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Chronic clonidine treatment and its termination: Effects of penile erection and ejaculation in the dog
L i f e Sciences, Vol. P r i n t e d in t h e U S A
CHRONIC
51, pp.
CLONIDINE
1999-2007
Pergamon
Press
T R E A T M E N T AND ITS T E R M I N A T I O N : E F F E C T S
ON P E N I L E E R E C T I O N AND E J A C U L A T I O N
IN T H E
DOG
Akihiko Yonczawa .1, Shunsuke Kawamura 1, Ryuichiro Ando l, • 9 Takcshi ~lhdano2, Kcnsukc Klsara - and Yukio Kimura3 Center for l~d3oratory Animal Science 1 and Dcpartment of Pharmacology2, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Aoba-ku, Sendal 981, and Towada Urology Hospital3, 13-2 Shimotai, Sanbongi, Towada City (/34, Japan (Received
in final
form O c t o b e r
12,
1992)
Summary
The cffects of chronic administration (4 wceks) of the alpha-2 adrenoceptor agonist clonidine (CL) and its tcrmination on penile erection and ejaculation were investigated in male dogs. Penile erection and cjaculation were elicited by manual penile stimulation (for 5 min). CL (l(I ug/kg/hr, s.c.) was delivered via osmotic minipump (Alza, 2ML-4). 3 or 7 days after the minipump implantation, CL caused a significant decrease in the amount of ejaculate produced by the genital stimulation without affecting the erectile potency. Ejaculatory ability returned to pretreatment levels despite continued CL administration, becoming evident in tests 14 days after initiation of treatment. Further, chronic CL (23 days) antagonized the inhibitory effects of acute administration of CL (0.05 mg/kg, i.p.). These data indicatc tolcrancc to continued delivery of low doses as well as to acute administration of a higher dose. In the acute drug experiments, the cjaculatory inhibition elicited by CL (0.05 mg/kg, i.p.) was completely antagonized by p r e t r c a t m c n t with y o h i m b i n e (0.05 and 0.10 mg/kg, i.p.), an alpha-2 adrenoceptor antagonist, but not with naloxone (1.(I mg/kg, i.p.), an opioid receptor antagonist. Furthcrmorc, DG-5128 (1.0 and 2.0 mg/kg, i.p.), a selective alpha-2 adrcnoccptor antagonist that poorly penetrates the blood-brain barrier, failed to antagonize the CL-induccd cjaculatory inhibition. This study suggests that functional alterations in the central alpha-2 adrenoceptor mechanism may be related to the changes in the cjaculatory capacity during chronic treatment with CL. Clinical investigations have indicated that sexual dysfunction is a relatively common complication of antihypertensive drug thcrap}: Clonidine (CL), a prototypc of centrally acting antihypertensive drug, has been noted to produce sexual dysfunction in both human and experimental animals. For example, the disorders associated in man include impotence, decreased libido and impaired ejaculation (1, 2). Experimental evidence indicated that systemically and centrally administered CL inhibits sexual behavior in rats (3, 4, 5) and the genital reflex such as penile * To whom correspondence should be addressed
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2000
Clonidine
and M a l e Sexual
Function
Vol.
51, No.
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erection and ciaculation in dogs (6, 7). CL has also been reported to inhibit penilc reflexes (crcction and seminal emission ex copula) in rats following acute systemic administration (4). CL exhibits a c o m p l c x p h a r m a c o l o g i c a l profile. Many of the effccts of CL, such as hypotcnsion and sedation, arc considered to be a result of stimulation of alpha-2 adrenoceptors within the central nervous system (8). An additional argument that the copulatory effects of CL are due to stimulation of central alpha-2 adrenoceptors comes from the observation that administration into thc cerebral ventricles or the medial preoptic area is associated with dysfunction (5). The fact that CLinduced inhibition of sexual behavior in rats (3, 5) and of the genital responses in dogs (6, 7) were completely antagonized by pretrcatment with yohimbine, an alpha-2 adrenoceptor antagonist, suggesting that the inhibitory effect of CL on male sexual functions lnay be also due to acti;'ation o n the central alpha-2 adrenoceptors. Biochemical and clcctrophysiological studies have shown that chronic administration of CL produced a down-regulation of the alpha-2 adrenoccptor mechanism in the ccntral nervous system (9, 10). It is also known that abrupt cessation of chronic treatment with CL caused a withdrawal syndrome, including a rapid rise in blood pressure and an increase in heart rate (8). However, there is little available information concerning the effect on male sexual function during and after chronic treatment with CL. The present studies wcrc undcrtaken to determine thc cffcct of chronic treatment (4 wccks) with CL and its termination on penile erection and ejaculation in male dogs. Further, we cxarnincd the putative p h a r m a c o l o g i c a l interaction of CL with alpha-2 adrenoccptor and opioid receptor mechanisms in the inhibition of ejaculation.
Materials and Methods Animals Fiftcen adult male mongrel dogs wcighing 10 to 16 kg wcrc uscd. All animals wcrc tcsted fl)r the reliable occurrence of erection and ejaculation in response to manual stimulation of the penis. Subsequently, the animals were randomly assigned to one of the following groups: (1) Chronic CLtreatment group, using an osmotic minipump (see below); (2) Sham-operated group; (3) Acute drugtreatment group. They were housed individually under a constant temperature (22-24 C) and humidity (50-60 %) and under a light-dark cycle (light on : 6:00 a.m. - 8:00 p.m.), which wcre automatically controlled in a dog's room. Water and standard dog food (CD-l, CLEA, Japan) were available tit all times except during the experimental sessions.
Drugs Drugs used in this study were clonidinc HCI (CL: Sigma), naloxonc HCI (Sigma), yohimbinc HCI (Nakarai Tesque) and DG-5128 (donated by Daiichi Pharmaceutical Co., Ltd., Tokyo). CL and naloxone were dissolved in physiologic saline. Yohimbine and DG-5128 were dissolved in sterile distilled water. In a chronic study, CL was infused for 4 weeks at a constant rate of 10 gg/kg/hr via subcutaneously implanted osmotic minipumps (Alza, model 2ML-4, mean output, 2.5 gl/hr). In an acute drug-treatment stud}; till four drugs were injected intraperitoneally (i.p.) in a w)lume of 3 ml/animals. The doses of the drugs were calculated as the salt.
Assessment of penile erection and ejaculation Penile ercction and ejaculation were clicited by continuous manual stimulation of the penis,
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which applied light pressure and gently rubbed the body of the penis just behind the bulbus glandis. All animals were stimulated for 5 min without restraints. This stimulation could easily produce a rapid penile tumescence and erection maintained throughout the entire period. A first ejaculation was observed within 0.5 rain and semen was expelled intermittently during the genital stimulation. The expelled semen from the urethra was collected with a pre-weighed plastic beaker during a period of the stimulation and then weighed on an electro-millibalance (Sartorius Type 1413). Our previous study showed that the amount of ejaculate produced by manual penile stimulation varied considerably among individual animals (11). Therefore, each animal served as his own control by being subjected to regular semen collection in order to accurately assess the drug effects on ejaculation. The time interval between semen collections was 3-7 days. To determine the basal value of ejaculate, the semen collection was made 3-5 times befi)rc the drug treatment and the mean weight of ejaculates was calculated in each animal (pre-drug value). All data for ejaculation were represented as a percentage of the pre-drug value. To assess penile erection, thc changes of crectile potency were simultaneously observed and were given one of four scores according to thc grade of occurrence based on the fl)llowing criteria: 3 - full erection, 2 - weak, 1 - slight increase of the size of glans and exposure o f the glans, .0 - no reaction. In the present study, drug treatment, manual penile stimulation and evaluation of penile crection were carricd out by the samc obscrver.
Chronic treatment with CL After detcrmination of the basal ejaculate value (pre-drug valuc), the animals were implanted with 'primed' osmotic minipumps under the skin of the back. Incision was closed with interrupted suture that was removed at 4 weeks after implantation. Implantation and removing of the pumps were performcd under sodium pentobarbital anaesthesia (30 mg/kg, i.p.). Our preliminary data showed that chronic infusion of a low dose of CL (1 ~tg/kg/hr, for 4 weeks) and physiologic saline did not affect the erectile and ejaculatory response in the dog. In the present experiment, therefore, a relatively high dose of CL (10 ~tg/kg/hr, for 4 weeks) was employed and sham-operated animals received the same operation without implantation of the pump. To determine whether CL-induced ejaculatory inhibition results from the delayed ejaculation, the manual stimulation to the penis was extended for 5 rain (total 10 mini. To determine the development of tolerance to the ejaculatory inhibition in dogs treated chronically with CL, CL (0.05 mg/kg) was acutely injected i.p. 23 days after initiation of CL treatment. In this experiment, the number of vomits was measured for 20 rain after acutc injcction of CL. The sedative effect of acutely administered CL was also evaluated by the gross behavior of the animals.
Acute drug experiment After dctcrmination of the basal ejaculate value (pre-drug value), each dog was tested twice over 4-5 days with CL ({).(15 mg/kg, i.p) to confirm its ejaculatory inhibition. Subsequently, the effects of pretreatment with two alpha-2 adrenoceptor antagonists, yohimbine and DG-5128, or an opioid antagonist, naloxone, on CL-induced ejaculatory inhibition were investigated in the same animals. Test 1 - effects of yohimbine pretreatment; the order of administration was CL (0.05 mg/kg), yohimbine (0.05 and 0.10 mg/kg), yohimbine (0.05 mg/kg) + CL, and yohimbine (0.10 mg/kg) + CL. Test 2 - effects of DG-5128 pretreatment; the order of administration was CL (0.05 mg/kg), DG5128 (1.0 and 2.0 mg/kg), DG-5128 (1.0 mg/kg) + CL, and DG-5128 (2.0 mg/kg) + CL. Test 3 effects of naloxone treatment; the order of administration was CL (0.05 mg/kg), naloxone (1.0 mg/kg), and CL + naloxone. The animals received all treatments in order of test 1, test 2 and test 3. The experimental sessions on a given animal were 4-5 day intervals. In the present stud}', the effects of the drugs on CL-induced eiaculatory inhibition were determined 3 hr after CL treatment, since it was previously confirmed that the selective inhibition of ejaculation without affecting the erectile potency in male dogs occurred at this time (6). Yohimbine (0.05 and 0.10 mg/kg) and DG-5128 (1.0
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and 2.0 mg/kg) were injected i.p. 15-20 rain before CL. Naloxone (1.0 mg/kg) was iniectcd i.p. 2.5 hr after CL. Statistical
analysis
Data arc presented as the mcan + S.E.M. Two-way analysis of variance (ANOVA) was performed on the ejaculation data. When ANOVA was significant (p<0.05), Student's t-test or Duncan's Multiple Range test were used. Willcoxon's test was used in statistical comparisons for the erection data. In all comparisons, a p value less than 0.05 was used as the criterion for statistical significance.
Results
P r e - d r u g e x p e r i m e n t . The weight of ejaculate produced by manual penile stimulation (for 5 min) varied considerably among individual dogs. The basal value (pre-drug value) in each animal obtained by regular semen collection (3-5 times at 3-7 day intervals) ranged from 1.23 to 8.79 g. However, the time to time variations in the weight of ejaculate is little changed in each of 15 dogs.
200
•
....
control
~
clonidine
A l:
3
0 Q.
-R
150
/ o
3. o
a .... ,
|
l ...........
............... ',, ........
_.-
o
Ill ,-ill w
5o
::
pre
3
:
:::: .:.:,:.:,........:.:.:.:.:.:,:+:....:.:,:.:,:.:.:.:.:,,..::: ::::::::::::::::::::::::::::::::::::::]
7
,
,
14
21
days
, [ 28
after
cessation
J
j
i
,
36hr
7
14
days
Fig. 1 The effect of chronic trcatment with CL and its tcrmination on ejaculatory response in the dog. Ejaculation was elicited by manual stimulation of the penis (for 5 rain). CL (10 ~tg/kg/hr, s.c.) was delivered via osmotic minipump (Alza 2ML-4). Each point represents the mean ~--S.E.M. of 5 dogs. The symbol(*) indicates p<0.05 compared to the sham operated control animals.
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Clonidine
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The averages of coefficients of variation for chronically CL-treated group (n=5), sham operated group (n=5) and acutely drug-treated group (n=5) were 13.17, 8.04 and 11.62 %, respectively. A rapid penile tumescence was also observed by the manual penile stimulation and all animals showed a full erection within 1.0 min.
Changes in the capacity of penile erection and ejaculation during and after chronic treatment with CL. Figure 1 shows the time course changes in the amount of ejaculate produced by manual penile stimulation (for 5 min) during and after chronic treatment with CL. 3 or 7 days after the minipump implantation, CL significantly inhibited the ejaculatory response in the dog; the amount of ejaculate produced by the genital stimulation showed about 60 % reduction in the pre-drug value. This ciaculatory inhibition causcd by CL was not due to retrograde cjaculation, since sperm was not found in the urine drawn from the urinary bladder. In addition, the amount of ejaculate did not return to pretreatment levels despite prolongation of the genital stimulation (for 10 rain: about 50 % of the pre-drug value). However, the ejaculatory ability returned to prctreatment levels despite continued CL administration, becoming evident in tests 14 days after initiation of treatment, and its maintenance lasted for 4 weeks.
150
-
A
m • c : o Q. m >
M~
100
50
0 MJ ,-,.,
I
I
I
control
CL-treated
Fig. 2 The effect of acute administration of CL (0.05 mg/kg, i.p.) on ejaculatory response in chronically CL-treated and sham operated control dogs. Test was done 23 days after initiation of chronic CL treatment and sham operation. Ejaculation was elicited 3 hr after acute administration of CL by manual penile stimulation (for 5 rain). Data are presented as a percentagc of semen weight 21 days after initiation of treatment. Each value represents the mean--+S.E.M, of 5 dogs. The symbol (*) indicates p<0.05 compared to the sham operated control animals.
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As shown in figure 2, chronic treatment with CL (on day 23) failed to decrease the amount of ejaculate produced by acute injection of CL (0.05 mg/kg, i.p.), which results in a marked inhibition of ejaculation in the intact dog. In addition, acute injection of CL caused vomits more than once and a sedation in the intact dog, but these effects were not observed in the chronically CL-treated dog. Cessation of chronic CL treatment with removing the osmotic minipump produced a tendency to increase the amount of ejaculate, which was not statistically significant (Fig. 1). In contrast, the sham operated animals had no change in the ejaculatory ability during the entire period. Chronic treatment with CL and its termination had also little effect on thc erectile potency in male dogs; Mean score was 3.0 except for the scorc 3 and 7 days after CL treatment (mean scores = 2.6, not significant: Willcoxon's test).
Effects of two alpha-2 adrenoceptor antagonists, yohimbine and DG-5128, and opioid antagonist, naloxone, on the CL-induced ejaculatory inhibition (Table 1) : Acute injection of CL (0.05 mg/kg, i.p.) to male dogs produced a marked reduction in the amount of ciaculate elicited by thc genital stimulation (3 hr post-iniection) without affecting the erectile potency. In agreement with a previous report (11), yohimbine (0.05 and 0.10 mg/kg) alonc incrcascd thc amount of ejaculate in a dose-related manncr. As shown in Table 1, pretrcatmcnt with yohimbinc completely prevented the decrcasc in the amount of cjaculatc clicitcd by, CL. Particularly, 0.11) mg/kg dose o f y o h i m b i n c r c v c r s c d t h e C L - i n d u c e d c j a c u l a t o r y i n h i b i t i o n . In contrast, DG-5128, a peripherally acting alpha-2 adrcnoccptor antagonist, had no effect on the CL-induccd cjaculatory inhibition in the dog. rib determine thc contribution of endogenous opioid system to the cjaculatory inhibition elicited by CL, the animals ,,','ere trcatcd with the opioid antagonist naloxonc. Although thc treatment with naloxone tendcd to prevent thc CL-induccd ejaculatory inhibition, this effect was not statistically significant.
Discussion Thc results of the prescnt study demonstrated that chronic administration of CL had an inhibitory effect on male sexual function in the dog, especially ejaculatory response. 3 or 7 days aftcr the osmotic minipump implantation, CL causcd a significant dccrcasc in the amount of cjaculatc during the genital stimulation, without affecting the crcctile potency. Thc absence of retrograde ejaculation suggcsts the possibility that thc decrease in the amount of cjaculatc could bc induced by impairment of seminal emission and/or cjaculation. In addition, thc ciaculatory inhibition clicitcd by CL was not duc to the delayed onset of cjaculation, because the amount of ejaculate did not rcturn to the normal level despite prolongation of the genital stimulation (for l/) min). Thc ejaculatory inhibition as a result of the chronic CL treatment is consistcnt with the result of its acutc administration. However, our previous study revealed that the acute administration of this drug markedly attenuated the erectile potency induccd by the genital stimulation (6). This suggests that the continuous administration of CL through an osmotic minipump may produce a selective effect on thc process of manifestation of ejaculation, rathcr than penile erection, in male dogs. In the prcscnt study, although CL inhibited the ejaculatory response in normal dogs, such an inhibition may bc causcd in hypertcnsive patients. Thc major finding of thc prescnt study is that tolerancc devclops to the cjaculatory inhibition induced by CL. Ejaculatory ability rcturncd to prctrcatmcnt levels dcspitc c o n t i n u e d CL administration, becoming evident in tests 14 days after initiation of treatmcnt. Furthermorc, chronic CL (23 days) antagonized the inhibitory effects of acute administration of CL (0.05 mg/kg, i.p.). Therefore, the development of tolerance duc to the chronic CL treatment is found not only for analgesia (12) and physiological phenomena of the cardiovascular systcm (13, 14) or others, but also
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for sexual function, cspecially ejaculatory inhibition. Vomiting or sedation induced by acute administration of the drug also did not observe in the chronic CL treatment group in the present study.
TABLE
I
The effect of two alpha-2 adrcnoceptor antagonists, yohimbinc and DG-5128, and an opioid antagonist, naloxonc on CL-induccd cjaculatory inhibition in dogs
Ejaculatory response Treatment
Test 1 CL Yohimbine (0.05 (0.10 Yohimbine (0.05 (0.10
(% o f p r e - d r u g
value)
25.21 + 151.84 -+ 187.99 + 101.23-+ 156.21 +
9.87 17.83 29.62 17.33 * 16.l(I *
Test 2 CL DG-5128 (l.0 mg/kg) (2.0 mg/kg) DG-2158 (1.0 mg/kg) + CL (2.(/mg/kg) + CL
39.18 104.62 1(13.29 36.35 38.41
15.51 8.75 18.26 22.64 18.93
Test 3 CL Naloxonc (1.0 mg/kg) CL + Naloxone (1.0 mg/kg)
28.45 -+ 13.11 107.34 -+ 13.04 55.34 + 16.71
mg/kg) mg/kg) mg/kg) + CL mg/kg) + CL
+ + + -+ -+
Ejaculation was elicited 3 hr after administration of CL (0.05 mg/kg, i.p.) by manual penile stimulation (for 5 rain). Each value presents the mean + S.E.M. of 5 dogs. The animals were received all treatments in order of test 1, test 2 and test 3. The experimental sessions on a given animal were 4-5 day intervals. Yohimbine and DG5128 were injected i.p. 15-20 rain before CL treatment. Naloxone was injected i.p. 2.5 hr after CL treatment. The symbol (*) indicates p<0.01 compared to CL-treated animals.
The antihypcrtensive effect of alpha-2 adrenoccptor agonists including CL is known to bc attenuated by naloxone (15), an opioid receptor antagonist, and the antiserum of beta-endorphin (16). In vitro studies also demonstrated that CL enhances the release of bcta-endorphin immunoreactivity from slices of brain stem in rats (17). Behavioral studies have shown that the intracerebroventricular administration of a small dosc of bcta-endorphin markedly inhibits the sexual behavior in male rats (18), and that tolerance develops for the analgesic effect of the drag. Although these findings suggest the possibility that endogenous opioid may bc involved in the CL-induced decrease in the amount of ejaculate and the development of tolerance in this study, such an involvement is denied because thc
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treatment with naloxone exerted no significant antagonizing effect on the CL- induced cjaculatory inhibition. This is supported by the earlier findings in rats that pretrcatment with naloxone failed to alter CL-induced inhibition of copulatory behavior or penile reflexes (4). In contrast, the pretreatment with yohimbine, an alpha-2 adrenoceptor antagonist, resulted in a complete blockade of the ejaculatory inhibition caused by CL, suggesting that direct activation of the alpha-2 adrenoceptor mechanism may produce a decrease in the amount of ejaculate. In particular, the activation on the central alpha-2 adrenoceptors seem to be related to this phenomenon for the following reasons: (1) The intracerebroventricular administration of alpha-methylnoradrenaline, an alpha-2 adrenoceptor agonist, causes a decrease in the amount of ejaculate elicited by the genital stimulation in dogs, as does that of CL (7). (2) The ejaculatory inhibition elicited by the central administration of CL is completely antagonized by pretreatmcnt with yohimbine, but not with prazosin, an alpha-1 adrcnoceptor antagonist (7). (3) In this study', the prctreatmcnt with yohimbine (ll. 10 mg/kg) reversed the CL-induccd cjaculatory inhibition, whereas DG-5128, a peripherally acting alpha-2 adrcnoceptor antagonist (19), did not exert a vohimbine-like prominent blocking activity (4) The single administration of the alpha-2 adrenoceptor antagonists such as yohimbine, ruuwolscine (11) or idazoxan (unpublished data) but not DG-5128, markedly enhances the ejaculatory reflex in male dogs. (5) A high dose (1 mg/kg/day) of CL is required for the development of tolcruncc in the peripheral nervous system including cardiovascular system (14), Clinical studies reported that a relatively high dose of CL caused erectile dysfunction in patients (1, 2). In the prescnt study; however, the continuous administration of the drug at a rclativcly high dose (10 ,ug/kg/hr, for 4 weeks) induced no erectile dysfunction in male dogs. Although this discrepancy can not bc explained, the fact that penile erection in this study, was of the rcflcx type to be elicited by the genital stimulation should be taken into consideration. In conclusion, our findings indicate that chronic treatment with CL causes a decrease in the amount of ciaculatc in the initial stage, and that tolerance to the ejaculatory inhibition is developed. These findings suggest that functional changes in the central alpha-2 adrenoceptor mechanism may be mainly' inw)lved in the changes in the ejaculatory capacity during chronic treatment with CL.
Acknowledgements The authors are grateful to Daiichi Pharmaceutical Co., Ltd.(Tokyo, Japan) for their gift of DG-5128.
References 1. J.G. STEVENSON and G.S. UMSTEAD, Drug Intell. Clin. Pharm. 18 113-120 (1984). 2. J.M.A. SITSEN. Prescription drugs and sexual function In: Handbook of Sexology, edited by J.M.A. Sitsen, p 425-461. Elsevier Science Publishers B.V. Amsterdam. (1988). 3. J.T. CLARK, E.R. SMITH and J.M. DAVIDSON, Neuroendocrinology 41 36-43 (1985). 4. J.T. CLARK and E.R. SMITH, Neuroendocrinology 51 357-364 (199(I) 5. J.T. CLARK, Neuropharmacology. 30 373-382 (1991). 6. K. KISARA, A. YONEZAWA, T. TADANO and Y. KIMURA, Pharmacometrics (in Japanese) 33 285-290 (1987). 7. A. YONEZAWA, R. ANDO, T. TADANO, K. KISARA, A. MIYAMOTO and Z KIMURA, J. Pharmacobio-Dyn. 9 1032-1035 (1986). 8. W. HOEFKE. Clonidine In: Pharmacology of Antihypertensive Drugs, edited by A.SCRIABINE, p 55-78. Raven Press, New York. (1980).
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9. G. MAURA, G. BONANNN and M. RAITERI, Eur. J. Pharmacoh 112 105-110 (1985). 111. G. ENGBERG, M. ELAM and T.H. SVENSSON, Life Sci. 3() 235-24 3 (1982). 11. A. YONEZAWA, S. KAWAMURA, R. ANDO, T. TADANO, T. NOBUNAGA and Y. KIMURA, Life Sci. 48 PL 103-109 (199l). 12. W.T. CHANCE, Life Sci. 33 2241-2246 (1983). 13. J.A. MASTRIANNI and A.J. INGENITO, Pharmacol. Rcs. Commun. 17 865-872 (1985) 14. K. ISHI and R. KATO, J. Pharmacoh Exp. Thcr. 23l 685-(¢11 (19841. 15. C. FARSANG and G. KUNOS. Br. J. Pharmacol. 67 161-164 (1979). 16. M.A. PETFY and W. DE JONG, Neuropharmacology 23 643-648 (1984). 17. G. KUNOS and C. FARSANG, Science 211 82-84 (1981). 18. B.J. MEYERSON and L. TERENIUS, Eur. J. Pharmacol. 4~ 191-192 (19771. 19. M. HIROHASHI, M. TANAKA, I. SUZUKI and A. AKASHI, Arch. Int. Pharmacodyn. 306 75-86 (1990).
CHRONIC
51, pp.
CLONIDINE
1999-2007
Pergamon
Press
T R E A T M E N T AND ITS T E R M I N A T I O N : E F F E C T S
ON P E N I L E E R E C T I O N AND E J A C U L A T I O N
IN T H E
DOG
Akihiko Yonczawa .1, Shunsuke Kawamura 1, Ryuichiro Ando l, • 9 Takcshi ~lhdano2, Kcnsukc Klsara - and Yukio Kimura3 Center for l~d3oratory Animal Science 1 and Dcpartment of Pharmacology2, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Aoba-ku, Sendal 981, and Towada Urology Hospital3, 13-2 Shimotai, Sanbongi, Towada City (/34, Japan (Received
in final
form O c t o b e r
12,
1992)
Summary
The cffects of chronic administration (4 wceks) of the alpha-2 adrenoceptor agonist clonidine (CL) and its tcrmination on penile erection and ejaculation were investigated in male dogs. Penile erection and cjaculation were elicited by manual penile stimulation (for 5 min). CL (l(I ug/kg/hr, s.c.) was delivered via osmotic minipump (Alza, 2ML-4). 3 or 7 days after the minipump implantation, CL caused a significant decrease in the amount of ejaculate produced by the genital stimulation without affecting the erectile potency. Ejaculatory ability returned to pretreatment levels despite continued CL administration, becoming evident in tests 14 days after initiation of treatment. Further, chronic CL (23 days) antagonized the inhibitory effects of acute administration of CL (0.05 mg/kg, i.p.). These data indicatc tolcrancc to continued delivery of low doses as well as to acute administration of a higher dose. In the acute drug experiments, the cjaculatory inhibition elicited by CL (0.05 mg/kg, i.p.) was completely antagonized by p r e t r c a t m c n t with y o h i m b i n e (0.05 and 0.10 mg/kg, i.p.), an alpha-2 adrenoceptor antagonist, but not with naloxone (1.(I mg/kg, i.p.), an opioid receptor antagonist. Furthcrmorc, DG-5128 (1.0 and 2.0 mg/kg, i.p.), a selective alpha-2 adrcnoccptor antagonist that poorly penetrates the blood-brain barrier, failed to antagonize the CL-induccd cjaculatory inhibition. This study suggests that functional alterations in the central alpha-2 adrenoceptor mechanism may be related to the changes in the cjaculatory capacity during chronic treatment with CL. Clinical investigations have indicated that sexual dysfunction is a relatively common complication of antihypertensive drug thcrap}: Clonidine (CL), a prototypc of centrally acting antihypertensive drug, has been noted to produce sexual dysfunction in both human and experimental animals. For example, the disorders associated in man include impotence, decreased libido and impaired ejaculation (1, 2). Experimental evidence indicated that systemically and centrally administered CL inhibits sexual behavior in rats (3, 4, 5) and the genital reflex such as penile * To whom correspondence should be addressed
Copyright
© 1992
0 0 2 4 - 3 2 0 5 / 9 2 $5.00 + .00 P e r g a m o n Press Ltd All r i g h t s
reserved.
2000
Clonidine
and M a l e Sexual
Function
Vol.
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erection and ciaculation in dogs (6, 7). CL has also been reported to inhibit penilc reflexes (crcction and seminal emission ex copula) in rats following acute systemic administration (4). CL exhibits a c o m p l c x p h a r m a c o l o g i c a l profile. Many of the effccts of CL, such as hypotcnsion and sedation, arc considered to be a result of stimulation of alpha-2 adrenoceptors within the central nervous system (8). An additional argument that the copulatory effects of CL are due to stimulation of central alpha-2 adrenoceptors comes from the observation that administration into thc cerebral ventricles or the medial preoptic area is associated with dysfunction (5). The fact that CLinduced inhibition of sexual behavior in rats (3, 5) and of the genital responses in dogs (6, 7) were completely antagonized by pretrcatment with yohimbine, an alpha-2 adrenoceptor antagonist, suggesting that the inhibitory effect of CL on male sexual functions lnay be also due to acti;'ation o n the central alpha-2 adrenoceptors. Biochemical and clcctrophysiological studies have shown that chronic administration of CL produced a down-regulation of the alpha-2 adrenoccptor mechanism in the ccntral nervous system (9, 10). It is also known that abrupt cessation of chronic treatment with CL caused a withdrawal syndrome, including a rapid rise in blood pressure and an increase in heart rate (8). However, there is little available information concerning the effect on male sexual function during and after chronic treatment with CL. The present studies wcrc undcrtaken to determine thc cffcct of chronic treatment (4 wccks) with CL and its termination on penile erection and ejaculation in male dogs. Further, we cxarnincd the putative p h a r m a c o l o g i c a l interaction of CL with alpha-2 adrenoccptor and opioid receptor mechanisms in the inhibition of ejaculation.
Materials and Methods Animals Fiftcen adult male mongrel dogs wcighing 10 to 16 kg wcrc uscd. All animals wcrc tcsted fl)r the reliable occurrence of erection and ejaculation in response to manual stimulation of the penis. Subsequently, the animals were randomly assigned to one of the following groups: (1) Chronic CLtreatment group, using an osmotic minipump (see below); (2) Sham-operated group; (3) Acute drugtreatment group. They were housed individually under a constant temperature (22-24 C) and humidity (50-60 %) and under a light-dark cycle (light on : 6:00 a.m. - 8:00 p.m.), which wcre automatically controlled in a dog's room. Water and standard dog food (CD-l, CLEA, Japan) were available tit all times except during the experimental sessions.
Drugs Drugs used in this study were clonidinc HCI (CL: Sigma), naloxonc HCI (Sigma), yohimbinc HCI (Nakarai Tesque) and DG-5128 (donated by Daiichi Pharmaceutical Co., Ltd., Tokyo). CL and naloxone were dissolved in physiologic saline. Yohimbine and DG-5128 were dissolved in sterile distilled water. In a chronic study, CL was infused for 4 weeks at a constant rate of 10 gg/kg/hr via subcutaneously implanted osmotic minipumps (Alza, model 2ML-4, mean output, 2.5 gl/hr). In an acute drug-treatment stud}; till four drugs were injected intraperitoneally (i.p.) in a w)lume of 3 ml/animals. The doses of the drugs were calculated as the salt.
Assessment of penile erection and ejaculation Penile ercction and ejaculation were clicited by continuous manual stimulation of the penis,
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which applied light pressure and gently rubbed the body of the penis just behind the bulbus glandis. All animals were stimulated for 5 min without restraints. This stimulation could easily produce a rapid penile tumescence and erection maintained throughout the entire period. A first ejaculation was observed within 0.5 rain and semen was expelled intermittently during the genital stimulation. The expelled semen from the urethra was collected with a pre-weighed plastic beaker during a period of the stimulation and then weighed on an electro-millibalance (Sartorius Type 1413). Our previous study showed that the amount of ejaculate produced by manual penile stimulation varied considerably among individual animals (11). Therefore, each animal served as his own control by being subjected to regular semen collection in order to accurately assess the drug effects on ejaculation. The time interval between semen collections was 3-7 days. To determine the basal value of ejaculate, the semen collection was made 3-5 times befi)rc the drug treatment and the mean weight of ejaculates was calculated in each animal (pre-drug value). All data for ejaculation were represented as a percentage of the pre-drug value. To assess penile erection, thc changes of crectile potency were simultaneously observed and were given one of four scores according to thc grade of occurrence based on the fl)llowing criteria: 3 - full erection, 2 - weak, 1 - slight increase of the size of glans and exposure o f the glans, .0 - no reaction. In the present study, drug treatment, manual penile stimulation and evaluation of penile crection were carricd out by the samc obscrver.
Chronic treatment with CL After detcrmination of the basal ejaculate value (pre-drug valuc), the animals were implanted with 'primed' osmotic minipumps under the skin of the back. Incision was closed with interrupted suture that was removed at 4 weeks after implantation. Implantation and removing of the pumps were performcd under sodium pentobarbital anaesthesia (30 mg/kg, i.p.). Our preliminary data showed that chronic infusion of a low dose of CL (1 ~tg/kg/hr, for 4 weeks) and physiologic saline did not affect the erectile and ejaculatory response in the dog. In the present experiment, therefore, a relatively high dose of CL (10 ~tg/kg/hr, for 4 weeks) was employed and sham-operated animals received the same operation without implantation of the pump. To determine whether CL-induced ejaculatory inhibition results from the delayed ejaculation, the manual stimulation to the penis was extended for 5 rain (total 10 mini. To determine the development of tolerance to the ejaculatory inhibition in dogs treated chronically with CL, CL (0.05 mg/kg) was acutely injected i.p. 23 days after initiation of CL treatment. In this experiment, the number of vomits was measured for 20 rain after acutc injcction of CL. The sedative effect of acutely administered CL was also evaluated by the gross behavior of the animals.
Acute drug experiment After dctcrmination of the basal ejaculate value (pre-drug value), each dog was tested twice over 4-5 days with CL ({).(15 mg/kg, i.p) to confirm its ejaculatory inhibition. Subsequently, the effects of pretreatment with two alpha-2 adrenoceptor antagonists, yohimbine and DG-5128, or an opioid antagonist, naloxone, on CL-induced ejaculatory inhibition were investigated in the same animals. Test 1 - effects of yohimbine pretreatment; the order of administration was CL (0.05 mg/kg), yohimbine (0.05 and 0.10 mg/kg), yohimbine (0.05 mg/kg) + CL, and yohimbine (0.10 mg/kg) + CL. Test 2 - effects of DG-5128 pretreatment; the order of administration was CL (0.05 mg/kg), DG5128 (1.0 and 2.0 mg/kg), DG-5128 (1.0 mg/kg) + CL, and DG-5128 (2.0 mg/kg) + CL. Test 3 effects of naloxone treatment; the order of administration was CL (0.05 mg/kg), naloxone (1.0 mg/kg), and CL + naloxone. The animals received all treatments in order of test 1, test 2 and test 3. The experimental sessions on a given animal were 4-5 day intervals. In the present stud}', the effects of the drugs on CL-induced eiaculatory inhibition were determined 3 hr after CL treatment, since it was previously confirmed that the selective inhibition of ejaculation without affecting the erectile potency in male dogs occurred at this time (6). Yohimbine (0.05 and 0.10 mg/kg) and DG-5128 (1.0
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and 2.0 mg/kg) were injected i.p. 15-20 rain before CL. Naloxone (1.0 mg/kg) was iniectcd i.p. 2.5 hr after CL. Statistical
analysis
Data arc presented as the mcan + S.E.M. Two-way analysis of variance (ANOVA) was performed on the ejaculation data. When ANOVA was significant (p<0.05), Student's t-test or Duncan's Multiple Range test were used. Willcoxon's test was used in statistical comparisons for the erection data. In all comparisons, a p value less than 0.05 was used as the criterion for statistical significance.
Results
P r e - d r u g e x p e r i m e n t . The weight of ejaculate produced by manual penile stimulation (for 5 min) varied considerably among individual dogs. The basal value (pre-drug value) in each animal obtained by regular semen collection (3-5 times at 3-7 day intervals) ranged from 1.23 to 8.79 g. However, the time to time variations in the weight of ejaculate is little changed in each of 15 dogs.
200
•
....
control
~
clonidine
A l:
3
0 Q.
-R
150
/ o
3. o
a .... ,
|
l ...........
............... ',, ........
_.-
o
Ill ,-ill w
5o
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pre
3
:
:::: .:.:,:.:,........:.:.:.:.:.:,:+:....:.:,:.:,:.:.:.:.:,,..::: ::::::::::::::::::::::::::::::::::::::]
7
,
,
14
21
days
, [ 28
after
cessation
J
j
i
,
36hr
7
14
days
Fig. 1 The effect of chronic trcatment with CL and its tcrmination on ejaculatory response in the dog. Ejaculation was elicited by manual stimulation of the penis (for 5 rain). CL (10 ~tg/kg/hr, s.c.) was delivered via osmotic minipump (Alza 2ML-4). Each point represents the mean ~--S.E.M. of 5 dogs. The symbol(*) indicates p<0.05 compared to the sham operated control animals.
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The averages of coefficients of variation for chronically CL-treated group (n=5), sham operated group (n=5) and acutely drug-treated group (n=5) were 13.17, 8.04 and 11.62 %, respectively. A rapid penile tumescence was also observed by the manual penile stimulation and all animals showed a full erection within 1.0 min.
Changes in the capacity of penile erection and ejaculation during and after chronic treatment with CL. Figure 1 shows the time course changes in the amount of ejaculate produced by manual penile stimulation (for 5 min) during and after chronic treatment with CL. 3 or 7 days after the minipump implantation, CL significantly inhibited the ejaculatory response in the dog; the amount of ejaculate produced by the genital stimulation showed about 60 % reduction in the pre-drug value. This ciaculatory inhibition causcd by CL was not due to retrograde cjaculation, since sperm was not found in the urine drawn from the urinary bladder. In addition, the amount of ejaculate did not return to pretreatment levels despite prolongation of the genital stimulation (for 10 rain: about 50 % of the pre-drug value). However, the ejaculatory ability returned to prctreatment levels despite continued CL administration, becoming evident in tests 14 days after initiation of treatment, and its maintenance lasted for 4 weeks.
150
-
A
m • c : o Q. m >
M~
100
50
0 MJ ,-,.,
I
I
I
control
CL-treated
Fig. 2 The effect of acute administration of CL (0.05 mg/kg, i.p.) on ejaculatory response in chronically CL-treated and sham operated control dogs. Test was done 23 days after initiation of chronic CL treatment and sham operation. Ejaculation was elicited 3 hr after acute administration of CL by manual penile stimulation (for 5 rain). Data are presented as a percentagc of semen weight 21 days after initiation of treatment. Each value represents the mean--+S.E.M, of 5 dogs. The symbol (*) indicates p<0.05 compared to the sham operated control animals.
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As shown in figure 2, chronic treatment with CL (on day 23) failed to decrease the amount of ejaculate produced by acute injection of CL (0.05 mg/kg, i.p.), which results in a marked inhibition of ejaculation in the intact dog. In addition, acute injection of CL caused vomits more than once and a sedation in the intact dog, but these effects were not observed in the chronically CL-treated dog. Cessation of chronic CL treatment with removing the osmotic minipump produced a tendency to increase the amount of ejaculate, which was not statistically significant (Fig. 1). In contrast, the sham operated animals had no change in the ejaculatory ability during the entire period. Chronic treatment with CL and its termination had also little effect on thc erectile potency in male dogs; Mean score was 3.0 except for the scorc 3 and 7 days after CL treatment (mean scores = 2.6, not significant: Willcoxon's test).
Effects of two alpha-2 adrenoceptor antagonists, yohimbine and DG-5128, and opioid antagonist, naloxone, on the CL-induced ejaculatory inhibition (Table 1) : Acute injection of CL (0.05 mg/kg, i.p.) to male dogs produced a marked reduction in the amount of ciaculate elicited by thc genital stimulation (3 hr post-iniection) without affecting the erectile potency. In agreement with a previous report (11), yohimbine (0.05 and 0.10 mg/kg) alonc incrcascd thc amount of ejaculate in a dose-related manncr. As shown in Table 1, pretrcatmcnt with yohimbinc completely prevented the decrcasc in the amount of cjaculatc clicitcd by, CL. Particularly, 0.11) mg/kg dose o f y o h i m b i n c r c v c r s c d t h e C L - i n d u c e d c j a c u l a t o r y i n h i b i t i o n . In contrast, DG-5128, a peripherally acting alpha-2 adrcnoccptor antagonist, had no effect on the CL-induccd cjaculatory inhibition in the dog. rib determine thc contribution of endogenous opioid system to the cjaculatory inhibition elicited by CL, the animals ,,','ere trcatcd with the opioid antagonist naloxonc. Although thc treatment with naloxone tendcd to prevent thc CL-induccd ejaculatory inhibition, this effect was not statistically significant.
Discussion Thc results of the prescnt study demonstrated that chronic administration of CL had an inhibitory effect on male sexual function in the dog, especially ejaculatory response. 3 or 7 days aftcr the osmotic minipump implantation, CL causcd a significant dccrcasc in the amount of cjaculatc during the genital stimulation, without affecting the crcctile potency. Thc absence of retrograde ejaculation suggcsts the possibility that thc decrease in the amount of cjaculatc could bc induced by impairment of seminal emission and/or cjaculation. In addition, thc ciaculatory inhibition clicitcd by CL was not duc to the delayed onset of cjaculation, because the amount of ejaculate did not rcturn to the normal level despite prolongation of the genital stimulation (for l/) min). Thc ejaculatory inhibition as a result of the chronic CL treatment is consistcnt with the result of its acutc administration. However, our previous study revealed that the acute administration of this drug markedly attenuated the erectile potency induccd by the genital stimulation (6). This suggests that the continuous administration of CL through an osmotic minipump may produce a selective effect on thc process of manifestation of ejaculation, rathcr than penile erection, in male dogs. In the prcscnt study, although CL inhibited the ejaculatory response in normal dogs, such an inhibition may bc causcd in hypertcnsive patients. Thc major finding of thc prescnt study is that tolerancc devclops to the cjaculatory inhibition induced by CL. Ejaculatory ability rcturncd to prctrcatmcnt levels dcspitc c o n t i n u e d CL administration, becoming evident in tests 14 days after initiation of treatmcnt. Furthermorc, chronic CL (23 days) antagonized the inhibitory effects of acute administration of CL (0.05 mg/kg, i.p.). Therefore, the development of tolerance duc to the chronic CL treatment is found not only for analgesia (12) and physiological phenomena of the cardiovascular systcm (13, 14) or others, but also
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for sexual function, cspecially ejaculatory inhibition. Vomiting or sedation induced by acute administration of the drug also did not observe in the chronic CL treatment group in the present study.
TABLE
I
The effect of two alpha-2 adrcnoceptor antagonists, yohimbinc and DG-5128, and an opioid antagonist, naloxonc on CL-induccd cjaculatory inhibition in dogs
Ejaculatory response Treatment
Test 1 CL Yohimbine (0.05 (0.10 Yohimbine (0.05 (0.10
(% o f p r e - d r u g
value)
25.21 + 151.84 -+ 187.99 + 101.23-+ 156.21 +
9.87 17.83 29.62 17.33 * 16.l(I *
Test 2 CL DG-5128 (l.0 mg/kg) (2.0 mg/kg) DG-2158 (1.0 mg/kg) + CL (2.(/mg/kg) + CL
39.18 104.62 1(13.29 36.35 38.41
15.51 8.75 18.26 22.64 18.93
Test 3 CL Naloxonc (1.0 mg/kg) CL + Naloxone (1.0 mg/kg)
28.45 -+ 13.11 107.34 -+ 13.04 55.34 + 16.71
mg/kg) mg/kg) mg/kg) + CL mg/kg) + CL
+ + + -+ -+
Ejaculation was elicited 3 hr after administration of CL (0.05 mg/kg, i.p.) by manual penile stimulation (for 5 rain). Each value presents the mean + S.E.M. of 5 dogs. The animals were received all treatments in order of test 1, test 2 and test 3. The experimental sessions on a given animal were 4-5 day intervals. Yohimbine and DG5128 were injected i.p. 15-20 rain before CL treatment. Naloxone was injected i.p. 2.5 hr after CL treatment. The symbol (*) indicates p<0.01 compared to CL-treated animals.
The antihypcrtensive effect of alpha-2 adrenoccptor agonists including CL is known to bc attenuated by naloxone (15), an opioid receptor antagonist, and the antiserum of beta-endorphin (16). In vitro studies also demonstrated that CL enhances the release of bcta-endorphin immunoreactivity from slices of brain stem in rats (17). Behavioral studies have shown that the intracerebroventricular administration of a small dosc of bcta-endorphin markedly inhibits the sexual behavior in male rats (18), and that tolerance develops for the analgesic effect of the drag. Although these findings suggest the possibility that endogenous opioid may bc involved in the CL-induced decrease in the amount of ejaculate and the development of tolerance in this study, such an involvement is denied because thc
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treatment with naloxone exerted no significant antagonizing effect on the CL- induced cjaculatory inhibition. This is supported by the earlier findings in rats that pretrcatment with naloxone failed to alter CL-induced inhibition of copulatory behavior or penile reflexes (4). In contrast, the pretreatment with yohimbine, an alpha-2 adrenoceptor antagonist, resulted in a complete blockade of the ejaculatory inhibition caused by CL, suggesting that direct activation of the alpha-2 adrenoceptor mechanism may produce a decrease in the amount of ejaculate. In particular, the activation on the central alpha-2 adrenoceptors seem to be related to this phenomenon for the following reasons: (1) The intracerebroventricular administration of alpha-methylnoradrenaline, an alpha-2 adrenoceptor agonist, causes a decrease in the amount of ejaculate elicited by the genital stimulation in dogs, as does that of CL (7). (2) The ejaculatory inhibition elicited by the central administration of CL is completely antagonized by pretreatmcnt with yohimbine, but not with prazosin, an alpha-1 adrcnoceptor antagonist (7). (3) In this study', the prctreatmcnt with yohimbine (ll. 10 mg/kg) reversed the CL-induccd cjaculatory inhibition, whereas DG-5128, a peripherally acting alpha-2 adrcnoceptor antagonist (19), did not exert a vohimbine-like prominent blocking activity (4) The single administration of the alpha-2 adrenoceptor antagonists such as yohimbine, ruuwolscine (11) or idazoxan (unpublished data) but not DG-5128, markedly enhances the ejaculatory reflex in male dogs. (5) A high dose (1 mg/kg/day) of CL is required for the development of tolcruncc in the peripheral nervous system including cardiovascular system (14), Clinical studies reported that a relatively high dose of CL caused erectile dysfunction in patients (1, 2). In the prescnt study; however, the continuous administration of the drug at a rclativcly high dose (10 ,ug/kg/hr, for 4 weeks) induced no erectile dysfunction in male dogs. Although this discrepancy can not bc explained, the fact that penile erection in this study, was of the rcflcx type to be elicited by the genital stimulation should be taken into consideration. In conclusion, our findings indicate that chronic treatment with CL causes a decrease in the amount of ciaculatc in the initial stage, and that tolerance to the ejaculatory inhibition is developed. These findings suggest that functional changes in the central alpha-2 adrenoceptor mechanism may be mainly' inw)lved in the changes in the ejaculatory capacity during chronic treatment with CL.
Acknowledgements The authors are grateful to Daiichi Pharmaceutical Co., Ltd.(Tokyo, Japan) for their gift of DG-5128.
References 1. J.G. STEVENSON and G.S. UMSTEAD, Drug Intell. Clin. Pharm. 18 113-120 (1984). 2. J.M.A. SITSEN. Prescription drugs and sexual function In: Handbook of Sexology, edited by J.M.A. Sitsen, p 425-461. Elsevier Science Publishers B.V. Amsterdam. (1988). 3. J.T. CLARK, E.R. SMITH and J.M. DAVIDSON, Neuroendocrinology 41 36-43 (1985). 4. J.T. CLARK and E.R. SMITH, Neuroendocrinology 51 357-364 (199(I) 5. J.T. CLARK, Neuropharmacology. 30 373-382 (1991). 6. K. KISARA, A. YONEZAWA, T. TADANO and Y. KIMURA, Pharmacometrics (in Japanese) 33 285-290 (1987). 7. A. YONEZAWA, R. ANDO, T. TADANO, K. KISARA, A. MIYAMOTO and Z KIMURA, J. Pharmacobio-Dyn. 9 1032-1035 (1986). 8. W. HOEFKE. Clonidine In: Pharmacology of Antihypertensive Drugs, edited by A.SCRIABINE, p 55-78. Raven Press, New York. (1980).
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9. G. MAURA, G. BONANNN and M. RAITERI, Eur. J. Pharmacoh 112 105-110 (1985). 111. G. ENGBERG, M. ELAM and T.H. SVENSSON, Life Sci. 3() 235-24 3 (1982). 11. A. YONEZAWA, S. KAWAMURA, R. ANDO, T. TADANO, T. NOBUNAGA and Y. KIMURA, Life Sci. 48 PL 103-109 (199l). 12. W.T. CHANCE, Life Sci. 33 2241-2246 (1983). 13. J.A. MASTRIANNI and A.J. INGENITO, Pharmacol. Rcs. Commun. 17 865-872 (1985) 14. K. ISHI and R. KATO, J. Pharmacoh Exp. Thcr. 23l 685-(¢11 (19841. 15. C. FARSANG and G. KUNOS. Br. J. Pharmacol. 67 161-164 (1979). 16. M.A. PETFY and W. DE JONG, Neuropharmacology 23 643-648 (1984). 17. G. KUNOS and C. FARSANG, Science 211 82-84 (1981). 18. B.J. MEYERSON and L. TERENIUS, Eur. J. Pharmacol. 4~ 191-192 (19771. 19. M. HIROHASHI, M. TANAKA, I. SUZUKI and A. AKASHI, Arch. Int. Pharmacodyn. 306 75-86 (1990).