Stimulatory effects of quinelorane on yawning and penile erection in the rat

Life Sciences, Vol. 54, No. 7, pp. 507-514, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/94 $6.O0 + .00

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STIMULATORY EFFECTS OF QUINELORANE ON YAWNING AND PENILE ERECTION IN THE RAT Paul C. Doherty and Petra A. Wisler Lilly Research Laboratories A Division of Eli Lilly and Company, Lilly Corporate Center Indianapolis, IN 46285 ( R e c e i v e d in final f o r m N o v e m b e r 29, 1993)

Summary Quinelorane, a highly selective D2 dopamine agonist, was assessed for its ability to induce the penile erection/stretch-yawn syndrome. Quinelorane (0.1 - 100 pg/kg s.c.) or saline vehicle was administered to adult male Sprague-Dawley rats just prior to a 30 min. observation period. Significant dose-related increases in erections were observed in the drug treated animals at 3-100 pg/kg. Yawning was also increased at 3-100 ~tg/kg, with highest levels occurring at 10 i.tg/kg. Defecation was stimulated between 10 and 100 pg/kg. The stimulatory effects of 30 #g/kg of quinelorane on erection, yawning and defecation were blocked by haloperidol (0.1-0.3 mg/kg) but not by domperidone (0.11.0 mg/kg). No significant effects of quinelorane on seminal emission were observed. These findings indicate that in addition to its stimulatory effects on sexual activity, quinelorane also acts on D2 receptors in the central nervous system to stimulate erection in the penile erection/stretch-yawn model. Quinelorane is a highly selective D2 dopamine (DA) agonist (15) that has been shown to stimulate sexual activity in male and female rats (12, 13). In males, quinelorane reduces intromission and ejaculation latencies as well as the number of intromissions required for ejaculation. These effects are mediated by interactions with D2 receptors within the central nervous system (CNS; 12). The effects of DA and DA agonists on sexual activity have often been attributed to changes in motivation/reward (1,4,23,26). However, DA receptor agonists also influence erectile function. For example, the mixed D1/D 2 agonist, apomorphine, increases the number of erections observed in the restrained supine rat (ex copula penile reflex tests). In addition, apomorphine affects several aspects of copulatory behavior in the male rat that have been associated with erectile capacity, including intromission rate, the percentage of mounts with intromission, and ejaculation latency. These effects are observed after subcutaneous injection, intracerebroventricular administration, or by direct infusion into the medial preoptic area (MPOA; 18,24) Administration of apomorphine also increases the occurrence of spontaneous erections in rats (16), rhesus monkeys (27) and men (7,20,33). Unlike apomorphine, quinelorane inhibits erection and stimulates seminal emission in rat penile reflex tests after subcutaneous administration (5) or direct infusion into the MPOA (2). However, a moderate dose of quinelorane has been shown to

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reduce reflex latency and increase the number of intense erections and penile movements during such tests in rats when administered into the paraventricular nucleus of the hypothalamus (PVN; 10). In addition, quinelorane increases the display of erections in rhesus monkeys after intra-muscular administration and does so by stimulating central DA receptors (28). The purpose of the present experiments was threefold. First, we wished to determine if quinelorane would induce erection in the penile erection/stretch-yawn syndrome (PE/SYS), a behavioral response that may more closely parallel the effects of DA agonists on erection in rhesus monkeys and men. Second, we wanted to assess the participation of D2 receptors in the CNS in this response. Finally, since quinelorane administration has been associated with seminal emission in penile reflex tests (2,5), and since the suggestion has been made that erection in PE/SYS is occurring as a reflexive response to drug induced emission (6), we have also examined the degree to which penile erection can be associated with seminal emission in PE/SYS after quinelorane administration. Methods Adult male Sprague-Dawley rats (250-275 g) purchased from Charles River Breeding Laboratories (Portage, MI) were housed in suspended wire cages (2-4 animals/cage) in temperature controlled rooms on a standard 12h L: 12h D lighting schedule (lights on 6.00 h) with food and water freely available. Drug studies were begun one week after initial receipt to allow the animals to adjust to their new surroundings. In addition, the animals were exposed to the observation cages during this time period to reduce the effects that a novel environment might have on the expression of these behaviors (9). The observation cages consisted of clear plastic shoe box type cages (4x6x8 in) with clear plastic filter top bonnets (3x6x8 in) placed on top. Mirrors were placed behind each of the cages to allow a clear view of the animals during erection and grooming. Quinelorane was dissolved in saline while the DA antagonists were dissolved in a 0.1 mM acetic acid / 0.1 mM ascorbic acid vehicle (VEH) and were given 30 min prior to quinelorane (30 p_g/kg) or saline administration. Drugs were administered in a volume of 1.0 ml/kg. Behavioral testing was performed between 9.00 and 13.30 h according to an adaptation of previously described methods (16). Stretching, yawning and penile erection were quantified by a trained observer during the thirty minute period immediately after quinelorane or saline injection. The number of fecal pellets (boluses) left in the cage by the animal during the thirty minute test period were also recorded. Penile erection was defined as a period of pelvic thrusting followed by genital grooming and the display of the engorged penis. Since an animal may spend a considerable portion of the thirty minutes grooming, it was necessary to visually confirm the presence of penile enlargement and engorgement to score the behavior as an erectile response. EXPERIMENT ONE: Twenty animals were used in a repeated dosing study. The animals were injected subcutaneously in the nape of the neck with saline vehicle or quinelorane (0.1,0.3, 1.0, 3.0, 10.0, 30.0, and 100.0 l.tg/kg) just prior to being placed in the observation cages. Each dose of quinelorane, one dose per test, was administered in ascending order with a two to three day lapse between each test. EXPERIMENT TWO: Two separate groups of ninety-six animals were examined for the ability of the centrally acting D2 antagonist, haloperidol (0.03, 0.1 and 0.3 mg/kg), or the peripherally selective D2 antagonist, domperidone (0.1, 0.3, and 1.0 mg/kg), to antagonize the stimulatory effects of quinelorane on penile erection. Since erection was the primary focus of this study, the dose of quinelorane (30 t.tg/kg) that showed maximal

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stimulation of penile erection was chosen. In these experiments, all possible treatment combinations were administered to subgroups of the animals on separate days using a modified Latin Square design to counterbalance the effects of dose of the antagonists with time and day of administration. EXPERIMENT THREE: The effects of quinelorane on penile erection and seminal emission were assessed using a counterbalanced design in six independent groups of animals (8 animals per group). Behavioral observations were conducted as previously described except that the number of emissions and the time to onset of each of the behaviors were also recorded. The cages were mounted on a glass shelf with mirrors placed behind and below to allow a more complete view of the animals genital region during emission, erection and grooming. STATISTICAL ANALYSIS: The data were analyzed using non-parametric methods. Significant treatment effects were first assessed in Friedman's two way analysis of variance for the repeated dosing study, in a non-parametric two by two factorial design for ranked data for the dopamine antagonist studies (8,32) and in the Kruskall-Wallis test for the study examining seminal emission and erection. Differences in the percentage of animals showing a response in this last experiment were assessed using Fisher's Exact Probability test. A posteriori comparisons were performed using the Wilcoxon signed rank test or the Mann-Whitney U-test. Such comparisons were made only between the response to each dose of drug and the appropriate vehicle response. A probability level of less than 0.05 (in two tails) was required as evidence of significant treatment effects. Results Subcutaneous administration of quinelorane was associated with significant increases in yawning and penile erection (Figure 1). Dose related increases in the erectile response were observed between 3 and 100 #g/kg, peaking at 30 #g/kg. The number of yawns sharply was increased between 3 and 100 #g/kg and with the highest levels occurring at 10 #g/kg. Quinelorane also induced significant alterations in the stretch and defecation responses. The number of stretches was significantly increased at 0.1 p.g/kg and decreased to control levels at 30 and 100 !~g/kg. In contrast, effects of quinelorane on the number of defecations showed a biphasic response. Defecation was significantly inhibited from control levels at 0.1 #g/kg, and stimulated at doses above 10 #g/kg. Administration of 0.1 and 0.3 mg/kg of the centrally acting D2 antagonist, haloperidol, inhibited erections (Fig. 2) and defecations induced by 30 I.tg/kg of quinelorane. Yawning was significantly inhibited from control levels only at the 0.3 mg/kg dose of haloperidol. The peripherally selective D2 antagonist, domperidone, in doses from 0.1 to 1 mg/kg, had no effect on the quinelorane-induced behavioral responses. No significant effects of either of the D2 antagonists were found on the level of behavior observed after saline injection. Administration of 10 and 30 ~tg/kg of quinelorane in the tests for seminal emission was associated with a significant increase in the number of erections and a decrease in the latency to the first erection (Table 1). Although some emission was observed with all doses of quinelorane, no significant effects of drug treatment on seminal emission occurred. In addition, in only one instance could emission be directly associated with erection. Emission was most often observed as a separate event completely displaced in time from the erectile response. Yawning and defecation were also increased and the time to onset of each of these behaviors was decreased by quinelorane administration (3-30 #g/kg for yawning, 10-100 #g/kg for defecation; data not shown).

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Discussion The results of the present experiments demonstrate that quinelorane can increase the occurrence of spontaneous erections in the male rat. This effect occurred within the dose range that is stimulatory to sexual activity (12) suggesting that the activation of penile erection may contribute to the effects of quinelorane on male sexual behavior. The blockade of the erectile response by the centrally acting antagonist, haloperidol, but not by domp.eridone in doses that do not alter CNS function in the rat (11), demonstrates that the effects of quinelorane on erections occur through stimulation of specific D2 receptors located within the CNS. In this paradigm as well as in tests of sexual activity, quinelorane is apparently more potent than other dopamine agonists

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Ouineiorane and Penile Erection

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The effects of haloperidol (left panels) and domperidone (right panels) on quineloraneinduced erections (upper panels), yawns (middle panels) and defecations (lower panels). Quinelorane (30 l.tg/kg) was injected 30 min after haloperidol or domperidone treatment. * - Significantly different from within dose saline control, p < 0.05. 1 - Significantly different from vehicle+quinelorane-treated control, p < 0.05.

1.0

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Table 1. The Effects of Quinelorane on Erection and Seminal Emission. Groups of adult male rats

(n = 8) were injected with various doses of quinelorane or vehicle and observed for the display of erection and seminal emission over the next thirty minutes and for the timing of the initial occurrence of these events (Latency). Latencies are reported in minutes. % - The percentageof animals within each group that displayed the response. Values (except %) are expressedas Mean + SEM. ERECTIONS Dose

Latency

EMISSIONS

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%

Latency

Number

%

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100

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75.0t

* - Significantly different from Vehicle treated group, p < 0.05. t - Significantly different from Vehicle treated group, p < 0.01. including quinpirole, apomorphine, and pergolide (12,16). However, the threshold dose for stimulation of the erectile response was higher than the doses needed to induce significant changes in tests of copulatory behavior (12). This shift in threshold corresponds with differences in the doses which restore mounting behavior versus those that restore ejaculations in adult non-maters (12). These results suggest that higher doses of quinelorane may be required to induce the erectile response than those needed to increase sexual drive. However, we cannot exclude the possibility that during standard tests of copulatory behavior, exposure of a sexually experienced male to an estrous female may shift the sensitivity of the quinelorane response. The presence of a female does enhance the display of erections after quinelorane treatment in rhesus monkeys (28). Quinelorane and apomorphine induce similar responses in PE/SYS (16), but strikingly different effects in penile reflex tests (2,4,10,24,25). Their similarity of action in PE/SYS appears to be due to stimulation of D2 receptors (15,16). In contrast, much of the difference in the activity of these two drugs on ex copula penile reflexes may involve the low affinity of quinelorane for the D1 receptor (15). It has recently been proposed that the brain exerts a dual influence on spinal centers involved in erection; disinhibition and excitation (30). According to this scheme, reduced erection latencies in penile reflex tests reflect disinhibition while increased numbers of erections represent excitation. Bazzett et al., have proposed that D2 and D1 receptor stimulation in the MPOA may correlate with disinhibition and excitation, respectively, of erections in penile reflex tests. Thus, apomorphine by stimulating both D1 and D2 receptors can decrease erection latency (disinhibition) and increase erections (excitation). Quinelorane, on the other hand, through selective stimulation of D2 receptors, decreases erection latency (2,19). It apparently does so both in tests of penile reflexes and in PE/SYS. The latter conclusion is limited, however, by the fact that a major effect of quinelorane on erection in PE/SYS is to increase the proportion of animals displaying erections during a set time period. The increase in the number of erections that is also observed may be mediated by stimulation of D2 receptors at sites other than the MPOA, such as the PVN (22). Infusion of both apomorphine (25) and quinelorane (10) into the PVN decreases erection latency and stimulates erections and emission in penile reflex testing.

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The suggestion that seminal emission plays a role in the erections observed in PE/SYS has arisen from several lines of evidence. First, quinelorane increases seminal emission and decreases the number of erections in penile reflex tests (2,5). It also decreases ejaculatory thresholds in tests of copulatory behavior (5,12). Furthermore, the display of erections in penile reflex tests is inhibited after the occurrence of emission, a phenomenon that has been likened to the post-ejaculatory refractory period. In addition, dopamine agonist induced erections in PE/SYS occur with a frequency that parallels the number of ejaculations in tests of copulatory behavior rather than the number of intromissions preceding ejaculation. It has seemed logical, therefore, to assume that erections observed in PE/SYS might be occurring as a reflexive response to emission (6,31), similar to the induction of coital reflexes after stimulation of the urethral bulb with saline (21), rather than a direct stimulation of erection. The experiment presented herein represents the first time that the occurrence of seminal emission during PE/SYS has been examined in a systematic fashion. It has been our experience, using not only quinelorane but also a variety of other dopamine agonists, that emissions occur infrequently (Doherty and Wisler, unpublished observations), and more often than not, the animal neither grooms in response to the emission nor does it display an erection. The lack of significant coincidence of erection with emission in the present experiment indicates that it is unlikely that emission is either a cause of, or a necessary requirement for erection in PE/SYS. PE/SYS has been widely used as a drug screen (3,16). Examination of its relationship to the effects of hormones and neurotransmitters on sexual activity has occurred only recently (17,31). The demonstration that this response is androgen dependent (17), but that acute treatment with dihydrotestosterone propionate does not restore the response (3), as can be shown for erections in penile reflex testing (31), demonstrates that hormones influence each of these models of the erectile response through interactions with different areas of the CNS. There appears to be a high degree of correlation, however, between the ability of dopamine agonists to induce erections in PE/SYS with similar results that have been observed in the rhesus monkey and in man (3,7,16,20,27,33). It may also be relevant that emission has not been reported in association with dopamine agonist induced erections in either rhesus monkeys or men. Thus, this behavioral model appears to be a useful paradigm for the development of tools in the diagnosis and treatment of erectile dysfunction (14,17). The ability of dopamine agonists to increase defecation in the rat has not been widely reported. Since domperidone failed to alter the effects of quinelorane on defecation, the results of the present study demonstrate that this response is mediated by DA receptors within the CNS. The stimutatory effects of quinelorane on defecation commence with doses that induce peak numbers of erections but at which yawning is rapidly decreasing. Thus, the defecation response may be mediated by DA receptors at different sites from those at which yawning and penile erection are induced. Potential sites of action include neurons in the MPOA/anterior hypothalamic region that influence marking behaviors like urination and defecation (9), or nigro-striatal DA neurons. Defecation has recently been correlated with changes in the levels of dopamine and its metabolites in the corpus striatum (29). In summary, the results of the present study show that quinelorane increases the occurrence of spontaneous erections in the male rat through a direct interaction with D2 receptors in the CNS. Moreover, the erectile response observed appears to be unrelated to any effects of quinelorane on seminal emission. However, this response may be related to both the increased copulatory efficiency and decreased ejaculatory threshold that have been seen previously with this compound in tests of male sexual activity (12). Though we are unable, on the basis of the results of this study, to precisely

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localize CNS structures involved in this response, one potential site may be the PVN. Implantation of quinelorane into this nucleus has been reported to increase the number of intense erections and penile movements in penile reflex tests (10). Reference~ 1. L.M. ALDERSON AND M.J. BAUM Brain Res. 218189-206 (1981). 2. T.J BAZZETT, R.C. EATON, J.T THOMPSON, V.P. MARKOWSKI, L.A. LUMLEY.AND E.M. HULL Life Sci. 482309-2316 (1991). 3. H.G. BERENDSON AND A.J. GOWER Neuroendocrinology 4._22185-190 (1986). 4. D. BITRAN AND E.M. HULL Neurosci. Biobehav. Rev. 11365-389 (1987). 5. D. BITRAN, J.T. THOMPSON, E.M. HULL AND B.D. SACHS Pharmacol. Biochem. Behav. 34453-458 (1989). 6 CLARK, J.T. Methods in Neurosci. 1{432-53 (1993) 7 P. DANJOU, L. ALEXANDRE, D. WAROT, L. LACOMBLEZ AND A.J. PUECH Br. J. Pharmacol. 2 6 7 3 3 - 7 3 9 (1988). 8. P.C. DOHERTY, A.BARTKE, M.P. HOGAN, H. KLEMCKE AND M.S. SMITH Endocrinology 1._! 820-826. 9. P.C. DOHERTY Physiol. Behav. 501047-1050 (1991). 10. R.C. EATON, V.P. MARKOWSKI, L.A. LUMLEY, J.T. THOMPSON, J. MOSES AND E.M. HULL. Pharmacol. Biochem. Behav. 39177-181 (1991). 11. J.M. FARAH JR., K.T. DEMAREST AND K.E. MOORE. Life Sci. 331561-1566 (1983). 12. M.M. FOREMAN ANDJ.L. HALL J. NeuralTrans. 68153-170 (1987). 13. M.M. FOREMAN AND J.L. HALL Psychopharmacol. 9"196-100 (1987). 14. M.M. FOREMAN AND J.C. WERNICKE Sem. Urol. _8107-112(1990). 15. M.M. FOREMAN, R.W FULLER, M.D. HYNES, J.S. GIDDA, C.L. NICHOLS, J.M. SCHAUS, E.C. KORNFELD, AND J.A. CLEMENS J. Pharmacol. Exp. Ther. 250 237-235 (1989). 16. A.J. GOWER, H.H.G. BERENDSON, M.M. PRINCEN AND C.L.E BROEKKAMP Eur. J. Pharmacol. 10381-89 (1984). 17. J.P.W. HEATON, S.J. VARRIN AND A. MORALES J. Urol. 1451099-1102 (1991). 18. E.M HULL, D. BITRAN, E.A. PEHEK, R.K. WARNER, L.C. BAND, AND G.M. HOLMES. Brain Res. 37073-81 (1986). 19. E. M. HULL, R.K. WARNER, T.J. BAZZETT, R.C. EATON, J.T. THOMPSON AND L.L. SCALLETTA J. Pharmacol. Exp. Ther. 251423-427 (1989). 20. S. LAL, Y. TESFAYE, J. THAVUNDAYIL, T.R. THOPMSON, M.E. KIELY, N.P.V. NAIR, A. GRASSINO AND B. DUBROVSKY 1__33329-339 (1989). 21. L. MARSON AND K.E. McKENNA Brain Res. 515303-308 (1990). 23. M.R. MELIS, A. ARGIOLAS AND G.L. GESSA Brain Res. 41598-104 (1987). 23. J.B. MITCHELL AND J. STEWART Brain Res. 491116-127 (1989). 24. E.A. PEHEK, J.T. THOMPSON, R.C. EATON, T.J. BAZZETT AND E.M. HULL Pharmacol. Biochem. Behav. 3"1221-208 (1988). 25. E.A. PEHEK, J.T. THOMPSON AND E.M. HULL Brain Res. 500325-332 (1989). 26. J.G. PFAUS, G DAMSMA, G.G. NOMIKOS, D.G. WENKSTERN, C.D. BLAHA, A.G. PHILLIPS AND H.C. FIBIGER Brain Res. 530345-348 (1990). 27. S.D. POMERANTZ Pharmacol. Biochem. Behav. 35659-664 (1990). 28. S.D. POMERANTZ Pharmacol. Biochem. Behav. 35659-664 (1991). 29. A.N. PRADHAN AND S. ARUNASMITHA Physiol. Behav. 5._0_0135-138(1991). 30. B.D. SACHS AND D.BITRAN. Brain Res. 52899-108 (1990). 31. B.D. SACHS. AND R.I. MEISEL. The Physiology of Reproduction, vol 2, E. Knobil, J.D. Neill, L.L. Ewing (eds.), 1393-1423, Karger, New York, (1988). 32. C.J. SCHEIRER, W.S. RAY AND N. HARE Biometrics 32429-434 (1976). 33. R.T. SEGRAVES, M. BARI, K. SEGRAVES AND P. SPIRNAK J. Urol. 14,51174-1175 (1991).