptly reversed the response to TSV (100 g) when infused during the relaxation phase. Representative tracing of four strips obtained from 1 donor.
and L-NAME, reduce the relaxations of cavernosal tissue caused by electrical field stimulation and muscarinic receptor activation.17–21 The findings that L-NAME markedly reduced the relaxations elicited by TSV and L-arginine significantly reversed this inhibition indicate that TSV relaxed HCC tissues through the release of NO. The involvement of NO in the TSV-induced HCC relaxations was further confirmed by the finding that ODQ, a potent and selective inhibitor of NO-stimulated soluble guanylyl cyclase,22 virtually abolished the relaxations. Similar to the findings in rabbits,6 these results provide evidence that NO and cyclic guanosine monophosphate mediate the relaxant response elicited by TSV in human cavernosal tissue. The erectile tissues have been shown to be densely innervated by adrenergic excitatory, cholinergic inhibitory and NANC inhibitory nerve fibers.1 The biochemical18 and immunohistochemical23 demonstration of NO synthase within cavernosal nerves, together with the findings that nerve-evoked relaxation is preserved after the removal of endothelium,24 indicates that the most important source of NO in penile tissue is neuronal, appointing a pivotal role for NO in erectile function. It remains possible, however, that the endothelium provides an important additional source of NO for the initiation and maintenance of penile erection. The muscarinic receptor antagonist atropine failed to affect the TSV-induced HCC relaxations, indicating that the venom does not relax this preparation by activating cholinergic fibers. The classic sodium channel blocker TTX ties together in a highly efficient manner various parts of the sodium channel. As a result, the passage of sodium ions through the channel is physically prevented and a conduction blockade results.25 TTX virtually abolished the TSV-induced HCC relaxations, strongly indicating that NO is generated within, and released from, NANC nerve fibers. InUROLOGY 57 (4), 2001
terestingly, TTX also promptly reversed the response to TSV when infused during the established relaxation phase, suggesting a persistent activation of the sodium channels elicited by TSV. 7-NI has been shown to be a potent inhibitor of neuronal NO synthase because of its potent antinociceptive effect26 and its ability to inhibit brain NO synthase activity.26,27 The infusion of 7-NI markedly reduced the HCC relaxations induced by TSV, indicating activation of brain NO synthase in the cavernosal tissue. However, 7-NI also caused small reductions in the relaxations elicited by both ACh and bradykinin because of nonspecific action on endothelium NO synthase. Indeed, 7-NI inhibits NO synthase activity in the homogenates of endothelial cells,26,28 although it has no effect on arterial blood pressure.26 Priapism is most commonly seen in children as a complication of systemic scorpion envenomation. Since our findings demonstrated a potent local effect of the scorpion toxin, it is possible that the amount of venom injected by the Tityus serrulatus scorpion (approximately 0.7 mg)29 may not be sufficient to achieve effective corpus cavernosum concentrations in an adult. Indeed, child envenomation is often accompanied by other systemic symptoms, and adult envenomation is characterized mainly by a restricted local response. CONCLUSIONS We have demonstrated that TSV relaxes the HCC through the release of NO from NANC nerves as a consequence of sodium channel activation. The purification of the responsible toxins should provide a valuable tool in the further understanding of the mechanisms underlying NANC nerve activation in human penile erectile tissue. The finding that sodium channel blockers and NO synthase inhibitors abolish both toxin-induced and electrical field stimulation-induced relaxation of HCC19 indicates a therapeutic potential for these drugs in 819
the treatment of priapism. Although the former is extremely toxic to be used clinically, this limitation does not apply to specific inhibitors of neuronal NO synthase. Indeed, nonspecific inhibitors of NO synthase have already been used in different clinical settings.30 REFERENCES 1. Andersson K-E, and Wagner G: Physiology of penile erection. Physiol Rev 75: 191–236, 1995. 2. Lerner SE, Melman A, and Christ GJ: A review of erection dysfunction: new insights and more questions. J Urol 149: 1246 –1255, 1993. 3. Burnett AL, Lowenstein CJ, Bredt DS, et al: Nitric oxide: a physiologic mediator of penile erection. Science 257: 401– 403, 1992. 4. Finberg JPM, Levy S, and Vardi Y: Inhibition of nerve stimulation-induced vasodilation in corpora cavernosa of the pithed rat by blockade of nitric oxide synthase. Br J Pharmacol 108: 1038 –1042, 1993. 5. Knispel HH, Goessl C, and Beckmann R: Nitric oxide mediates neurogenic relaxation induced in rabbit cavernous smooth muscle by electric field stimulation. Urology 40: 471– 476, 1996. 6. Teixeira CE, Bento AC, Lopes-Martins RAB, et al: Tityus serrulatus scorpion venom relaxes the isolated rabbit corpus cavernosum by activating NANC nitrergic nerve fibers. Br J Pharmacol 123: 435– 442, 1998. 7. Hayashida H, Okamura T, Tomoyoshi T, et al: Neurogenic nitric oxide mediates relaxation of canine corpus cavernosum. J Urol 155: 1122–1127, 1996. 8. Okamura T, Ayajiki K, and Toda N: Monkey corpus cavernosum relaxation mediated by NO and other relaxing factor derived from nerves. Am J Physiol 274: H1075–H1081, 1998. 9. Leone AM, Wiklund NP, Hokfelt T, et al: Release of nitric oxide by nerve stimulation in the human urogenital tract. Neuroreport 24: 733–736, 1994. 10. Couraud F, and Jover E: Mechanism of action of scorpion toxins, in Tu AT (Ed): Handbook of Natural Toxins. New York, Marcel Dekker, 1984, vol 2, pp 659 – 678. 11. Simard J, and Watt DD: Venoms and toxins, in Polis GA (Ed): The Biology of Scorpions. Standford, Standford University Press, 1990, pp 414 – 444. 12. Bawaskar HS: Diagnostic cardiac premonitory signs and symptoms of red scorpion sting. Lancet 1: 552–554, 1982. 13. Amitai Y, Mines Y, Aker M, et al: Scorpion sting in children: a review of 51 cases. Clin Pediatr 24: 136 –140, 1985. 14. Hershkovich Y, Elitsur Y, Margolis CZ, et al: Criteria map audit of scorpion envenomation in Negev, Israel. Toxicon 23: 845– 854, 1985. 15. Freire-Maia L: Peripheral effects of Tityus serrulatus scorpion venom. J Toxicol 14: 423– 435, 1995. 16. Vane JR: The use of isolated organs for detecting active
820
substances in the circulating blood. Br J Pharmacol Chemother 23: 360 –373, 1964. 17. Ignarro LJ, Bush PA, Buga GM, et al: Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle. Biochem Biophys Res Commun 170: 843– 850, 1990. 18. Bush PA, Gonzalez NE, and Ignarro LJ: Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum. Biochem Biophys Res Commun 186: 308 –314, 1992. 19. Holmquist F, Andersson K-E, and Hedlund H: Characterization of inhibitory neurotransmission in the isolated corpus cavernosum from rabbit and man. J Physiol (Lond) 449: 295–311, 1992. 20. Pickard RS, Powell PH, and Zar MA: The effect of inhibitors of nitric oxide biosynthesis and cyclic GMP formation on nerve-evoked relaxation of human cavernosal smooth muscle. Br J Pharmacol 104: 755–759, 1991. 21. Rajfer J, Aronson WJ, Bush PA, et al: Nitric oxide as mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med 326: 90 –94, 1992. 22. Garthwaite J, Southam E, Boulton CL, et al: Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-alquinoxalin-1-one]. Mol Pharmacol 48: 184 –188, 1995. 23. Burnett AL, Tillman SL, Chang TSK, et al: Immunohistochemical localization of nitric oxide synthase in the autonomic innervation of the human penis. J Urol 150: 73–76, 1993. 24. Trigo-Rocha F, Hsu G-L, Donatucci CF, et al: The role of cyclic adenosine monophosphate, cyclic guanosine monophosphate, endothelium and nonadrenergic, noncholinergic neurotransmission in canine penile erection. J Urol 149: 872– 877, 1993. 25. Smythies JR, Benington F, and Morin RD: Model for the action of tetrodotoxin and batrachotoxin. Nature 231: 188 – 190, 1971. 26. Moore PK, Wallace P, Gaffen ZA, et al: Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects. Br J Pharmacol 110: 219 –224, 1993. 27. Mayer B, Klatt P, Werner ER, et al: Molecular mechanisms of inhibition of porcine brain nitric oxide synthase by the antinociceptive drug 7-nitro-indazole. Neuropharmacology 33: 1253–1259, 1994. 28. Wolff DJ, Lubeskie A, and Umansky C: The inhibition of the constitutive bovine endothelial nitric oxide synthase by imidazole and indazole agents. Arch Biochem Biophys 314: 360 –366, 1994. 29. Bu¨cherl W: Classification, biology, and venom extraction of scorpions, in Bu¨cherl W, and Buckley EE (Eds): Venomous Animals and Their Venoms. New York, Academic Press, 1971, vol 3, pp 317–347. 30. Cotter G, Kaluski E, Blatt A, et al: L-NMMA (a nitric oxide synthase inhibitor) is effective in the treatment of cardiogenic shock. Circulation 101: 1358 –1361, 2000.
UROLOGY 57 (4), 2001