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Nitric Oxide Mediates Penile Erection in Cats
0022-534 7/94/1511-0234$03.00/0 THE JOURNAL OF UROLOGY Copyright© 1994 by AMERICAN UROLOGICAL ASSOCIATION, INC.
Vol. 151, 234-237, January 1994
Printed in U.S.A.
NITRIC OXIDE MEDIATES PENILE ERECTION IN CATS RUN WANG, FLOYD R. DOMER, SURESH C_ SIKKA, PHILIP J. KADOWITZ AND WAYNE J. G. HELLSTROM* From the Departments of Urology and Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana
ABSTRACT
The present study was undertaken to investigate the in vivo effects of nitric oxide (NO) mediating agents injected intracavernosally on penile erection in cats. All NO donors increased the cavernosal pressure and penile length in a dose-dependent manner. The maximal effects on cavernosal pressure and penile length induced by s-nitrosocysteine (NO-CYS) and s-nitroso-n-acetylpenicillamine (SNAP), respectively, were 8-fold and 5-fold increases in pressure, and 45% and 34% increases in length when compared with baseline values. These changes were comparable to that caused by the control drug combination (papaverine, phentolamine and prostaglandin E 1 ). The effects of acetylcholine (ACh) and substance P on cavernosal pressure and penile length were less than those obtained with the control drug combination, NO-CYS (p <0.01), or SNAP (p <0.05). Nw-nitro-1arginine-methyl-ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, significantly decreased the effects of NO-CYS, ACh and substance Pon penile erection. This in vivo study with NO donors and an NOS inhibitor suggests that NO is a mediator of penile erection in cats. KEY WORDS: penile erection, cats, nitric oxide
Penile erection involves parasympathetic, neuronally mediated relaxation of the blood vessels and the trabecular meshwork of smooth muscle that constitutes the corpora cavernosa. 1 The relaxation of the cavernous smooth muscle plays a critical role in erection, which is largely nerve-mediated by a nonadrenergic, noncholinergic (NANC) mechanism; 1·2 however, endothelium-dependent cholinergic neurotransmission may also mediate penile erection. 3 Recent studies have shown that nitric oxide (NO) is the major neuronal mediator of erection. 4 Several in vitro studies have demonstrated that NO is responsible for the relaxation of rabbit and human corpus cavernosum smooth muscle strips. 5- 12 In vivo studies in rats 4 and dogs 3 also demonstrated that NO is the principal neurotransmitter in cavernous smooth muscle relaxation. However, in vivo characterization of the role of NO in penile erection requires a comparison of the efficacy of NO in a well-established animal model with that of the most commonly used agents causing penile erection in man. Our previous studies have demonstrated that the adult cat is a useful and reliable model for studying penile erection in response to intracavernous injections of vasoactive agents. 13·14 The present study was undertaken to investigate the in vivo erectile response caused by (1) the agents that release NO directly, s-nitrosocysteine (NO-CYS) and s-nitroso-n-acetylpenicillamine (SNAP), and (2) the agents known to act via activation of nitric oxide synthase (NOS), acetylcholine (ACh) and substance P. These effects were compared with a standard reference (combination of papaverine, prostaglandin E1 and phentolamine) commonly used in clinical practice for treatment of impotence. Also, the inhibiting actions of Nw-nitro-1arginine-methyl-ester (L-NAME), an inhibitor of NOS, were investigated. MATERIALS AND METHODS
Twenty-six mature male cats weighing 4.0 to 6.0 kg. were sedated with ketamine hydrochloride (10 to 15 mg./kg. intramuscularly) and anesthetized with sodium pentobarbital (30 mg./kg. intravenously). A vertical, circumcision-like incision
was made to expose the two ventral corpora cavernosa and the dorsal corpus spongiosum. A 30-gauge needle was placed into the right corpus to permit administration of the drug into the penis. A 25-gauge needle was placed midway into the left corpus for the measurement of intracavernous pressure. Systemic arterial and intracavernous pressures (mm.Hg) were measured with Statham P23 transducers connected to a Grass model 7 polygraph zeroed at the right atrial level, as described previously.13·14 Penile length (mm.) was measured with a ruler. NO-CYS and SNAP were prepared by Dr. L. J. lgnarro (Department of Pharmacology, UCLA Medical School, Los Angeles, California). Dilutions of NO-CYS from stock were prepared by use of degassed, N 2 purged, cold methanol. Dilutions of SNAP were prepared in normal saline. Acetylcholine, substance P and L-NAME were purchased from Sigma Chemical Co. (St. Louis, Missouri) and dissolved in normal saline. The papaverine, PGE1 and phentolamine were prepared and used as previously described. 13 ·14 For each animal in the group, one of the agents (NO-CYS, ACh, substance P, or SNAP) was injected (200 µl. volume) intracavernosally in incremental doses. The dose producing the maximal effect was selected for comparison. L-NAME (20 mg.) was then injected intracavernosally followed by the injections of the above selected dose of the agent at 3- and 30 minuteintervals to observe the block by L-NAME. At the conclusion of each experiment, the control combination of papaverine, PGE1 and phentolamine was administered for quantitative comparison. 13 ' 14 All data were expressed as mean ± the standard error of the mean and analyzed by Student's t test for single-group comparison and by one-way analysis of variance for multiple-group comparisons. The value of p <0.05 was considered statistically significant. RESULTS
Accepted for publication July 13, 1993. * Requests for reprints: Department of Urology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112. Supported by BRSG Grant No. 1090-1-02-110 and in part by NIH Grant HL 15580.
Intracavernous injection of NO-CYS (0.02 to 0.8 µM.) caused a dose-dependent increase in cavernosal pressure and penile length (fig. 1, A and B). The maximal effects were observed with a dose of 0.4 µM. There was an 8-fold increase in cavernosal pressure (133.0 ± 17.3 mm.Hg) and a 45% increase in penile length (30.2 ± 1.6 mm.) when compared with preinjection baseline values (17.1 ± 2.1 mm.Hg for cavernosal pressure and 20.8 ± 2.1 mm. for penile length). This maximal response was
234
235
I";ITRIC OXIDE ~1XEDIATES PENILE ERECTION Il:-J CATS NO-CYS
with the control combination (p <0.01). The effects of ACh and substance P on cavernosal pressure and penile length were also less than those with NO-CYS (p <0.01) and SNAP
SNAP 160
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FIG. 1. Dose-response effects of NO-CYS and SNAP on cavernosal pressure and penile length. NO-CYS and SNAP were injected in increasing doses from 0.02 to 0.8 µM. for NO-CYS (a, b, n = 8) and from 0.3 to 2 µg. for SNAP (c, d, n = 5). C denotes control combination (1.65 mg. papaverine, 25 µg. phentolamine, and 0.5 µg. PGE 1 ) administered at end of experiment. Asterisks denote level of significance compared with baseline. * p <0.05, ** p <0.01.
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Injection of L-NAME blocked the penile responses to NOCYS, ACh and substance P in a time-dependent manner (fig. 3.), but had no effect on the response to the control combination. SNAP was not tested in this study. The duration (in minutes) of the peak pressure and total duration of drug effect were significantly shorter with NO-CYS (3.3 ± 2.0 and 11.0 ± 4.9), SNAP (1.0 ± 0.4 and 6.5 ± 1.2), ACh (2.8 ± 1.6 and 10.0 ± 2.3) and substance P (3.5 ± 1.4 and 13.0 ± 1.1) than with the control combination (30.0 ± 8.0 and 43.0 ± 12.0) (p <0.01). There were no significant differences between NO-CYS, SNAP and ACh, or substance P. All of the agents except L-NAME reduced the systemic arterial pressure when the maximal penile response was obtained (table). Only the decrease induced by ACh and the control combination was statistically significant. L-NAME slightly increased systemic arterial pressure, but not significantly. Systemic pressures always returned to normal levels within 2 minutes after injection of these agents. DISCUSSION
It has been demonstrated that there are at least three neuroeffector pathways that control smooth muscle tone of the corpus cavernosum: adrenergic, cholinergic and NANC. 1 • 2 The neuronal chemical mediator of erection has not been clarified. 2 • 15 In recent in vitro studies with animal and human corpus cavernosa, relaxation evoked by electrical field stimulation was blocked by NOS inhibitors. 5- 12 These studies suggested that NO was a mediator of cavernosal muscular relaxation. In the presence of muscarinic and adrenergic blockade, transmural electrical stimulation of corpus cavernosal tissue in which the endothelium had been mechanically or chemically disrupted elicited NANC neurogenic relaxation. This response was significantly blocked by substances that interfere with the synthesis or the effects of N0. 16 These studies strongly support
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FIG. 2. Dose-response effects of ACh and substance Pon cavernosal pressure and penile length. Acetylcholine and substance P were injected in incremental doses from 25 to 200 µg. for ACh (a, b, n = 5) and from 5 to 60 µg. for substance P (c, d, n = 5). C denotes control combination (1.65 mg. papaverine, 25 µg. phentolamine, and 0.5 µ.g. PGE 1 ) administered at end of experiment. Asterisks denote level of significance compared with baseline. * p <0.05, ** p <0.01.
similar to that obtained with the control combination (fig. 1, A and B). Administration of SNAP (0.3 to 2 µg.) increased the cavernosal pressure and penile length in 5 of 8 cats, with the maximal effects at a dose of 1.0 µg. These maximal effects on cavernosal pressure (70.6 ± 17.5 mm.Hg) and penile length (27.5 ± 1.3 mm.) were 5-fold higher and 34% longer than those of the baseline and were not statistically different from the control (fig. 1, C and D). Three of 8 cats had no change in cavernosal pressure or penile length after the injection of SNAP, but did respond to the control combination. Acetylcholine (25 to 200 µg.) and substance P (5 to 60 µg.) also increased the cavernosal pressure and penile length in the cats, with the maximal effects observed with 50 µg. ACh (fig. 2, A and B) and 40 µg. substance P (fig. 2, C and D). The maximal effects were significantly less than those obtained
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FIG. 3. Effect of L-NAME blocking penile response to different agents. B = baseline, M = maximal effect of agents, L(3) = injection of agents 3 minutes after administration of L-NAME, L(30) = injection of agents 30 minutes after administration of L-NAME; n = 8 for NOCYS, n = 5 for ACh and substance P.
236
NITRIC OXIDE MEDIATES PENILE ERECTION IN CATS
Effect of vasoagents on systemic arterial pressure (mm. Hg) after injection into the penis Before Injection After Injection
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156.7 ± 8.3 137.5 ± 11.3
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the hypothesis that NO is a NANC dilator neurotransmitter in the corpus cavernosum. Though in vitro studies have demonstrated the relaxation effect of NO on cavernosal tissue, in vivo studies are needed to characterize the role of NO in causing penile erection when injected intracavernosally. Because NO is very labile, with a lifetime of only seconds, 10 its use as a pharmacological agent is impractical. But NO donors that can either directly release NO or cause the formation of NO by activating NOS may be useful. A preliminary study by Stief et al. has shown that intracavernosal injection of SIN-1 (an NO donor) induced penile erection in human corpora. 17 Our previous study also demonstrated that an NO donor, sodium nitroprusside (SNP), caused penile erection following intracavernous injection in cats. 14 The present study extends these observations to other NO donors (NOCYS, SNAP, ACh and substance P). Injection of NO-CYS and SNAP caused vascular smooth muscle relaxation by directly releasing N0. 18 Perhaps through the same NO-mediating pathway, NO-CYS and SNAP caused penile erection in cats when injected intracavernosally. The maximal effect on cavernosal pressure and penile length by NO-CYS was comparable to that caused by the control combination, while the maximal effect on cavernosal pressure caused by SNAP was only 60% of that caused by control combination and only effective in 63% (5 of 8) cats. We do not know why 3 of 8 cats had no response to the injection of SNAP. However, these three nonresponders did respond to the control drug combination. This finding may suggest that NO-CYS has better potential for inducing penile erection than SNAP. The duration of maximal pressure and the total duration of drug effect were much shorter for NO-CYS and SNAP than for the control combination. This finding raises the concern that a functional erection produced by NO-CYS and SNAP may be of limited duration. This short-lasting response may be the result of a short-lasting release of NO after injecting NO-CYS and SNAP into the penis and may also represent a different erectile response in different animals, since our unpublished study in primates provided more than 30 minutes of erection with the use of these agents. Acetylcholine and substance P produce relaxation of vascular smooth muscle by stimulating the activation of NOS and the release of N0. 19 Acetylcholine has been shown to cause cavernosal smooth muscle relaxation by the NO-mediating pathway as well. 9- 12 Although the present study demonstrated an increase in cavernosal pressure and penile length in cats after injection of ACh and substance P, the maximal effects of these agents were significantly less than those with the control combination, NO-CYS, or SNAP. In vitro study also showed that ACh and substance P caused less than 60% of the maximal relaxation in human and rabbit cavernosal tissue. Both NO and SNP (which release NO), however, caused greater than 95% of the maximal relaxation. 9 Administration of L-NAME, an inhibitor of NOS, blocks the enzymatic formation of NO from L-arginine. 20 L-NAME significantly blocked the effects of NO-CYS, ACh and substance P in a time-dependent manner. For this reason, the inhibitory effect of L-NAME may be delayed, as observed in the present study (fig. 3), before L-NAME completely inhibited the formation of NO, which is required for smooth muscle tone. It is also possible that the erection caused by NO donors may be based on the NO level contained in the penile tissues. However, the effects of L-NAME on the response to NO-CYS were not expected and may indicate that the response to NO-CYS may,
in part, involve the activation of NOS and the release of NO from L-arginine. The mechanism by which L-NAME inhibits the response to NO-CYS in the feline penis is uncertain and requires further study. Although all NO donors and control drug combinations used in this study reduced the systemic arterial pressure, NO-CYS, SNAP and substance P had fewer adverse effects on systemic arterial pressure since only the decrease induced by ACh and the control combination was statistically significant. By contrast, L-NAME slightly, but not significantly, increased systemic blood pressure after injection into the penis. Caution should be exercised when selecting the doses and methods of injection. In our preliminary study we found the adverse effect on systemic blood pressure could be overcome with the use of a rubber band around the penile base for 30 seconds during injection of the drug. In conclusion, this in vivo feline study supports the hypothesis that NO is a mediator of penile erection. The agents that release NO have a better effect on penile erection in cats than the agents that stimulate the endogenous formation of NO. NO-CYS has a comparable effect on penile erection in cats to that of the control combination and, thus, may have better potential for clinical use. L-NAME can significantly inhibit the effect of NO-CYS, ACh and substance P on the corpus cavernosum in a time-dependent manner. However, this study only assessed in vivo characteristics of some NO donors on penile erection in cats. Caution should be exercised with reference to potential use of these NO donors for clinical trials since the safety and long-term effects of these agents have not been studied at all. Acknowledgement. The authors would like to thank Todd R. Higuera, Department of Pharmacology, for his assistance in preparing the drugs. The authors also want to thank June Banks Evans and Michon Breisacher Shinn for the evaluation and preparation of this manuscript. REFERENCES
1. Saenz de Tejada, I., Blanco, R., Goldstein, I., Azadzoi, K., de las Morenas, A., Krane, R. J. and Cohen, R. A.: Cholinergic neurotransmission in human corpus cavernosum. I. Responses of isolated tissue. Am. J. Physiol. 254: H459, 1988. 2. Saenz de Tejada, I.: In the physiology of erection, signposts to impotence. Contemporary Urology, 4: 52, 1992. 3. Trigo-Rocha, F., Hsu, G. L., Donatucci, C. F. and Lue, T. F.: The role of cyclic adenosine monophosphate, cyclic guanosine monophosphate, endothelium and nonadrenergic, noncholinergic neurotransmission in canine penile erection. J. Urol., 149: 872, 1993. 4. Burnett, A. L., Lowenstein, C. J., Bredt, D.S., Chang, T. S. K. and Snyder, S. H.: Nitric oxide: a physiologic mediator of penile erection. Science, 257: 401, 1992. 5. Ignarro, L. J., Bush, P. A., Buga, G. M., Wood, K. S., Fukuto, J. M. and Rajfer, J.: Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle. Biochem. Biophys. Res. Commun., 1 70: 843, 1990. 6. Pickard, R. S., Powell, P. H. and Zar, M. A.: The effect of inhibitors of nitric oxide biosynthesis and cyclic GMP formation on nerveevoked relaxation of human cavernosal smooth muscle. Br. J. Pharmacol., 104: 755, 1991. 7. Knispel, H. H., Goessl, C. and Beckmann, R.: Basal and acetylcholine-stimulated nitric oxide formation mediates relaxation of rabbit cavernous smooth muscle. J. Urol., 146: 1429, 1991. 8. Rajfer, J., Aronson, W. J., Bush, P.A., Dorey, F. J. and Ignarro, L. J.: Nitric oxide as a mediator of relaxation of the corpus caver-
NITRIC OXIDE MEDIATES PENILE ERECTION IN CATS
9.
10.
11. 12. 13.
14.
nosum in response to nonadrenergic, noncholinergic neurotransmission. N. Engl. J. Med., 326: 90, 1992. Azadzoi, K. M., Kim, N., Brown, M. L., Goldstein, I., Cohen, R. A. and Saenz de Tejada, I.: Endothelium-derived nitric oxide and cyclooxygenase products modulate corpus cavemosum smooth muscle tone. J. Urol., 147: 220, 1992. Bush, P. A., Aronson, W. J., Buga, G. M., Rajfer, J. and Ignarro, L. J.: Nitric oxide is a potent relaxant of human and rabbit corpus cavernosum. J. Urol., 14 7: 1650, 1992. Knispel, H. H., Goessl, C. and Beckmann, R.: Nitric oxide mediates relaxation in rabbit and human corpus cavernosum smooth muscle. Urol. Res., 20: 253, 1992. Knispel, H. H., Goessl, C. and Beckmann, R.: Nitric oxide mediates neurogenic relaxation induced in rabbit cavernous smooth muscle by electric field stimulation. Urology, 40: 471, 1992. Hellstrom, W. J. G., Wang, R., Kadowitz, P. J. and Domer, F. R.: Potassium channel agonists cause penile erection in cats. Int. J. Impotence Res., 4: 35, 1992. Wang, R., Higuera, T. R., Sikka, S. C., Minkes, R. K., Bellan, J. A., Kadowitz, P. J., Domer, F. R. and Hellstrom, W. J. G.: Penile erections induced by vasoactive intestinal peptide and sodium nitroprusside. Ural. Res., 21: 75, 1993.
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15. Keast, J. R.: A possible neural source of nitric oxide in the rat penis. Neuroscience Lett., 143: 69, 1992. 16. Kim, N., Azadzoi, K. M., Goldstein, I. and Saenz de Tejada, I.: A nitric oxide-like factor mediates non-adrenergic non-cholinergic neurogenic relaxation of penile corpus cavernosum smooth muscle. J. Clin. Invest., 88: 112, 1991. 17. Stief, C. G., Holmquist, F., Allhoff, E. P., Andersson, K.-E. and Jonas, U.: Preliminary report on the effect of the nitric oxide (NO) donor SIN-1 on human cavernous tissue in vivo. World J. Ural., 9: 237, 1991. 18. Ignarro, L. J., Lippton, H., Edwards, J.C., Baricos, W. H., Hyman, A. L., Kadowitz, P. J. and Gruetter, C. A.: Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of Snitrosothiols as active intermediates. J. Pharmacol. Exp. Ther., 218: 739, 1981. 19. McMahon, T. J. and Kadowitz, P. J.: Analysis of responses to substance P in the pulmonary vascular bed of the cat. Am. J. Physiol., 264: H394, 1993. 20. McMahon, T. J., Hood, J. S., Bellan, J. A. and Kadowitz, P. J.: Nw-nitro-L-arginine methyl ester selectively inhibits pulmonary vasodilator responses to acetylcholine and bradykinin. J. Appl. Physiol., 75: 2026, 1991.
Vol. 151, 234-237, January 1994
Printed in U.S.A.
NITRIC OXIDE MEDIATES PENILE ERECTION IN CATS RUN WANG, FLOYD R. DOMER, SURESH C_ SIKKA, PHILIP J. KADOWITZ AND WAYNE J. G. HELLSTROM* From the Departments of Urology and Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana
ABSTRACT
The present study was undertaken to investigate the in vivo effects of nitric oxide (NO) mediating agents injected intracavernosally on penile erection in cats. All NO donors increased the cavernosal pressure and penile length in a dose-dependent manner. The maximal effects on cavernosal pressure and penile length induced by s-nitrosocysteine (NO-CYS) and s-nitroso-n-acetylpenicillamine (SNAP), respectively, were 8-fold and 5-fold increases in pressure, and 45% and 34% increases in length when compared with baseline values. These changes were comparable to that caused by the control drug combination (papaverine, phentolamine and prostaglandin E 1 ). The effects of acetylcholine (ACh) and substance P on cavernosal pressure and penile length were less than those obtained with the control drug combination, NO-CYS (p <0.01), or SNAP (p <0.05). Nw-nitro-1arginine-methyl-ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, significantly decreased the effects of NO-CYS, ACh and substance Pon penile erection. This in vivo study with NO donors and an NOS inhibitor suggests that NO is a mediator of penile erection in cats. KEY WORDS: penile erection, cats, nitric oxide
Penile erection involves parasympathetic, neuronally mediated relaxation of the blood vessels and the trabecular meshwork of smooth muscle that constitutes the corpora cavernosa. 1 The relaxation of the cavernous smooth muscle plays a critical role in erection, which is largely nerve-mediated by a nonadrenergic, noncholinergic (NANC) mechanism; 1·2 however, endothelium-dependent cholinergic neurotransmission may also mediate penile erection. 3 Recent studies have shown that nitric oxide (NO) is the major neuronal mediator of erection. 4 Several in vitro studies have demonstrated that NO is responsible for the relaxation of rabbit and human corpus cavernosum smooth muscle strips. 5- 12 In vivo studies in rats 4 and dogs 3 also demonstrated that NO is the principal neurotransmitter in cavernous smooth muscle relaxation. However, in vivo characterization of the role of NO in penile erection requires a comparison of the efficacy of NO in a well-established animal model with that of the most commonly used agents causing penile erection in man. Our previous studies have demonstrated that the adult cat is a useful and reliable model for studying penile erection in response to intracavernous injections of vasoactive agents. 13·14 The present study was undertaken to investigate the in vivo erectile response caused by (1) the agents that release NO directly, s-nitrosocysteine (NO-CYS) and s-nitroso-n-acetylpenicillamine (SNAP), and (2) the agents known to act via activation of nitric oxide synthase (NOS), acetylcholine (ACh) and substance P. These effects were compared with a standard reference (combination of papaverine, prostaglandin E1 and phentolamine) commonly used in clinical practice for treatment of impotence. Also, the inhibiting actions of Nw-nitro-1arginine-methyl-ester (L-NAME), an inhibitor of NOS, were investigated. MATERIALS AND METHODS
Twenty-six mature male cats weighing 4.0 to 6.0 kg. were sedated with ketamine hydrochloride (10 to 15 mg./kg. intramuscularly) and anesthetized with sodium pentobarbital (30 mg./kg. intravenously). A vertical, circumcision-like incision
was made to expose the two ventral corpora cavernosa and the dorsal corpus spongiosum. A 30-gauge needle was placed into the right corpus to permit administration of the drug into the penis. A 25-gauge needle was placed midway into the left corpus for the measurement of intracavernous pressure. Systemic arterial and intracavernous pressures (mm.Hg) were measured with Statham P23 transducers connected to a Grass model 7 polygraph zeroed at the right atrial level, as described previously.13·14 Penile length (mm.) was measured with a ruler. NO-CYS and SNAP were prepared by Dr. L. J. lgnarro (Department of Pharmacology, UCLA Medical School, Los Angeles, California). Dilutions of NO-CYS from stock were prepared by use of degassed, N 2 purged, cold methanol. Dilutions of SNAP were prepared in normal saline. Acetylcholine, substance P and L-NAME were purchased from Sigma Chemical Co. (St. Louis, Missouri) and dissolved in normal saline. The papaverine, PGE1 and phentolamine were prepared and used as previously described. 13 ·14 For each animal in the group, one of the agents (NO-CYS, ACh, substance P, or SNAP) was injected (200 µl. volume) intracavernosally in incremental doses. The dose producing the maximal effect was selected for comparison. L-NAME (20 mg.) was then injected intracavernosally followed by the injections of the above selected dose of the agent at 3- and 30 minuteintervals to observe the block by L-NAME. At the conclusion of each experiment, the control combination of papaverine, PGE1 and phentolamine was administered for quantitative comparison. 13 ' 14 All data were expressed as mean ± the standard error of the mean and analyzed by Student's t test for single-group comparison and by one-way analysis of variance for multiple-group comparisons. The value of p <0.05 was considered statistically significant. RESULTS
Accepted for publication July 13, 1993. * Requests for reprints: Department of Urology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112. Supported by BRSG Grant No. 1090-1-02-110 and in part by NIH Grant HL 15580.
Intracavernous injection of NO-CYS (0.02 to 0.8 µM.) caused a dose-dependent increase in cavernosal pressure and penile length (fig. 1, A and B). The maximal effects were observed with a dose of 0.4 µM. There was an 8-fold increase in cavernosal pressure (133.0 ± 17.3 mm.Hg) and a 45% increase in penile length (30.2 ± 1.6 mm.) when compared with preinjection baseline values (17.1 ± 2.1 mm.Hg for cavernosal pressure and 20.8 ± 2.1 mm. for penile length). This maximal response was
234
235
I";ITRIC OXIDE ~1XEDIATES PENILE ERECTION Il:-J CATS NO-CYS
with the control combination (p <0.01). The effects of ACh and substance P on cavernosal pressure and penile length were also less than those with NO-CYS (p <0.01) and SNAP
SNAP 160
(p <0.05).
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FIG. 1. Dose-response effects of NO-CYS and SNAP on cavernosal pressure and penile length. NO-CYS and SNAP were injected in increasing doses from 0.02 to 0.8 µM. for NO-CYS (a, b, n = 8) and from 0.3 to 2 µg. for SNAP (c, d, n = 5). C denotes control combination (1.65 mg. papaverine, 25 µg. phentolamine, and 0.5 µg. PGE 1 ) administered at end of experiment. Asterisks denote level of significance compared with baseline. * p <0.05, ** p <0.01.
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28
Injection of L-NAME blocked the penile responses to NOCYS, ACh and substance P in a time-dependent manner (fig. 3.), but had no effect on the response to the control combination. SNAP was not tested in this study. The duration (in minutes) of the peak pressure and total duration of drug effect were significantly shorter with NO-CYS (3.3 ± 2.0 and 11.0 ± 4.9), SNAP (1.0 ± 0.4 and 6.5 ± 1.2), ACh (2.8 ± 1.6 and 10.0 ± 2.3) and substance P (3.5 ± 1.4 and 13.0 ± 1.1) than with the control combination (30.0 ± 8.0 and 43.0 ± 12.0) (p <0.01). There were no significant differences between NO-CYS, SNAP and ACh, or substance P. All of the agents except L-NAME reduced the systemic arterial pressure when the maximal penile response was obtained (table). Only the decrease induced by ACh and the control combination was statistically significant. L-NAME slightly increased systemic arterial pressure, but not significantly. Systemic pressures always returned to normal levels within 2 minutes after injection of these agents. DISCUSSION
It has been demonstrated that there are at least three neuroeffector pathways that control smooth muscle tone of the corpus cavernosum: adrenergic, cholinergic and NANC. 1 • 2 The neuronal chemical mediator of erection has not been clarified. 2 • 15 In recent in vitro studies with animal and human corpus cavernosa, relaxation evoked by electrical field stimulation was blocked by NOS inhibitors. 5- 12 These studies suggested that NO was a mediator of cavernosal muscular relaxation. In the presence of muscarinic and adrenergic blockade, transmural electrical stimulation of corpus cavernosal tissue in which the endothelium had been mechanically or chemically disrupted elicited NANC neurogenic relaxation. This response was significantly blocked by substances that interfere with the synthesis or the effects of N0. 16 These studies strongly support
24
D 20
20
16
0
25
50 100 200
(µg)
16 ·
C
Dose
ACl:I
H!O
0
5
20
40
60
C
(µg)
FIG. 2. Dose-response effects of ACh and substance Pon cavernosal pressure and penile length. Acetylcholine and substance P were injected in incremental doses from 25 to 200 µg. for ACh (a, b, n = 5) and from 5 to 60 µg. for substance P (c, d, n = 5). C denotes control combination (1.65 mg. papaverine, 25 µg. phentolamine, and 0.5 µ.g. PGE 1 ) administered at end of experiment. Asterisks denote level of significance compared with baseline. * p <0.05, ** p <0.01.
similar to that obtained with the control combination (fig. 1, A and B). Administration of SNAP (0.3 to 2 µg.) increased the cavernosal pressure and penile length in 5 of 8 cats, with the maximal effects at a dose of 1.0 µg. These maximal effects on cavernosal pressure (70.6 ± 17.5 mm.Hg) and penile length (27.5 ± 1.3 mm.) were 5-fold higher and 34% longer than those of the baseline and were not statistically different from the control (fig. 1, C and D). Three of 8 cats had no change in cavernosal pressure or penile length after the injection of SNAP, but did respond to the control combination. Acetylcholine (25 to 200 µg.) and substance P (5 to 60 µg.) also increased the cavernosal pressure and penile length in the cats, with the maximal effects observed with 50 µg. ACh (fig. 2, A and B) and 40 µg. substance P (fig. 2, C and D). The maximal effects were significantly less than those obtained
...,
..d !:l!.l
i:1 I!)
...J
.....w ...... d
Q,)
p..,
-ss -
JO 25 20
15
B
M
L(30)
FIG. 3. Effect of L-NAME blocking penile response to different agents. B = baseline, M = maximal effect of agents, L(3) = injection of agents 3 minutes after administration of L-NAME, L(30) = injection of agents 30 minutes after administration of L-NAME; n = 8 for NOCYS, n = 5 for ACh and substance P.
236
NITRIC OXIDE MEDIATES PENILE ERECTION IN CATS
Effect of vasoagents on systemic arterial pressure (mm. Hg) after injection into the penis Before Injection After Injection
*p
<0.05,
** p
NO-CYS
SNAP
Ach
Substance P
Control
L-NAME
156.7 ± 8.3 137.5 ± 11.3
171.3 ± 4.3 165.0 ± 6.5
155.8 ± 8.5 81.7 ± 10.5**
158.0 ± 13.7 131.0 ± 15.3
160.7 ± 8.7 125.0 ± 15.0*
162.7 ± 7.1 173.6 ± 7.2
<0.01.
the hypothesis that NO is a NANC dilator neurotransmitter in the corpus cavernosum. Though in vitro studies have demonstrated the relaxation effect of NO on cavernosal tissue, in vivo studies are needed to characterize the role of NO in causing penile erection when injected intracavernosally. Because NO is very labile, with a lifetime of only seconds, 10 its use as a pharmacological agent is impractical. But NO donors that can either directly release NO or cause the formation of NO by activating NOS may be useful. A preliminary study by Stief et al. has shown that intracavernosal injection of SIN-1 (an NO donor) induced penile erection in human corpora. 17 Our previous study also demonstrated that an NO donor, sodium nitroprusside (SNP), caused penile erection following intracavernous injection in cats. 14 The present study extends these observations to other NO donors (NOCYS, SNAP, ACh and substance P). Injection of NO-CYS and SNAP caused vascular smooth muscle relaxation by directly releasing N0. 18 Perhaps through the same NO-mediating pathway, NO-CYS and SNAP caused penile erection in cats when injected intracavernosally. The maximal effect on cavernosal pressure and penile length by NO-CYS was comparable to that caused by the control combination, while the maximal effect on cavernosal pressure caused by SNAP was only 60% of that caused by control combination and only effective in 63% (5 of 8) cats. We do not know why 3 of 8 cats had no response to the injection of SNAP. However, these three nonresponders did respond to the control drug combination. This finding may suggest that NO-CYS has better potential for inducing penile erection than SNAP. The duration of maximal pressure and the total duration of drug effect were much shorter for NO-CYS and SNAP than for the control combination. This finding raises the concern that a functional erection produced by NO-CYS and SNAP may be of limited duration. This short-lasting response may be the result of a short-lasting release of NO after injecting NO-CYS and SNAP into the penis and may also represent a different erectile response in different animals, since our unpublished study in primates provided more than 30 minutes of erection with the use of these agents. Acetylcholine and substance P produce relaxation of vascular smooth muscle by stimulating the activation of NOS and the release of N0. 19 Acetylcholine has been shown to cause cavernosal smooth muscle relaxation by the NO-mediating pathway as well. 9- 12 Although the present study demonstrated an increase in cavernosal pressure and penile length in cats after injection of ACh and substance P, the maximal effects of these agents were significantly less than those with the control combination, NO-CYS, or SNAP. In vitro study also showed that ACh and substance P caused less than 60% of the maximal relaxation in human and rabbit cavernosal tissue. Both NO and SNP (which release NO), however, caused greater than 95% of the maximal relaxation. 9 Administration of L-NAME, an inhibitor of NOS, blocks the enzymatic formation of NO from L-arginine. 20 L-NAME significantly blocked the effects of NO-CYS, ACh and substance P in a time-dependent manner. For this reason, the inhibitory effect of L-NAME may be delayed, as observed in the present study (fig. 3), before L-NAME completely inhibited the formation of NO, which is required for smooth muscle tone. It is also possible that the erection caused by NO donors may be based on the NO level contained in the penile tissues. However, the effects of L-NAME on the response to NO-CYS were not expected and may indicate that the response to NO-CYS may,
in part, involve the activation of NOS and the release of NO from L-arginine. The mechanism by which L-NAME inhibits the response to NO-CYS in the feline penis is uncertain and requires further study. Although all NO donors and control drug combinations used in this study reduced the systemic arterial pressure, NO-CYS, SNAP and substance P had fewer adverse effects on systemic arterial pressure since only the decrease induced by ACh and the control combination was statistically significant. By contrast, L-NAME slightly, but not significantly, increased systemic blood pressure after injection into the penis. Caution should be exercised when selecting the doses and methods of injection. In our preliminary study we found the adverse effect on systemic blood pressure could be overcome with the use of a rubber band around the penile base for 30 seconds during injection of the drug. In conclusion, this in vivo feline study supports the hypothesis that NO is a mediator of penile erection. The agents that release NO have a better effect on penile erection in cats than the agents that stimulate the endogenous formation of NO. NO-CYS has a comparable effect on penile erection in cats to that of the control combination and, thus, may have better potential for clinical use. L-NAME can significantly inhibit the effect of NO-CYS, ACh and substance P on the corpus cavernosum in a time-dependent manner. However, this study only assessed in vivo characteristics of some NO donors on penile erection in cats. Caution should be exercised with reference to potential use of these NO donors for clinical trials since the safety and long-term effects of these agents have not been studied at all. Acknowledgement. The authors would like to thank Todd R. Higuera, Department of Pharmacology, for his assistance in preparing the drugs. The authors also want to thank June Banks Evans and Michon Breisacher Shinn for the evaluation and preparation of this manuscript. REFERENCES
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