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Age-related change in serotonin-mediated prejunctional inhibition of rat vas deferens
European Journal o[ Pharmacology, 132 (1986) 39-46
39
Elsevier EJP 00584
Age-related change in serotonin-mediated prejunctional inhibition of rat vas deferens H i d e k i M o r i t o k i *, T a k e s h i I w a m o t o , J u n K a n a y a , Y u k i o I s h i d a a n d H i r o s h i F u k u d a * Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, UniversiO, of Tokushima, Shomachi, Tokushima 770, Japan
Received 6 August 1986, accepted 9 September 1986
The effect of age on the serotonin (5-HT)-induced prejunctional inhibition of twitch contractions induced in rat vas deferens by single-pulse field stimulation at 0.1 Hz was studied. The inhibitory effect of 5-HT gradually decreased with increasing age of the rats (4-15 weeks old) and was no longer detectable in preparations from rats over 15 weeks old. Moreover, on a further increase in age to 45 weeks, 5-HT conversely enhanced the twitch response of the vas deferens. The 5-HT2 antagonists ketanserin and mianserin potentiated the 5-HT-induced inhibition of contractions of the vas deferens of young rats (8-15 weeks old), and attenuated the amplifying effect of 5-HT on the responses of preparations from old rats (95 weeks old). Thus unmasking of the inhibition depended on the age of rats. This change occurred earlier in life in the prostatic portion of the vas deferens than in the epididymal portion. A functional decrease in 5-HT-mediated prejunctional inhibition and the resultant increase in amplification of the twitch response by 5-HT are probably responsible for the age-related decrease in prejunctional inhibition. Rat vas deferens; 5-HT; Prejunctional inhibition; Aging; Ketanserin 1. Introduction Serotonin (5-HT) has been reported to inhibit twitch contractions of rat vas deferens by a prejunctional mechanism (Kapur and Mottram, 1979). However, in our experiments 5-HT had a variable effect on rat vas deferens: in some preparations, it slightly inhibited the twitch response with a transient increase in the basal tone whereas in other preparations, it did not inhibit the twitch response but augmented it (Moritoki et al., in press). The discrepancy between our findings and those of Kapur and Mottram (1979) could be due to a difference in the age of the rats or in the portions of vas deferens examined. The effects of age on the responses of arteries to vasoactive substances have been well established (Cohen and Berkowitz,
1974; Ericsson and Lundholm, 1975; Fieisch and Hooker, 1976; Schoeffter and Stoclet, 1982, 1983) but little is known about changes in adrenergic transmission with age. There are a few reports of an age-related decrease in prejunctional a:-receptor-mediated inhibition (Docherty and O'Malley, 1983; Hyland and Docherty, 1985). In the present experiments, we studied the influence of age on the prejunctional inhibition by 5-HT of the twitch response of rat vas deferens caused by field stimulation, and the potentiating effect of ketanserin on the inhibitory action of 5-HT. A preliminary account of this work was presented at the I U P A R 9th International Congress of Pharmacology (Moritoki, 1984).
2. Materials and methods * To whom all correspondence should be addressed. * Present address: Department of PharmacologyII, School of Medicine, University of Osaka, Osaka 530, Japan. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Male Wistar rats aged 4 weeks ( + 3 days), 8 weeks ( + 7 days), 15 weeks ( + 10 days), 45 weeks
40 (40-50 weeks) and 95 weeks (90-105 weeks) were used. The vas deferens was excised and separated from connective tissue under a dissecting microscope. In some experiments, the vas deferens was cut into epididymal, middle and prostatic portions constituting 50%, 10% and 40%, respectively, of its total length. The vas deferens was set up in a 10 ml organ bath containing Krebs solution of the following composition (mM): NaC1 115.3, KCI 4.7, CaC12 1.6, MgSO 4 1.2, N a H C O 3 25.0, K H z P O 4 1.2 and glucose 11.1. The medium was maintained at 35°C and bubbled with 5% CO 2 in 02 . The resting tension of the preparation was maintained at 200 rag. After equilibration of the preparation for 60 min, field stimulation of 0.5 ms duration was applied at a frequency of 0.1 Hz from a stimulator (Nihon Kohoden SEN 3201) with a constant voltage modulator through a pair of platinum electrodes. One electrode was attached to one end of the preparation and the other, the reference electrode was placed around the preparation. Contractions were recorded isometrically with a force displacement transducer (Nihon Kohoden SD-1TH). The twitch contractions elicited by field stimulation were abolished by either tetrodotoxin (10 7 M) or guanethidine (3 × 10 -6 M), indicating that the response was neurogenic. A dose-response curve was obtained by cumulative addition of 5-HT in a volume of 10-70 btl to the 10 ml organ bath. Ketanserin and other antagonists were applied 10 min before 5-HT. In tests on the amplifying effect of 5-HT on contractions induced by norepinephrine (NE) or ATP, 5-HT was added 5 rain before the agonists. The significance of differences between values was examined with the unpaired t-test and a P value of less than 0.05 was considered as significant. The drugs used were 5-hydroxytryptamine creatinine sulfate (5-HT, Sigma Chemical Co., St. Louis, MO), norepinephrine bitartrate (Sigma), ATP (2 Na salt, Sigma), ketanserin (Janssen Pharmaceutica, Beerse, Belgium), mianserin hydrochloride (Research Biochemicals Inc., Wayland, MA), cyproheptadine hydrochloride (Merck Sharp & Dohme Research Laboratories, West point, PA), methysergide (Sandoz, Switzerland) and prazosin (Taito-Pfeizer, Tokyo, Japan).
3. Results
3.1. Effect of aging on twitch contractions and 5HT-induced contractions The twitch contractions of rat whole vas deferens induced by field stimulation at 0.1 Hz increased with increasing age of the rats, reaching a maximum in preparations from rats aged 8 weeks. With a further increase in age, the twitch response decreased to an almost steady level from 15 weeks on (fig. 1). In unstimulated preparations, the contractions caused by 5-HT at a concentration of 10 5 M, which was the highest concentration applied to inhibit the twitch response, were less than the twitch height and disappeared rapidly (within 1 min) in the continuous presence of 5-HT. These contractions were not dependent on the age of the rats, being greatest in preparations from 8 and 95 week old rats and negligible in those from 4 week old rats (fig. 1). 5-HT at concentrations above 3 × 10 5 M caused intense contractions that were accompanied by wave-like movement and were resistant to ketanserin (3 x 10 7 M) but antagonized by prazosin with a pA 2 value of 8.03.
3.2. Effect of age on inhibition by 5-HT of the twitch response 5-HT inhibited the twitch response to field stimulation of whole vas deferens from young rats although at concentrations above 3 × 10 _7 M it caused a transient, slight increase in the basal tone. As shown in figs. 2 and 3, the inhibitory effect of 5-HT was greatest in vas deferens of 4 week old rats, in which 10 5 M 5-HT caused 82.4 + 3.8% inhibition (n = 5). However, with increasing age of the rats, the inhibitory effect of 5-HT gradually decreased and the dose-response curve was shifted upwards, The inhibitory effect of 10 5 M 5-HT in vas deferens from 8 week old rats had decreased to 49.8 + 2.6% (n = 8) from the maximal value of 82.4% at 4 weeks, without alteration in the threshold concentration for inhibition. With preparations from 15 week old rats 5-HT did not have a dose-dependent effect: with an increase in concentration, inhibition increased to a maxi-
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Fig. 1. Tension developed with 5-HT and field stimulation of whole vas deferens from rats of different ages. Open columns: Twitch tensions induced by field stimulation at 0.1 Hz. Dotted columns: contractile tension produced by 10 5 M 5-HT, i.e. the highest concentration applied to the electrically stimulated preparations. The ordinate indicates the tension developed in rag/whole vas deferens. Ages are shown in weeks. Values are means±S.E.M, of values for 10-15 preparations. NS: not significant, ** P < 0.001, significance of difference from the value for vas deferens from 8 week old rats.
m u m of o n l y 15.3 + 1.4% (n = 5) at 3 × ] 0 - 7 M t h e n d e c r e a s e d at c o n c e n t r a t i o n s of ] 0 - 6 M a n d 3 × 10 6 M, a n d i n c r e a s e d a g a i n at a c o n c e n t r a -
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Fig. 2. Selected tracing from a record showing the effect of aging on the action of 5-HT in rat vas deferens. The preparations were stimulated with single-pulse field stimulation (0.1 Hz, 0.5 ms duration). 5-HT was added at the times indicated by dots. Age of rats is shown in weeks.
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5-HT (-log M) Fig. 3. Changes with age in the effect of 5-HT on the twitch responses of rat whole vas deferens, and the effect of ketanserin. The ordinates indicate changes in the twitch response (inhibition, downwards: augmentation, upwards), as percentages of the initial responses. Open circles, control; closed circles, in the presence of 3 × 10 -v M ketanserin. Age is shown in weeks (w). Values are means + S.E.M. of values for 5-11 preparations. NS, not significantly different from the respective control value (unpaired t-test).
42 tion of 1 0 - s M. W i t h p r e p a r a t i o n s from 45 week o l d rats c o n c e n t r a t i o n s of 5 - H T of up to 3 x 10 7 M d i d n o t affect the twitch response and, conversely, c o n c e n t r a t i o n s a b o v e 10 . 6 M a u g m e n t e d the response. I n p r e p a r a t i o n s f r o m 95 week old rats 5 - H T greatly a m p l i f i e d the twitch response, a c o n c e n t r a t i o n of 10 `-5 M causing 300.0_+ 45.1% (n = 8) a m p l i f i c a t i o n . The i n h i b i t o r y a n d a m p l i f y i n g effects of 5 - H T b o t h r e m a i n e d u n c h a n g e d when the vas deferens was desensitized to the c o n t r a c t i n g a c t i o n of 5-HT.
vas deferens from 4 week old rats (fig. 3). H o w ever, in vas deferens of 8 week old rats, it potentia t e d the i n h i b i t i o n b y 5 - H T to a l m o s t the same level as that o b s e r v e d with 5 - H T alone in vas deferens f r o m 4 week old rats. In vas deferens f o r m 15 week o l d rats, the relative p o t e n t i a t i n g effect of k e t a n s e r i n was greater t h a n at 8 weeks. In p r e p a r a t i o n s from 40 week old rats, 5 - H T augm e n t e d the twitch response while ketanserin not o n l y s u p p r e s s e d the a m p l i f i e d twitch response b u t also u n m a s k e d the i n h i b i t o r y effect of 5-HT. In p r e p a r a t i o n s from old rats (95 weeks old), k e t a n s e r i n a t t e n u a t e d the a m p l i f y i n g effect of 5 - H T b u t d i d not uncover the p r e j u n c t i o n a l inhibition. Essentially similar results were o b t a i n e d with m i a n s e r i n (3 × 10 v M) a n d c y p r o h e p t a d i n e (3 ×
3.3. Effect of ketanserin on preparations from rats of various ages K e t a n s e r i n 3 × 10 - v M d i d n o t affect the 5H T - i n d u c e d i n h i b i t i o n of the twitch response in
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Fig. 4. Changes with age in the effect of 5-HT on the twitch responses of the epididymal and prostatic portions of rat vas deferens, and the effect of ketanserin. Explanations are as for fig. 3. Values are means+S.E.M, for 6 preparations. NS, not significant, * P < 0.05, ** P < 0.0i, significance of difference from the respective control value (unpaired t-test).
43 10 - I M), b u t not with m e t h y s e r g i d e (3 × 10 - 6 M) or p r a z o s i n (10 - 7 M).
3.4. Regional difference in effect of age W e then e x a m i n e d the effects of 5 - H T on the e p i d i d y m a l a n d p r o s t a t i c p o r t i o n s of the vas deferens from rats of various ages. Essentially similar a g e - d e p e n d e n t a l t e r a t i o n s in the effect of 5 - H T were o b s e r v e d in b o t h p o r t i o n s of the vas deferens. In b o t h p o r t i o n s of vas p r e p a r a t i o n s from 8 week old rats, 5 - H T i n h i b i t e d the twitch response a n d k e t a n s e r i n p o t e n t i a t e d the inhibition. However, with increasing age, the a m p l i f i c a t i o n o c c u r r e d earlier (at 15 weeks) in the p r o s t a t i c p o r t i o n (fig. 4). In the p r o s t a t i c p o r t i o n k e t a n s e r i n o n l y a t t e n u a t e d the a m p l i f y i n g effect of 5 - H T whereas, in the e p i d i d y m a l p o r t i o n , it also uncovered the p r e j u n c t i o n a l i n h i b i t i o n b y 5 - H T to s o m e extent, even when the p r e p a r a t i o n was from vas deferens of 95 week old rats.
3.5. Effect of 5 - H T on norepinephrine- and A TP-induced contractions T o d e t e r m i n e w h e t h e r the a u g m e n t i n g effect of 5 - H T on the vas deferens of old rats was the result of i n t e r a c t i o n of 5 - H T with t r a n s m i t t e r released in r e s p o n s e to nerve stimulation, we e x a m i n e d the effect of 10 -5 M 5 - H T on c o n t r a c t i o n s i n d u c e d b y n o r e p i n e p h r i n e ( N E ) a n d A T P . N E and A T P were a d d e d after the 5 - H T - i n d u c e d increase in basal tone h a d r e t u r n e d to the original level. The results are shown in table 1 . 5 - H T at a c o n c e n t r a tion of 10 5 M a m p l i f i e d the c o n t r a c t i o n s caused
b y N E a n d A T P in p r e p a r a t i o n s from b o t h y o u n g a n d old rats. These increases were n o t signific a n t l y different from the respective values for 95 week old rats. K e t a n s e r i n at a c o n c e n t r a t i o n of 3 × 10 7 M, which p o t e n t i a t e d the i n h i b i t o r y effect of 5-HT, caused greater a n t a g o n i s m of the a m p l i f y i n g effect of 5 - H T on the N E - i n d u c e d c o n t r a c t i o n than of the effect on the A T P - i n d u c e d c o n t r a c t i o n , irrespective of the age of the rats.
4. Discussion In our previous studies on the p r e j u n c t i o n a l effect of 5 - H T ( M o r i t o k i et al., in press), we could not c o n f i r m the r e p o r t of K a p u r a n d M o t t r a m (1979) that 5 - H T caused m a r k e d inhibition of twitch c o n t r a c t i o n s of rat vas deferens. This disc r e p a n c y might have been due to a difference in age of the rats used, because we found that in vas deferens from rats aged 4 weeks 5 - H T almost c o m p l e t e l y inhibited the twitch response, whereas in p r e p a r a t i o n s from rats aged over 20 weeks, 5 - H T did not inhibit the twitch response but a u g m e n t e d it. These findings indicate that the p r e j u n c t i o n a l inhibition of the twitch response b y 5 - H T is d e p e n d e n t on the age of rats. A similar age-related decrease in a2-receptorm e d i a t e d p r e j u n c t i o n a l inhibition of adrenergic transmission in rat vas deferens has been r e p o r t e d ( D o c h e r t y and O ' M a l l e y , 1983; H y l a n d a n d D o c h e r t y , 1985). T h e vascular responses to vasoactive substances, such as isoprenaline, c A M P a n d histamine, have also been shown to decrease with age (Cohen a n d Berkowitz, 1974; Fleisch and
TABLE 1 Amplifying effect of 5-HT on the responses to norepinephrine and ATP of whole vas deferens from young and old rats, and the effect of ketanserin. NE: 3×10 7 M norepinephrine, ATP: 10 4 M ATP, KS: 3×10-7 M ketanserin. Values indicate changes in the responses in the presence of 10 5 M 5-HT with and without ketanserin as percentages of the control. Values are means_+ S.E.M. of values from 6-7 preparations, unless otherwise indicated. NS, not significantly different from the value for preparations from 95 week old rats. NE, in the presence of 4 weeks 8 weeks 95 weeks
ATP, in the presence of
5-HT
5-HT with KS
5-HT
5-HT with KS
204.4--+ 16.2 (NS)
- 63.3 ± 24.8 - 11.7 ± 21.3 -37.7 (n = 2)
345.3 ± 32.6 (NS) 402.0 ± 33.5 (NS) 441.5_+76.1
49.0± 31.0 108.5 ± 16.7 51.6 (n = 2)
234.6 ± 26.5 (NS) 220.7± 12.7
44 Hooker, 1976; Moritoki et al., 1986; Schoeffter and Stoclet, 1982; Toda and Hayashi, 1979). In rat vas deferens, the reduction of 5-HT-induced inhibition with age could conceivably be secondary to an age-related increase in the twitch tension or 5-HT-induced contraction and not to a functional alteration. In rat aorta, NE-induced contractions were found to increase with age and the percentage relaxation by isoprenaline was shown to be inversely related to the initial tension (Cohen and Berkowitz, 1974; Toda and Hayashi, 1979). This was not so in the present case because the tension developed was not inversely related to the 5-HT-induced inhibition: the twitch tension in preparations from rats aged 15, 45 and 95 weeks was less than the tension in preparations from 6 and 8 week old rats (fig. 1). In addition, we have previously reported that age-related histamine-induced dilatation in rat mesenteric artery was not the result of increased initial tone as a result of aging (Moritoki et al., 1986). The question then arises of whether ketanserin potentiated the prejunctional inhibitory effect directly, or indirectly by attenuating the stimulatory effect of 5-HT. If ketanserin had a direct potentiating effect, it should shift the dose-response curve for 5-HT to a lower concentration range, and even in preparations from young rats (4 week old), where the amplifying effect of 5-HT on the twitch response was not yet observable, it should potentiate the inhibitory effect of 5-HT. But ketanserin did not have these effects: it did not potentiate the inhibitory effect of threshold concentrations of 5-HT (10 s-3 × 10 ~ M) in preparations from rats aged 8 and 15 weeks or potentiate the 5-HTinduced inhibition in preparations from 4 week old rats (fig. 3). In addition, ketanserin at concentrations necessary to potentiate the prejunctional inhibitory effect of 5-HT antagonized the contractile action of 5-HT (below 10-s M) and its amplifying effect on NE- or ATP-induced contractions. These results suggest that ketanserin potentiated the inhibitory effect of 5-HT indirectly by attenuating the postjunctional stimulatory effect of 5-HT. An amplifying action of 5-HT has been demonstrated in rabbit mesenteric artery (Moritoki and Su, 1981), femoral artery (Van Nueten et al., 1982)
and saphenous vein (Curro et al., 1978). Similar amplifying effects of al-agonists in potentiating nerve-evoked contractions by an action on postsynaptic receptors (Docherty and McGrath, 1984; MacDonald and McGrath, 1980) have been described. We have shown that in rat vas deferens, 5-HT augmented the twitch contractions by a mechanism mediated by 5-HT 2 receptors (Moritoki et al., in press). In the present experiments, ketanserin potentiated or unmasked the inhibitory effect of 5-HT, or attenuated its amplifying effect, depending on the age of the rats. It is assumed, based on these results and various considerations, that with increasing age of the rats, some amplifying effect of 5-HT became predominant, and that this either attenuated its prejunctional inhibitory effect or augmented the twitch response postjunctionally, depending on the age of the rats. However, in rat vas deferens, the potentiating effect of ~l-agonists and the presynaptic c~l-effect were found not to change with age (Docherty and McGrath, 1984). In addition, the present experiments showed that the amplifying effect of 5-HT on the contractile actions of N E and ATP did not change with age. These findings indicate that, although a postjunctional amplifying effect could at least to some extent attenuate the inhibition, it was presumably not the primary cause of the age-related decrease in inhibition. Moreover, it is unlikely that the contractile effect of 5-HT was responsible for the age-related changes because the contractile response to 5-HT was independent of age and because, even though the ratios of the 5-HT-induced tension to the twitch tension for preparations from rats aged 8 and 45 weeks were almost the same, 5-HT did not inhibit the twitch response of the preparation from rats aged 45 weeks. This contractile effect may be different from the amplifying effect, because after the vas deferens had been desensitized to the contractile action of 5-HT, 5-HT still amplified the twitch response. A much more likely explanation is therefore that with an increase in age, the postjunctional amplifying effect of 5-HT on the action of released transmitters is unmasked as a result of decreased prejunctional inhibition, and that ketanserin indirectly potentiated the 5-HT-induced prejunctional inhibition by attenuating the
45 p o s t j u n c t i o n a l effect. It is also p r o b a b l e that the decrease in i n h i b i t o r y f u n c t i o n b u t not the increase in the p o s t j u n c t i o n a l f u n c t i o n mediated by 5 - H T 2 receptor is m a i n l y due to the age-related loss of the 5 - H T - i n d u c e d inhibition. The adrenergic c o m p o n e n t was shown to pred o m i n a t e in the e p i d i d y m a l p o r t i o n of the vas deferens a n d the n o n - a d r e n e r g i c c o m p o n e n t to p r e d o m i n a t e in the prostatic p o r t i o n ( M c G r a t h , 1978). The age-related decrease in responses of the whole vas deferens to 5 - H T a n d the p o t e n t i a t i n g effect of ketanserin on the action of 5 - H T m a i n l y reflect changes in the e p i d i d y m a l p o r t i o n of the vas deferens. I n addition, 5 - H T also inhibited the twitch response of the prostatic p o r t i o n from y o u n g rats, suggesting that i n h i b i t o r y 5 - H T receptors control stimulatory n o n - a d r e n e r g i c f u n c t i o n a n d that this i n h i b i t o r y f u n c t i o n diminishes with age. The a m p l i f y i n g effect of 5 - H T in p r e p a r a t i o n s from aged rats, p r o b a b l y mediated by 5 - H T 2 receptors through a p o s t j u n c t i o n a l mechanism, was more d o m i n a n t in the prostatic p o r t i o n than in the e p i d i d y m a l p o r t i o n of the vas deferens. Loss of i n h i b i t o r y f u n c t i o n with age occurred earlier in the prostatic p o r t i o n than in the epididymal portion. The reason for the decrease in p r e j u n c t i o n a l i n h i b i t i o n by 5 - H T with age is u n k n o w n . However, there are several possible e x p l a n a t i o n s for this p h e n o m e n o n . It could be due to a decrease in n u m b e r or sensitivity of p r e j u n c t i o n a l 5 - H T receptors or an alteration in receptor-response linkage. With respect to these possibilities, loss of B-receptor-mediated vasodilatation in older rats was suggested to be due to a decrease in the n u m b e r of B-receptors (Schoeffter a n d Stoclet, 1982) or a reduction in B-receptor activity (Ericsson a n d L u n d h o l m , 1975). At present however there is no evidence for these possibilities in the case of the p r e j u n c t i o n a l 5 - H T receptor. T o summarize a n d conclude, we have presented evidence that the p r e j u n c t i o n a l i n h i b i t i o n mediated by 5 - H T in both the e p i d i d y m a l a n d prostatic portions of rat vas deferens decreases with age while, conversely, 5H T 2 receptor-mediated amplification of the twitch response becomes p r e d o m i n a n t as a result of decreased p r e j u n c t i o n a l inhibition. D e p e n d i n g o n the age of the rats, the 5 - H T z a n t a g o n i s t ketanserin
p o t e n t i a t e d or u n m a s k e d the p r e j u n c t i o n a l inhibitory effect of 5 - H T by a t t e n u a t i n g the amplifying effect. A n age-linked decrease in the p r e j u n c t i o n a l i n h i b i t i o n mediated by 5 - H T could result in adrenergic transmission being facilitated.
Acknowledgements We are grateful to Dr. J.M. Van Nueten, Janssen Pharmaceutica, Beerse, Belgium,for a gift of ketanserin, to Sandoz, Switzerland, for supplying methysergide, and to Taito-Pfeizer for supplying prazosin.
References Cohen, M.L. and B.A. Berkowitz, 1974, Age-related changes in vascular responsiveness to cyclic nucleotides and contractile agonists, J. Pharmacol, Exp. Ther. 191,147. Curro, F.A., S. Greenberg, T.J. Verbeuren and P.M. Vanhoutte, 1978, Interaction between alpha adrenergic and serotonergic activation of canine saphenous veins, J. Pharmacol Exp. Ther. 207, 936. Docherty, J.R. and K. O'Malley, 1983, An examination of age-related changes in pre- and postsynaptic a-adrenoceptors in the rat isolated vas deferens, European J. Pharmacol. 95, 171. Docherty, J,R. and J.C. McGrath, 1984, The actions of cirazoline on the rat vas deferens, Br. J. Pharmacol. 82, 9. Ericsson, E, and L. Lundholm, 1975, Adrenergic ,&receptor activity and cyclic AMP metabolism in vascular smooth muscle~ variation with age, Mech. Age. Develop. 4, 1. Fleisch, J.H. and C.S. Hooker, 1976, The relationship between age and relaxation of vascular smooth muscle in the rabbit and rat, Circ. Res. 38, 243. Hyland, L. and J.R. Docherty, 1985, Further examination of the effects of aging on the adrenoceptor responsiveness of the rat vas deferens, European J. Pharmacol. 110, 241. Kapur, H. and D.R. Mottram, 1979, A presynaptic inhibitory effect of 5-hydroxytryptamine on the electrically induced twitch response of the rat vas deferens, Biochem. Pharmacol. 28, 951. MacDonald, A. and J.C. McGrath, 1980, The distribution of adrenoceptors and other drug receptors between the two ends of the rat vas deferens as revealed by selective agonists and antagonists, Br. J. Pharmacol. 71,445. McGrath, J.C., 1978, Adrenergic and non-adrenergic components in the contractile response of the vas deferens to a single indirect stimulus, J. Physiol. 283, 23. Moritoki, H., 1984, Characteristics of the presynaptic inhibitory effect of 5-HT on rat vas deferens, IUPAR 9th International Congress of Pharmacology, London, 1984, Abstract 802P. Moritoki, H., H. Fukuda, J. Kanaya and Y. Ishida, Ketanserin
46 potentiates the prejunctional inhibitory effect of 5-hydroxytryptamine on rat vas deferens, J. Pharm. Pharmacol. (in press). Moritoki, H., E. Hosoki and Y. Ishida, 1986, Age-related decrease in endothelium-dependent dilator response to histamine in rat mesenteric artery, European J. Pharmacol. 126, 61. Moritoki. H. and C. Su, 1981, Potentiating effects of 5-hydroxytryptamine and histamine on nerve stimulation-induced contractions of the rabbit mesenteric artery, Arch. Int. Pharmacodyn. 252, 186. Schoeffter, P. and J.C. Stoclet, 1982, Age-related decrease of in vitro isoproterenol-induced cyclic A M P accumulation in rat aorta, European J. Pharmacol. 77, 183.
Schoeffter, P. and J.C. Stoclet, 1983, Ageing as a factor governing the effect of isoproterenol on the cyclic A M P level in isolated aorta from spontaneously hypertensive and normotensive rats, European J. Pharmacol+ 86, 481. Toda, N. and S. Hayashi, 1979, Age-dependent alteration in the response of isolated rabbit basilar arteries to vasoactive agents, J. Pharmacol. Exp. Ther. 211,716. Van Nueten, J.M., P.A.J. Janssen+ W. De Ridder and P.M. Vanhoutte, 1982, Interaction between 5-hydroxytryptamine and other vasoconstrictor substances in the isolated femoral artery of the rabbit; effect of ketanserin (R 41468), European J. Pharmacol. 77, 281.
39
Elsevier EJP 00584
Age-related change in serotonin-mediated prejunctional inhibition of rat vas deferens H i d e k i M o r i t o k i *, T a k e s h i I w a m o t o , J u n K a n a y a , Y u k i o I s h i d a a n d H i r o s h i F u k u d a * Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, UniversiO, of Tokushima, Shomachi, Tokushima 770, Japan
Received 6 August 1986, accepted 9 September 1986
The effect of age on the serotonin (5-HT)-induced prejunctional inhibition of twitch contractions induced in rat vas deferens by single-pulse field stimulation at 0.1 Hz was studied. The inhibitory effect of 5-HT gradually decreased with increasing age of the rats (4-15 weeks old) and was no longer detectable in preparations from rats over 15 weeks old. Moreover, on a further increase in age to 45 weeks, 5-HT conversely enhanced the twitch response of the vas deferens. The 5-HT2 antagonists ketanserin and mianserin potentiated the 5-HT-induced inhibition of contractions of the vas deferens of young rats (8-15 weeks old), and attenuated the amplifying effect of 5-HT on the responses of preparations from old rats (95 weeks old). Thus unmasking of the inhibition depended on the age of rats. This change occurred earlier in life in the prostatic portion of the vas deferens than in the epididymal portion. A functional decrease in 5-HT-mediated prejunctional inhibition and the resultant increase in amplification of the twitch response by 5-HT are probably responsible for the age-related decrease in prejunctional inhibition. Rat vas deferens; 5-HT; Prejunctional inhibition; Aging; Ketanserin 1. Introduction Serotonin (5-HT) has been reported to inhibit twitch contractions of rat vas deferens by a prejunctional mechanism (Kapur and Mottram, 1979). However, in our experiments 5-HT had a variable effect on rat vas deferens: in some preparations, it slightly inhibited the twitch response with a transient increase in the basal tone whereas in other preparations, it did not inhibit the twitch response but augmented it (Moritoki et al., in press). The discrepancy between our findings and those of Kapur and Mottram (1979) could be due to a difference in the age of the rats or in the portions of vas deferens examined. The effects of age on the responses of arteries to vasoactive substances have been well established (Cohen and Berkowitz,
1974; Ericsson and Lundholm, 1975; Fieisch and Hooker, 1976; Schoeffter and Stoclet, 1982, 1983) but little is known about changes in adrenergic transmission with age. There are a few reports of an age-related decrease in prejunctional a:-receptor-mediated inhibition (Docherty and O'Malley, 1983; Hyland and Docherty, 1985). In the present experiments, we studied the influence of age on the prejunctional inhibition by 5-HT of the twitch response of rat vas deferens caused by field stimulation, and the potentiating effect of ketanserin on the inhibitory action of 5-HT. A preliminary account of this work was presented at the I U P A R 9th International Congress of Pharmacology (Moritoki, 1984).
2. Materials and methods * To whom all correspondence should be addressed. * Present address: Department of PharmacologyII, School of Medicine, University of Osaka, Osaka 530, Japan. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Male Wistar rats aged 4 weeks ( + 3 days), 8 weeks ( + 7 days), 15 weeks ( + 10 days), 45 weeks
40 (40-50 weeks) and 95 weeks (90-105 weeks) were used. The vas deferens was excised and separated from connective tissue under a dissecting microscope. In some experiments, the vas deferens was cut into epididymal, middle and prostatic portions constituting 50%, 10% and 40%, respectively, of its total length. The vas deferens was set up in a 10 ml organ bath containing Krebs solution of the following composition (mM): NaC1 115.3, KCI 4.7, CaC12 1.6, MgSO 4 1.2, N a H C O 3 25.0, K H z P O 4 1.2 and glucose 11.1. The medium was maintained at 35°C and bubbled with 5% CO 2 in 02 . The resting tension of the preparation was maintained at 200 rag. After equilibration of the preparation for 60 min, field stimulation of 0.5 ms duration was applied at a frequency of 0.1 Hz from a stimulator (Nihon Kohoden SEN 3201) with a constant voltage modulator through a pair of platinum electrodes. One electrode was attached to one end of the preparation and the other, the reference electrode was placed around the preparation. Contractions were recorded isometrically with a force displacement transducer (Nihon Kohoden SD-1TH). The twitch contractions elicited by field stimulation were abolished by either tetrodotoxin (10 7 M) or guanethidine (3 × 10 -6 M), indicating that the response was neurogenic. A dose-response curve was obtained by cumulative addition of 5-HT in a volume of 10-70 btl to the 10 ml organ bath. Ketanserin and other antagonists were applied 10 min before 5-HT. In tests on the amplifying effect of 5-HT on contractions induced by norepinephrine (NE) or ATP, 5-HT was added 5 rain before the agonists. The significance of differences between values was examined with the unpaired t-test and a P value of less than 0.05 was considered as significant. The drugs used were 5-hydroxytryptamine creatinine sulfate (5-HT, Sigma Chemical Co., St. Louis, MO), norepinephrine bitartrate (Sigma), ATP (2 Na salt, Sigma), ketanserin (Janssen Pharmaceutica, Beerse, Belgium), mianserin hydrochloride (Research Biochemicals Inc., Wayland, MA), cyproheptadine hydrochloride (Merck Sharp & Dohme Research Laboratories, West point, PA), methysergide (Sandoz, Switzerland) and prazosin (Taito-Pfeizer, Tokyo, Japan).
3. Results
3.1. Effect of aging on twitch contractions and 5HT-induced contractions The twitch contractions of rat whole vas deferens induced by field stimulation at 0.1 Hz increased with increasing age of the rats, reaching a maximum in preparations from rats aged 8 weeks. With a further increase in age, the twitch response decreased to an almost steady level from 15 weeks on (fig. 1). In unstimulated preparations, the contractions caused by 5-HT at a concentration of 10 5 M, which was the highest concentration applied to inhibit the twitch response, were less than the twitch height and disappeared rapidly (within 1 min) in the continuous presence of 5-HT. These contractions were not dependent on the age of the rats, being greatest in preparations from 8 and 95 week old rats and negligible in those from 4 week old rats (fig. 1). 5-HT at concentrations above 3 × 10 5 M caused intense contractions that were accompanied by wave-like movement and were resistant to ketanserin (3 x 10 7 M) but antagonized by prazosin with a pA 2 value of 8.03.
3.2. Effect of age on inhibition by 5-HT of the twitch response 5-HT inhibited the twitch response to field stimulation of whole vas deferens from young rats although at concentrations above 3 × 10 _7 M it caused a transient, slight increase in the basal tone. As shown in figs. 2 and 3, the inhibitory effect of 5-HT was greatest in vas deferens of 4 week old rats, in which 10 5 M 5-HT caused 82.4 + 3.8% inhibition (n = 5). However, with increasing age of the rats, the inhibitory effect of 5-HT gradually decreased and the dose-response curve was shifted upwards, The inhibitory effect of 10 5 M 5-HT in vas deferens from 8 week old rats had decreased to 49.8 + 2.6% (n = 8) from the maximal value of 82.4% at 4 weeks, without alteration in the threshold concentration for inhibition. With preparations from 15 week old rats 5-HT did not have a dose-dependent effect: with an increase in concentration, inhibition increased to a maxi-
41
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Fig. 1. Tension developed with 5-HT and field stimulation of whole vas deferens from rats of different ages. Open columns: Twitch tensions induced by field stimulation at 0.1 Hz. Dotted columns: contractile tension produced by 10 5 M 5-HT, i.e. the highest concentration applied to the electrically stimulated preparations. The ordinate indicates the tension developed in rag/whole vas deferens. Ages are shown in weeks. Values are means±S.E.M, of values for 10-15 preparations. NS: not significant, ** P < 0.001, significance of difference from the value for vas deferens from 8 week old rats.
m u m of o n l y 15.3 + 1.4% (n = 5) at 3 × ] 0 - 7 M t h e n d e c r e a s e d at c o n c e n t r a t i o n s of ] 0 - 6 M a n d 3 × 10 6 M, a n d i n c r e a s e d a g a i n at a c o n c e n t r a -
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Fig. 2. Selected tracing from a record showing the effect of aging on the action of 5-HT in rat vas deferens. The preparations were stimulated with single-pulse field stimulation (0.1 Hz, 0.5 ms duration). 5-HT was added at the times indicated by dots. Age of rats is shown in weeks.
45 w
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5-HT (-log M) Fig. 3. Changes with age in the effect of 5-HT on the twitch responses of rat whole vas deferens, and the effect of ketanserin. The ordinates indicate changes in the twitch response (inhibition, downwards: augmentation, upwards), as percentages of the initial responses. Open circles, control; closed circles, in the presence of 3 × 10 -v M ketanserin. Age is shown in weeks (w). Values are means + S.E.M. of values for 5-11 preparations. NS, not significantly different from the respective control value (unpaired t-test).
42 tion of 1 0 - s M. W i t h p r e p a r a t i o n s from 45 week o l d rats c o n c e n t r a t i o n s of 5 - H T of up to 3 x 10 7 M d i d n o t affect the twitch response and, conversely, c o n c e n t r a t i o n s a b o v e 10 . 6 M a u g m e n t e d the response. I n p r e p a r a t i o n s f r o m 95 week old rats 5 - H T greatly a m p l i f i e d the twitch response, a c o n c e n t r a t i o n of 10 `-5 M causing 300.0_+ 45.1% (n = 8) a m p l i f i c a t i o n . The i n h i b i t o r y a n d a m p l i f y i n g effects of 5 - H T b o t h r e m a i n e d u n c h a n g e d when the vas deferens was desensitized to the c o n t r a c t i n g a c t i o n of 5-HT.
vas deferens from 4 week old rats (fig. 3). H o w ever, in vas deferens of 8 week old rats, it potentia t e d the i n h i b i t i o n b y 5 - H T to a l m o s t the same level as that o b s e r v e d with 5 - H T alone in vas deferens f r o m 4 week old rats. In vas deferens f o r m 15 week o l d rats, the relative p o t e n t i a t i n g effect of k e t a n s e r i n was greater t h a n at 8 weeks. In p r e p a r a t i o n s from 40 week old rats, 5 - H T augm e n t e d the twitch response while ketanserin not o n l y s u p p r e s s e d the a m p l i f i e d twitch response b u t also u n m a s k e d the i n h i b i t o r y effect of 5-HT. In p r e p a r a t i o n s from old rats (95 weeks old), k e t a n s e r i n a t t e n u a t e d the a m p l i f y i n g effect of 5 - H T b u t d i d not uncover the p r e j u n c t i o n a l inhibition. Essentially similar results were o b t a i n e d with m i a n s e r i n (3 × 10 v M) a n d c y p r o h e p t a d i n e (3 ×
3.3. Effect of ketanserin on preparations from rats of various ages K e t a n s e r i n 3 × 10 - v M d i d n o t affect the 5H T - i n d u c e d i n h i b i t i o n of the twitch response in
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Fig. 4. Changes with age in the effect of 5-HT on the twitch responses of the epididymal and prostatic portions of rat vas deferens, and the effect of ketanserin. Explanations are as for fig. 3. Values are means+S.E.M, for 6 preparations. NS, not significant, * P < 0.05, ** P < 0.0i, significance of difference from the respective control value (unpaired t-test).
43 10 - I M), b u t not with m e t h y s e r g i d e (3 × 10 - 6 M) or p r a z o s i n (10 - 7 M).
3.4. Regional difference in effect of age W e then e x a m i n e d the effects of 5 - H T on the e p i d i d y m a l a n d p r o s t a t i c p o r t i o n s of the vas deferens from rats of various ages. Essentially similar a g e - d e p e n d e n t a l t e r a t i o n s in the effect of 5 - H T were o b s e r v e d in b o t h p o r t i o n s of the vas deferens. In b o t h p o r t i o n s of vas p r e p a r a t i o n s from 8 week old rats, 5 - H T i n h i b i t e d the twitch response a n d k e t a n s e r i n p o t e n t i a t e d the inhibition. However, with increasing age, the a m p l i f i c a t i o n o c c u r r e d earlier (at 15 weeks) in the p r o s t a t i c p o r t i o n (fig. 4). In the p r o s t a t i c p o r t i o n k e t a n s e r i n o n l y a t t e n u a t e d the a m p l i f y i n g effect of 5 - H T whereas, in the e p i d i d y m a l p o r t i o n , it also uncovered the p r e j u n c t i o n a l i n h i b i t i o n b y 5 - H T to s o m e extent, even when the p r e p a r a t i o n was from vas deferens of 95 week old rats.
3.5. Effect of 5 - H T on norepinephrine- and A TP-induced contractions T o d e t e r m i n e w h e t h e r the a u g m e n t i n g effect of 5 - H T on the vas deferens of old rats was the result of i n t e r a c t i o n of 5 - H T with t r a n s m i t t e r released in r e s p o n s e to nerve stimulation, we e x a m i n e d the effect of 10 -5 M 5 - H T on c o n t r a c t i o n s i n d u c e d b y n o r e p i n e p h r i n e ( N E ) a n d A T P . N E and A T P were a d d e d after the 5 - H T - i n d u c e d increase in basal tone h a d r e t u r n e d to the original level. The results are shown in table 1 . 5 - H T at a c o n c e n t r a tion of 10 5 M a m p l i f i e d the c o n t r a c t i o n s caused
b y N E a n d A T P in p r e p a r a t i o n s from b o t h y o u n g a n d old rats. These increases were n o t signific a n t l y different from the respective values for 95 week old rats. K e t a n s e r i n at a c o n c e n t r a t i o n of 3 × 10 7 M, which p o t e n t i a t e d the i n h i b i t o r y effect of 5-HT, caused greater a n t a g o n i s m of the a m p l i f y i n g effect of 5 - H T on the N E - i n d u c e d c o n t r a c t i o n than of the effect on the A T P - i n d u c e d c o n t r a c t i o n , irrespective of the age of the rats.
4. Discussion In our previous studies on the p r e j u n c t i o n a l effect of 5 - H T ( M o r i t o k i et al., in press), we could not c o n f i r m the r e p o r t of K a p u r a n d M o t t r a m (1979) that 5 - H T caused m a r k e d inhibition of twitch c o n t r a c t i o n s of rat vas deferens. This disc r e p a n c y might have been due to a difference in age of the rats used, because we found that in vas deferens from rats aged 4 weeks 5 - H T almost c o m p l e t e l y inhibited the twitch response, whereas in p r e p a r a t i o n s from rats aged over 20 weeks, 5 - H T did not inhibit the twitch response but a u g m e n t e d it. These findings indicate that the p r e j u n c t i o n a l inhibition of the twitch response b y 5 - H T is d e p e n d e n t on the age of rats. A similar age-related decrease in a2-receptorm e d i a t e d p r e j u n c t i o n a l inhibition of adrenergic transmission in rat vas deferens has been r e p o r t e d ( D o c h e r t y and O ' M a l l e y , 1983; H y l a n d a n d D o c h e r t y , 1985). T h e vascular responses to vasoactive substances, such as isoprenaline, c A M P a n d histamine, have also been shown to decrease with age (Cohen a n d Berkowitz, 1974; Fleisch and
TABLE 1 Amplifying effect of 5-HT on the responses to norepinephrine and ATP of whole vas deferens from young and old rats, and the effect of ketanserin. NE: 3×10 7 M norepinephrine, ATP: 10 4 M ATP, KS: 3×10-7 M ketanserin. Values indicate changes in the responses in the presence of 10 5 M 5-HT with and without ketanserin as percentages of the control. Values are means_+ S.E.M. of values from 6-7 preparations, unless otherwise indicated. NS, not significantly different from the value for preparations from 95 week old rats. NE, in the presence of 4 weeks 8 weeks 95 weeks
ATP, in the presence of
5-HT
5-HT with KS
5-HT
5-HT with KS
204.4--+ 16.2 (NS)
- 63.3 ± 24.8 - 11.7 ± 21.3 -37.7 (n = 2)
345.3 ± 32.6 (NS) 402.0 ± 33.5 (NS) 441.5_+76.1
49.0± 31.0 108.5 ± 16.7 51.6 (n = 2)
234.6 ± 26.5 (NS) 220.7± 12.7
44 Hooker, 1976; Moritoki et al., 1986; Schoeffter and Stoclet, 1982; Toda and Hayashi, 1979). In rat vas deferens, the reduction of 5-HT-induced inhibition with age could conceivably be secondary to an age-related increase in the twitch tension or 5-HT-induced contraction and not to a functional alteration. In rat aorta, NE-induced contractions were found to increase with age and the percentage relaxation by isoprenaline was shown to be inversely related to the initial tension (Cohen and Berkowitz, 1974; Toda and Hayashi, 1979). This was not so in the present case because the tension developed was not inversely related to the 5-HT-induced inhibition: the twitch tension in preparations from rats aged 15, 45 and 95 weeks was less than the tension in preparations from 6 and 8 week old rats (fig. 1). In addition, we have previously reported that age-related histamine-induced dilatation in rat mesenteric artery was not the result of increased initial tone as a result of aging (Moritoki et al., 1986). The question then arises of whether ketanserin potentiated the prejunctional inhibitory effect directly, or indirectly by attenuating the stimulatory effect of 5-HT. If ketanserin had a direct potentiating effect, it should shift the dose-response curve for 5-HT to a lower concentration range, and even in preparations from young rats (4 week old), where the amplifying effect of 5-HT on the twitch response was not yet observable, it should potentiate the inhibitory effect of 5-HT. But ketanserin did not have these effects: it did not potentiate the inhibitory effect of threshold concentrations of 5-HT (10 s-3 × 10 ~ M) in preparations from rats aged 8 and 15 weeks or potentiate the 5-HTinduced inhibition in preparations from 4 week old rats (fig. 3). In addition, ketanserin at concentrations necessary to potentiate the prejunctional inhibitory effect of 5-HT antagonized the contractile action of 5-HT (below 10-s M) and its amplifying effect on NE- or ATP-induced contractions. These results suggest that ketanserin potentiated the inhibitory effect of 5-HT indirectly by attenuating the postjunctional stimulatory effect of 5-HT. An amplifying action of 5-HT has been demonstrated in rabbit mesenteric artery (Moritoki and Su, 1981), femoral artery (Van Nueten et al., 1982)
and saphenous vein (Curro et al., 1978). Similar amplifying effects of al-agonists in potentiating nerve-evoked contractions by an action on postsynaptic receptors (Docherty and McGrath, 1984; MacDonald and McGrath, 1980) have been described. We have shown that in rat vas deferens, 5-HT augmented the twitch contractions by a mechanism mediated by 5-HT 2 receptors (Moritoki et al., in press). In the present experiments, ketanserin potentiated or unmasked the inhibitory effect of 5-HT, or attenuated its amplifying effect, depending on the age of the rats. It is assumed, based on these results and various considerations, that with increasing age of the rats, some amplifying effect of 5-HT became predominant, and that this either attenuated its prejunctional inhibitory effect or augmented the twitch response postjunctionally, depending on the age of the rats. However, in rat vas deferens, the potentiating effect of ~l-agonists and the presynaptic c~l-effect were found not to change with age (Docherty and McGrath, 1984). In addition, the present experiments showed that the amplifying effect of 5-HT on the contractile actions of N E and ATP did not change with age. These findings indicate that, although a postjunctional amplifying effect could at least to some extent attenuate the inhibition, it was presumably not the primary cause of the age-related decrease in inhibition. Moreover, it is unlikely that the contractile effect of 5-HT was responsible for the age-related changes because the contractile response to 5-HT was independent of age and because, even though the ratios of the 5-HT-induced tension to the twitch tension for preparations from rats aged 8 and 45 weeks were almost the same, 5-HT did not inhibit the twitch response of the preparation from rats aged 45 weeks. This contractile effect may be different from the amplifying effect, because after the vas deferens had been desensitized to the contractile action of 5-HT, 5-HT still amplified the twitch response. A much more likely explanation is therefore that with an increase in age, the postjunctional amplifying effect of 5-HT on the action of released transmitters is unmasked as a result of decreased prejunctional inhibition, and that ketanserin indirectly potentiated the 5-HT-induced prejunctional inhibition by attenuating the
45 p o s t j u n c t i o n a l effect. It is also p r o b a b l e that the decrease in i n h i b i t o r y f u n c t i o n b u t not the increase in the p o s t j u n c t i o n a l f u n c t i o n mediated by 5 - H T 2 receptor is m a i n l y due to the age-related loss of the 5 - H T - i n d u c e d inhibition. The adrenergic c o m p o n e n t was shown to pred o m i n a t e in the e p i d i d y m a l p o r t i o n of the vas deferens a n d the n o n - a d r e n e r g i c c o m p o n e n t to p r e d o m i n a t e in the prostatic p o r t i o n ( M c G r a t h , 1978). The age-related decrease in responses of the whole vas deferens to 5 - H T a n d the p o t e n t i a t i n g effect of ketanserin on the action of 5 - H T m a i n l y reflect changes in the e p i d i d y m a l p o r t i o n of the vas deferens. I n addition, 5 - H T also inhibited the twitch response of the prostatic p o r t i o n from y o u n g rats, suggesting that i n h i b i t o r y 5 - H T receptors control stimulatory n o n - a d r e n e r g i c f u n c t i o n a n d that this i n h i b i t o r y f u n c t i o n diminishes with age. The a m p l i f y i n g effect of 5 - H T in p r e p a r a t i o n s from aged rats, p r o b a b l y mediated by 5 - H T 2 receptors through a p o s t j u n c t i o n a l mechanism, was more d o m i n a n t in the prostatic p o r t i o n than in the e p i d i d y m a l p o r t i o n of the vas deferens. Loss of i n h i b i t o r y f u n c t i o n with age occurred earlier in the prostatic p o r t i o n than in the epididymal portion. The reason for the decrease in p r e j u n c t i o n a l i n h i b i t i o n by 5 - H T with age is u n k n o w n . However, there are several possible e x p l a n a t i o n s for this p h e n o m e n o n . It could be due to a decrease in n u m b e r or sensitivity of p r e j u n c t i o n a l 5 - H T receptors or an alteration in receptor-response linkage. With respect to these possibilities, loss of B-receptor-mediated vasodilatation in older rats was suggested to be due to a decrease in the n u m b e r of B-receptors (Schoeffter a n d Stoclet, 1982) or a reduction in B-receptor activity (Ericsson a n d L u n d h o l m , 1975). At present however there is no evidence for these possibilities in the case of the p r e j u n c t i o n a l 5 - H T receptor. T o summarize a n d conclude, we have presented evidence that the p r e j u n c t i o n a l i n h i b i t i o n mediated by 5 - H T in both the e p i d i d y m a l a n d prostatic portions of rat vas deferens decreases with age while, conversely, 5H T 2 receptor-mediated amplification of the twitch response becomes p r e d o m i n a n t as a result of decreased p r e j u n c t i o n a l inhibition. D e p e n d i n g o n the age of the rats, the 5 - H T z a n t a g o n i s t ketanserin
p o t e n t i a t e d or u n m a s k e d the p r e j u n c t i o n a l inhibitory effect of 5 - H T by a t t e n u a t i n g the amplifying effect. A n age-linked decrease in the p r e j u n c t i o n a l i n h i b i t i o n mediated by 5 - H T could result in adrenergic transmission being facilitated.
Acknowledgements We are grateful to Dr. J.M. Van Nueten, Janssen Pharmaceutica, Beerse, Belgium,for a gift of ketanserin, to Sandoz, Switzerland, for supplying methysergide, and to Taito-Pfeizer for supplying prazosin.
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