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Evidence for the existence of prejunctional receptor sites for dopamine in the mouse vas deferens
Journal of the Autonomic Nervous System, 27 (1989) 221-228
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Elsevier JANS 00951
Evidence for the existence of prejunctional receptor sites for dopamine in the mouse vas deferens M.R. Carratfi
1, D.
Conte-Camerino 2, A. De Serio 2, E. Ferrari 3 and D. Mitolo-Chieppa
Institutes of 1 Pharmacology and 3 Neurology, Medical Faculty and 2 Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Bari (Italy) (Received 5 December 1988) (Revised version received 31 May 1989) (Accepted 6 June 1989)
Key words: Dopamine-receptor; Vas deferens Abstract The present work is focused on the effects of newly developed dopaminergic agonists and antagonists on the field-stimulated vas deferens. Both LY 171555 and SK&F 38393, relatively selective DA 2 and DA 1 receptor agonists, respectively, produced concentration-dependent inhibition of the field stimulation-evoked contractions in the mouse vas deferens; both compounds did not modify the baseline tone nor the contractile responses to exogenous noradrenaline. Control LY 171555 and SK&F 38393 concentration-response curves, were shifted rightward in a parallel manner in the presence of sulpiride (relatively specific DA 2 antagonist) and SCH 23390 (relatively specific DA 1 antagonist), respectively. Control concentration-response curves for dopaminergic agonists were not modified in the presence of specific blockers for H 1 and H E histamine receptors, serotonin receptors and a2-adrenoceptors. These preliminary findings are suggestive of the existence of two dopaminergic receptor types both presumably located prejunctionally.
Introduction
Two different types of dopamine receptors, D 1 and D 2 receptors, have been identified in brain [18,24,26,30,31]. According to Langer [19,20] the D 2 site is a presynaptic receptor situated on dopaminergic nerves with an inhibitory role on the release of various neurotransmitters. There is now substantial experimental evidence supporting the hypothesis that a dopaminergic neuronal system is present in peripheral tissues; peripheral dopamine receptors have been mainly characterized by the
Correspondence: M.R. Carrath, Institute of Pharmacology, Medical Faculty, University of Bari, Bari, Italy.
pharmacological effects of agonists and antagonists upon them. Some of these effects have been ascribed to interaction with dopamine receptors situated on the efferent autonomic innervation. The two types of peripheral dopamine receptors are generally designated as DA 1 and DA 2 to differentiate them from the receptor classification of D 1 and D 2 for dopamine receptors in the brain [18]. The first indication of the existence of a peripheral prejunctional dopamine receptor came from the experiments of Langer and coworkers [7] in the in vitro nictitating membrane preparation of the cat where dopamine was found to inhibit the release of noradrenaline via a dopamine receptor. Soon after this demonstration, the presence in mammalian organs of selective dopamine recep-
0165-1838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
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tors, mediating prejunctional inhibitory effects, has been postulated. Most emphasis was put on the cardiovascular system where the prejunctional regulation of noradrenaline release has been extensively reviewed. An inhibitory receptor selective for dopamine has been described in the heart [8,16]; blood vessels in which inhibitory prejunctional dopamine receptors have been suggested are the renal and mesenteric vasculature, portal vein and the human omental vein [3,4,10,22]. Type DA 1 dopamine receptors are found postjunctionally, predominantly on renal and mesenteric vascular beds and their activation leads to direct myorelaxation [12]. Type DA 2 dopamine receptors are apparently found prejunctionally on sympathetic axonal varicosities and their activation leads to reduced release of noradrenaline [19,20]. However, in vascular tissue some DA 2 receptors may be found also postjunctionally opposing the D A l - m e d i a t e d myorelaxation [23]. Another favourite model for mechanical and electrophysiological studies of autonomic nerve-muscle transmission among the scientists concerned with adrenergic mechanism, is represented by the vas deferens of rat, guinea pig and mouse, due to the findings of its unique content of adrenergic nerves and noradrenaline [8,32]. Controversy has surrounded the existence of specific pre- and postjunctional dopamine receptors in the rat vas deferens. Several authors have concluded that dopamine activates a population of postjunctional receptors distinct from those activated by noradrenaline [6,28,33] which had been previously denied by other authors [27]. Tayo [33,34] has proposed the existence of specific prejunctional receptors, whose activation by dopamine leads to inhibition of the twitches in the field-stimulated rat vas deferens; conversely other authors have not confirmed the existence of specific dopamine receptors located pre- and postjunctionally in the rat vas deferens [21]. In the past decade a limited number of agonists and antagonists able to discriminate between these two types of receptors existed; more recently relatively selective agonists and antagonists can be used to identify these receptors: for example, R ( + ) 1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol HC1 ( S K & F 38393) is a relatively potent agonist while R ( + )
8-chloro-2,3,4,5-tetrahydro-5-phenyl-(1H)-3-benzazepine-7-ol maleate (SCH 23390) is a relatively potent antagonist at DA I receptors [1,11,15,25]. For the DA 2 receptors, trans-(-)-4a-4,4a,5,6,7,8, 8 a , 9 - o c t a h y d r o - 5 - p r o p y l - ( 1 H)-pyrazolo-(3,4-g) quinoline hydrochloride (LY 171555), and sulpiride are relatively specific as agonist and antagonist, respectively [5]. Our interest in the dopamine receptors and in the effects they mediate on adrenergic transmission, led to study these newly developed dopaminergic agonists and antagonists on the field-stimulated mouse vas deferens; pharmacological receptor characterization is attempted on the basis of the determination of their relative potencies.
Materials and Methods
Male albino mice (30-35 g) were killed by cervical dislocation and exanguination. Both vasa deferentia were removed, carefully cleaned of adhering connective tissue, tied together in parallel and mounted in a 20-ml organ bath, containing a solution of the following composition (mM): NaC1 118; KC1 5.3; CaC12 2.5; N a H 2 P O 4 2 H 2 0 1.4; glucose 5; N a H C O 3 17.8; ascorbic acid 0.25. Magnesium was omitted in agreement with Hughes et al. [14], who observed more effective responses in its absence. The solution was maintained at 37 ° C and bubbled with 95% 02 and 5% CO 2. In all of the experiments propranolol (1 /xmol) was added to the bathing solution to block /3-adrenoceptors. The tissue was allowed to equilibrate for 45 rain before starting the experiment. Platinum electrodes were placed at the top and at the bottom of the organ bath and the vasa deferentia were stimulated with pulses of 1-3 ms duration delivered at 0.1 Hz at supramaximal voltage (30 V). Contractile responses were recorded by means of a microdynamometer recorder using a 1-g isometric transducer (Basile, Milano). Tetrodotoxin (0.63 #M) abolished the twitches elicited by these stimulation parameters, indicating that only nerve fibres were stimulate.d The above protocol of stimulation evoked a monophasic response, completely inhibited by either prazosin (3 /~M) or
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phentolamine (15 /~M), suggesting that only the noradrenaline-mediated component was activated. After twitch responses to field-stimulation had become constant, concentration-response curves for agonists were determined; the potencies of agonists were expressed as p D 2 values which are the negative log molar concentrations producing 50% of the maximum effect. In each preparation the concentration-response curves were constructed either as control or in the presence of 3 concentrations of antagonists (added 15 min before obtaining each curve); in this situation it was possible to calculate the pA 2 and the slopes of the Schild plots in each experiment. All the results are given as the mean + S.E.M. The means were statistically compared using Student's t-test [29]. The following drugs were used: ( - ) n o r a d r e n a line-D-bitartrate (Sigma); dopamine hydrochloride (Sigma); yohimbine hydrochloride (Sigma); propranolol hydrochloride (Sigma); phentolamine hydrochloride (Ciba); prazosin h y d r o c h l o r i d e (Pfizer); S K & F 38393 hydrochloride (Smith, Kline & French); LY 171555 hydrochloride (Lilly); SCH 23390 maleate (Schering); sulpiride (Sigma); clonidine hydrochloride (Boehringer); ranitidine hydrochloride (Glaxo); methysergide maleate (Sandoz); chlorpheniramine maleate (Essex). SCH 23390 was dissolved in 0.1 ml 2 N HC1 and was made up to volume with distilled water to give a stock solution of 10 -3 M. The solution was subsequently diluted with saline.
Results
Clonidine, dopamine, LY 171555 and S K & F 38393 produced concentration-dependent inhibition of field-stimulation-induced contractions of the mouse vas deferens. The potencies of the adrenergic and dopaminergic agonists were expressed as p D 2 values; the mean p D 2 values for clonidine, dopamine, LY 171555 and S K & F 38393 were respectively: 8.5 _ 0.14 (n = 6), 5.6 5:0.12 (n = 6), 8.8 5:0.07 (n = 6), 6.5 5:0.14 (n = 6) (Table 1). The rank order of agonist potencies was LY 171555 > clonidine > S K & F 38393 > dopamine.
TABLE I
Mean pD 2 oalues for agonists at prejunctional receptors in the field-stimulated mouse oas deferens n = n u m b e r of experiments.
Agonists
pD 2 (Mean + S.E.M.)
(n)
Clonidine Dopamine SK&F 38393 LY 171555
8.5 + 0.14 5.6 +0.12 * 6.5 +0.14 * 8.8 + 0.07
(6) (6) (6) (6)
• Significantly different from corresponding value for clonidine ( P < 0.05, paired t-test).
The effects of DA 1- and DA2-selective antagonists were studied on cumulative concentration-response curves to S K & F 38393 (selective DA 1 agonist), LY 171555 (selective DA 2 agonist) and clonidine (selective o~2 agonist). The selective DA 1 antagonist, SCH 23390, produced parallel rightward shift of the S K & F 38393 control concentration-response curve (Fig. 1); the calculated pA 2 value was 6.89 +_ 0.01 (n = 6); a plot of log (concentration ratio - 1 ) against log (antagonist concentrations) (1.2 X 1 0 - 7 M , 3.7 X 1 0 - 7 M , 7.4 X 10 -7 M) gave a linear regression with a slope of - 0 . 9 9 _+ 0.005. The selective DA 2 antagonist, sulpiride produced parallel rightward shift of the LY 171555 control concentration-response curve (Fig. 2); the calculated p A 2 value was 8.80 _ 0.08 (n = 6); a plot of log (concentration ratio - 1 ) against log (antagonist concentrations) (1.4 x 10 -8 M, 8.8 x 10 -8 M, 1.4 × 10 -7 M) gave a linear regression with a slope of - 0 . 9 6 + 0.009. Both SCH 23390 and sulpiride seem to be competitive antagonists; the antagonism was concentration-dependent and surmountable by higher concentrations of the agonist. In no case did SCH 23390 antagonize the inhibitory effect of LY 171555 even at concentration as high as 1.2 x 10 -3 M (Fig. 3); in no case did sulpiride antagonize the inhibitory effect produced by S K & F 38393 even at concentration as high as 1.5 x 10 -5 M (Fig. 4). Control concentration-response curve to the a2-adrenoceptor agonist, clonidine, was shifted
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Fig. 1. Concentration-response curve of inhibitory effects of SK&F 38393 (O) on the field-stimulation-evoked contractions of the mouse vas deferens and its antagonism by 1.2 x 10 -7 M (o), 3.7×10 -7 M (A), 7.4X 10 - 7 M ([3) SCH 23390. Abscissa: negative log of drug molar concentration. Ordinate: % of inhibition. Each point is a mean of 6 experiments.
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Fig. 2. Concentration-response curve of inhibitory effects of LY 171555 ( t ) on the field-stimulation-evoked contractions of the mouse vas deferens and its antagonism by 8.8 x 1 0 -9 M (o), 1 . 5 x 1 0 8 M (zx), 8 . 8 x 1 0 -8 M (t3) sulpiride. Abscissa: negative log of drug molar concentration. Ordinate: % of inhibition. Each point is a mean of 6 experiments.
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Fig. 6. Concentration-response curve of inhibitory effects of clonidine on the field-stimulation-evoked contractions of the mouse vas deferens. (0) control; (Lx) in the presence of 7.4 x 10 -7 M SCH 23390; ([3) in the presence of 8 . 8 x 1 0 - s M sulpiride. Abscissa: negative log of drug molar concentration. Ordinate: % of inhibition. Each point is a mean of 6 experiments.
225
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- 0 . 8 0 + 0 . 0 2 5 . Yohimbine failed to modify the concentration-response curve to LY 171555; only the S K & F 38393 control concentration-response curve was slightly impaired, but a Schild plot could not be constructed for yohimbine since the effects of this compound could only be observed at a single concentration (1.4 × 10 -4 M) on the maximum inhibitory response; lower concentrations of yohimbine were ineffective. Pretreatment with H 1- and H2-histamine receptor antagonists (2.4 X 10 -4 M chlorpheniramine and 7.4 × 1 0 - 4 M ranitidine), respectively, and with antiserotoninergic (2.2 × 10 -5 M methysergide) did not modify the concentration-response curves to the dopaminergic agonists used. Both LY 171555 and S K & F 38393 were tested on the resting tone of the unstimulated vas deferens; in no case did we observe modifications of the resting tone even at concentrations higher than those required to inhibit the field-stimulation-induced contractions. Both LY 171555 (from 1.9 X 1 0 - 9 M to 1.9 x 1 0 - 6 M ) and S K & F 38393 (from 1.7 x 10 -7 M to 1.7 × 10 -5 M) were tested on the exogenous noradrenaline; the contractile responses evoked by noradrenaline were never modified in the presence of both dopaminergic agonists (Fig. 8A, B).
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Fig. 7. Concentration-response curve of inhibitory effects of dopamine (e) on the field-stimulation-evoked contractions of the mouse vas deferens and its antagonism by ( 0 ) 4.3 x 10 - 9 M, (A) 8.6 x 10 -9 M, (r-q) 1.4× 10 -8 M yohimbine. Abscissa: negative log of drug molar concentration. Ordinate: % of inhibition. Each point is a mean of 6 experiments.
rightward in a parallel manner in the presence of the selective a2-adrenoceptor antagonist, yohimbine (Fig. 5); the calculated pA 2 value was 8.33 + 0.07 (n = 6); a plot of log (concentration ratio - 1) against log (antagonist concentrations) (4.3 x 1 0 - 9 M, 8.6 x 1 0 - 9 M, 1.4 X 10 -8) gave a linear regression with a slope of - 1 . 0 4 + 0.007. The control concentration-response curve to clonidine was not shifted by pretreatment with both sulpiride and SCH 23390, at least at those concentrations modifying the concentration-response curves to the dopaminergic agonists (Fig. 6). The control concentration-response curve to dopamine was shifted to the right in a parallel manner in the presence of yohimbine (Fig. 7); the calculated p A 2 value was 8.4 + 0.04 (n = 6); a plot of log (concentration ratio - 1 ) against log (antagonist concentrations) (4.3 × 1 0 - 9 M , 8 . 6 X 1 0 - 9 M , 1.4 X 10 -8 M) gave a linear regression with a slope
Discussion
Authors have denied over the past years the existence of dopamine receptors in the rat vas deferens [21,27]. By using the relatively specific
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Fig. 8. Concentration-response curve of contractile responses of the m o u s e vas deferens to exogenous noradrenaline. A: (e) control; ( 0 ) 1.9 X 10 - 8 M, (zx) 1.9 × 10 - 7 M, ([3) 1.9 x 10 - 6 M LY 171555. B: (e) control; ( 0 ) 1.7 x 10 -8 M, (A) 1.7 × 10 - 7 M, (D) 1.7 × 10 -6 M S K & F 38393. Abscissa: negative log of drug molar concentration. Ordinate: % of m a x i m u m contractile response. Each point is a mean of 5 experiments.
226
drugs for each category of peripheral dopamine receptors, we have observed in both cases an inhibition of field-stimulation-induced twitches in this tissue. Two main questions arose: (1) whether the inhibition was adrenergic or dopaminergic in nature; the effectivity or ineffectivity of the antagonists could answer this first question; (2) whether the inhibition was due to an interaction with pre- or postjunctional sites; experiments performed on the baseline tone of the unstimulated tissue and on the contractile responses to exogenous noradrenaline could answer this second question. All the agonists used produced an inhibition of field-stimulated vas deferens; by comparing the estimated p D 2 values, which can reflect differences in affinities or intrinsic efficacies, the rank order of agonist potencies was LY 171555 > clonidine > S K & F 38393 > dopamine. The relatively specific DA 1 antagonist, SCH 23390, and the relatively specific DA 2 antagonist, sulpiride, produced parallel, concentration-dependent shift to the right of the S K & F 38393 and LY 171555 concentration-response curves, respectively; in both cases the antagonism may be defined as competitive in type and it was surmountable by higher concentrations of agonists. The pA 2 estimates for SCH 23390 against S K & F 38393 was found to be 6.89 and the p A 2 estimates for sulpiride against LY 171555 was found to be 8.80; the two mean slopes of the regression lines were near the theoretical value of 1. In no case was the activity of LY 171555 influenced by SCH 23390, nor that of S K & F 38393 by sulpiride; the p A 2 values of both SCH 23390 and sulpiride against dopamine could not be calculated; only very high concentrations of both antagonists slightly counteracted the m a x i m u m inhibitory effects of dopamine. Both SCH 23390 and sulpiride were ineffective on the inhibitory responses produced by clonidine. The inhibition produced by dopamine was antagonized by yohimbine; the p A 2 value of yohimbine against dopamine was 8.40; however it was obvious why a-adrenergic blocking agents antagonized the inhibitory activity of dopamine, namely this amine has also an amimetic activity. The strongest evidence that both LY 171555 and S K & F 38393 do not act via
adrenergic receptors, was provided by the lack of effects of yohimbine on the LY 171555 and SK& F 38393 concentration-response curves. Only the S K & F 38393 concentration-response curve was slightly modified, but a Schild plot could not be constructed since the effects of yohimbine could only be observed at a single concentration (1.4 × 10 `-4 M) on the m a x i m u m inhibitory response. Interference of both LY 171555 and S K & F 38393 with other receptors was also studied: neither the antagonists for histamine receptors nor the antagonists for serotonin receptors were effective. Both LY 171555 and S K & F 38393 seem to be selective agonists for one type of receptor which could be defined as dopaminergic in type. Arguments can be also provided in favour of the prejunctional localization of both LY 171555- and S K & F 38393-activated sites; both compounds never modified the baseline tone of the tissue nor the contractile responses to exogenous noradrenaline. Therefore both LY 171555- and S K & F 38393-mediated effects seem to be due to an interaction with prejunctional sites. The discrepancy between our results and those of other authors [21,27] could be due in part to species differences and in part to the lack, until recently, of relatively selective d o p a m i n e r e c e p t o r agonists and antagonists. In addition to the physiological function of the prejunctional c~2-adrenoceptors on sympathetic nerve endings in the negative feedback regulation of noradrenaline release, a possible physiological function for the prejunctional dopamine receptors can be envisaged. It has been suggested that d o p a m i n e and prejunctional dopamine receptors participate in the negative feed-back in situations of prolonged nerve activity; under these conditions dopamine is released from noradrenergic nerves and acts on prejunctional DA receptors to decrease the noradrenaline release in order to save transmitter [13]. Another point which deserves to be discussed concerns with the hypothesis that noradrenaline and ATP are co-transmitters in the sympathetic nerves of rodent vasa [2]; a possible interference of dopamine or dopaminergic agonists with the purinergic component was not analyzed since the protocol of stimulation evoked a monophasic response completely inhibited by c~-adrenoceptor antagonists
227
(phentolamine or prazosin); whether dopamine or dopaminergic agonists may cause a prejunctional inhibition of ATP-release is open to further investigations, taking into account the notion that variation of the parameters of stimulation is useful to detect subtle drug effects and that the supposed purinergic component dominates during stimulation of a given frequency and duration [17].
Acknowledgements This work was supported by a grant from the Italian National Research Council (CNR No CT 85.497.07). We are grateful to Prof. G. Di Chiara and Dr. E. Ongini for the gift of LY 171555 and SCH 23390.
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12
13
14
15 16
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Elsevier JANS 00951
Evidence for the existence of prejunctional receptor sites for dopamine in the mouse vas deferens M.R. Carratfi
1, D.
Conte-Camerino 2, A. De Serio 2, E. Ferrari 3 and D. Mitolo-Chieppa
Institutes of 1 Pharmacology and 3 Neurology, Medical Faculty and 2 Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Bari (Italy) (Received 5 December 1988) (Revised version received 31 May 1989) (Accepted 6 June 1989)
Key words: Dopamine-receptor; Vas deferens Abstract The present work is focused on the effects of newly developed dopaminergic agonists and antagonists on the field-stimulated vas deferens. Both LY 171555 and SK&F 38393, relatively selective DA 2 and DA 1 receptor agonists, respectively, produced concentration-dependent inhibition of the field stimulation-evoked contractions in the mouse vas deferens; both compounds did not modify the baseline tone nor the contractile responses to exogenous noradrenaline. Control LY 171555 and SK&F 38393 concentration-response curves, were shifted rightward in a parallel manner in the presence of sulpiride (relatively specific DA 2 antagonist) and SCH 23390 (relatively specific DA 1 antagonist), respectively. Control concentration-response curves for dopaminergic agonists were not modified in the presence of specific blockers for H 1 and H E histamine receptors, serotonin receptors and a2-adrenoceptors. These preliminary findings are suggestive of the existence of two dopaminergic receptor types both presumably located prejunctionally.
Introduction
Two different types of dopamine receptors, D 1 and D 2 receptors, have been identified in brain [18,24,26,30,31]. According to Langer [19,20] the D 2 site is a presynaptic receptor situated on dopaminergic nerves with an inhibitory role on the release of various neurotransmitters. There is now substantial experimental evidence supporting the hypothesis that a dopaminergic neuronal system is present in peripheral tissues; peripheral dopamine receptors have been mainly characterized by the
Correspondence: M.R. Carrath, Institute of Pharmacology, Medical Faculty, University of Bari, Bari, Italy.
pharmacological effects of agonists and antagonists upon them. Some of these effects have been ascribed to interaction with dopamine receptors situated on the efferent autonomic innervation. The two types of peripheral dopamine receptors are generally designated as DA 1 and DA 2 to differentiate them from the receptor classification of D 1 and D 2 for dopamine receptors in the brain [18]. The first indication of the existence of a peripheral prejunctional dopamine receptor came from the experiments of Langer and coworkers [7] in the in vitro nictitating membrane preparation of the cat where dopamine was found to inhibit the release of noradrenaline via a dopamine receptor. Soon after this demonstration, the presence in mammalian organs of selective dopamine recep-
0165-1838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
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tors, mediating prejunctional inhibitory effects, has been postulated. Most emphasis was put on the cardiovascular system where the prejunctional regulation of noradrenaline release has been extensively reviewed. An inhibitory receptor selective for dopamine has been described in the heart [8,16]; blood vessels in which inhibitory prejunctional dopamine receptors have been suggested are the renal and mesenteric vasculature, portal vein and the human omental vein [3,4,10,22]. Type DA 1 dopamine receptors are found postjunctionally, predominantly on renal and mesenteric vascular beds and their activation leads to direct myorelaxation [12]. Type DA 2 dopamine receptors are apparently found prejunctionally on sympathetic axonal varicosities and their activation leads to reduced release of noradrenaline [19,20]. However, in vascular tissue some DA 2 receptors may be found also postjunctionally opposing the D A l - m e d i a t e d myorelaxation [23]. Another favourite model for mechanical and electrophysiological studies of autonomic nerve-muscle transmission among the scientists concerned with adrenergic mechanism, is represented by the vas deferens of rat, guinea pig and mouse, due to the findings of its unique content of adrenergic nerves and noradrenaline [8,32]. Controversy has surrounded the existence of specific pre- and postjunctional dopamine receptors in the rat vas deferens. Several authors have concluded that dopamine activates a population of postjunctional receptors distinct from those activated by noradrenaline [6,28,33] which had been previously denied by other authors [27]. Tayo [33,34] has proposed the existence of specific prejunctional receptors, whose activation by dopamine leads to inhibition of the twitches in the field-stimulated rat vas deferens; conversely other authors have not confirmed the existence of specific dopamine receptors located pre- and postjunctionally in the rat vas deferens [21]. In the past decade a limited number of agonists and antagonists able to discriminate between these two types of receptors existed; more recently relatively selective agonists and antagonists can be used to identify these receptors: for example, R ( + ) 1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol HC1 ( S K & F 38393) is a relatively potent agonist while R ( + )
8-chloro-2,3,4,5-tetrahydro-5-phenyl-(1H)-3-benzazepine-7-ol maleate (SCH 23390) is a relatively potent antagonist at DA I receptors [1,11,15,25]. For the DA 2 receptors, trans-(-)-4a-4,4a,5,6,7,8, 8 a , 9 - o c t a h y d r o - 5 - p r o p y l - ( 1 H)-pyrazolo-(3,4-g) quinoline hydrochloride (LY 171555), and sulpiride are relatively specific as agonist and antagonist, respectively [5]. Our interest in the dopamine receptors and in the effects they mediate on adrenergic transmission, led to study these newly developed dopaminergic agonists and antagonists on the field-stimulated mouse vas deferens; pharmacological receptor characterization is attempted on the basis of the determination of their relative potencies.
Materials and Methods
Male albino mice (30-35 g) were killed by cervical dislocation and exanguination. Both vasa deferentia were removed, carefully cleaned of adhering connective tissue, tied together in parallel and mounted in a 20-ml organ bath, containing a solution of the following composition (mM): NaC1 118; KC1 5.3; CaC12 2.5; N a H 2 P O 4 2 H 2 0 1.4; glucose 5; N a H C O 3 17.8; ascorbic acid 0.25. Magnesium was omitted in agreement with Hughes et al. [14], who observed more effective responses in its absence. The solution was maintained at 37 ° C and bubbled with 95% 02 and 5% CO 2. In all of the experiments propranolol (1 /xmol) was added to the bathing solution to block /3-adrenoceptors. The tissue was allowed to equilibrate for 45 rain before starting the experiment. Platinum electrodes were placed at the top and at the bottom of the organ bath and the vasa deferentia were stimulated with pulses of 1-3 ms duration delivered at 0.1 Hz at supramaximal voltage (30 V). Contractile responses were recorded by means of a microdynamometer recorder using a 1-g isometric transducer (Basile, Milano). Tetrodotoxin (0.63 #M) abolished the twitches elicited by these stimulation parameters, indicating that only nerve fibres were stimulate.d The above protocol of stimulation evoked a monophasic response, completely inhibited by either prazosin (3 /~M) or
223
phentolamine (15 /~M), suggesting that only the noradrenaline-mediated component was activated. After twitch responses to field-stimulation had become constant, concentration-response curves for agonists were determined; the potencies of agonists were expressed as p D 2 values which are the negative log molar concentrations producing 50% of the maximum effect. In each preparation the concentration-response curves were constructed either as control or in the presence of 3 concentrations of antagonists (added 15 min before obtaining each curve); in this situation it was possible to calculate the pA 2 and the slopes of the Schild plots in each experiment. All the results are given as the mean + S.E.M. The means were statistically compared using Student's t-test [29]. The following drugs were used: ( - ) n o r a d r e n a line-D-bitartrate (Sigma); dopamine hydrochloride (Sigma); yohimbine hydrochloride (Sigma); propranolol hydrochloride (Sigma); phentolamine hydrochloride (Ciba); prazosin h y d r o c h l o r i d e (Pfizer); S K & F 38393 hydrochloride (Smith, Kline & French); LY 171555 hydrochloride (Lilly); SCH 23390 maleate (Schering); sulpiride (Sigma); clonidine hydrochloride (Boehringer); ranitidine hydrochloride (Glaxo); methysergide maleate (Sandoz); chlorpheniramine maleate (Essex). SCH 23390 was dissolved in 0.1 ml 2 N HC1 and was made up to volume with distilled water to give a stock solution of 10 -3 M. The solution was subsequently diluted with saline.
Results
Clonidine, dopamine, LY 171555 and S K & F 38393 produced concentration-dependent inhibition of field-stimulation-induced contractions of the mouse vas deferens. The potencies of the adrenergic and dopaminergic agonists were expressed as p D 2 values; the mean p D 2 values for clonidine, dopamine, LY 171555 and S K & F 38393 were respectively: 8.5 _ 0.14 (n = 6), 5.6 5:0.12 (n = 6), 8.8 5:0.07 (n = 6), 6.5 5:0.14 (n = 6) (Table 1). The rank order of agonist potencies was LY 171555 > clonidine > S K & F 38393 > dopamine.
TABLE I
Mean pD 2 oalues for agonists at prejunctional receptors in the field-stimulated mouse oas deferens n = n u m b e r of experiments.
Agonists
pD 2 (Mean + S.E.M.)
(n)
Clonidine Dopamine SK&F 38393 LY 171555
8.5 + 0.14 5.6 +0.12 * 6.5 +0.14 * 8.8 + 0.07
(6) (6) (6) (6)
• Significantly different from corresponding value for clonidine ( P < 0.05, paired t-test).
The effects of DA 1- and DA2-selective antagonists were studied on cumulative concentration-response curves to S K & F 38393 (selective DA 1 agonist), LY 171555 (selective DA 2 agonist) and clonidine (selective o~2 agonist). The selective DA 1 antagonist, SCH 23390, produced parallel rightward shift of the S K & F 38393 control concentration-response curve (Fig. 1); the calculated pA 2 value was 6.89 +_ 0.01 (n = 6); a plot of log (concentration ratio - 1 ) against log (antagonist concentrations) (1.2 X 1 0 - 7 M , 3.7 X 1 0 - 7 M , 7.4 X 10 -7 M) gave a linear regression with a slope of - 0 . 9 9 _+ 0.005. The selective DA 2 antagonist, sulpiride produced parallel rightward shift of the LY 171555 control concentration-response curve (Fig. 2); the calculated p A 2 value was 8.80 _ 0.08 (n = 6); a plot of log (concentration ratio - 1 ) against log (antagonist concentrations) (1.4 x 10 -8 M, 8.8 x 10 -8 M, 1.4 × 10 -7 M) gave a linear regression with a slope of - 0 . 9 6 + 0.009. Both SCH 23390 and sulpiride seem to be competitive antagonists; the antagonism was concentration-dependent and surmountable by higher concentrations of the agonist. In no case did SCH 23390 antagonize the inhibitory effect of LY 171555 even at concentration as high as 1.2 x 10 -3 M (Fig. 3); in no case did sulpiride antagonize the inhibitory effect produced by S K & F 38393 even at concentration as high as 1.5 x 10 -5 M (Fig. 4). Control concentration-response curve to the a2-adrenoceptor agonist, clonidine, was shifted
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Fig. 1. Concentration-response curve of inhibitory effects of SK&F 38393 (O) on the field-stimulation-evoked contractions of the mouse vas deferens and its antagonism by 1.2 x 10 -7 M (o), 3.7×10 -7 M (A), 7.4X 10 - 7 M ([3) SCH 23390. Abscissa: negative log of drug molar concentration. Ordinate: % of inhibition. Each point is a mean of 6 experiments.
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Fig. 2. Concentration-response curve of inhibitory effects of LY 171555 ( t ) on the field-stimulation-evoked contractions of the mouse vas deferens and its antagonism by 8.8 x 1 0 -9 M (o), 1 . 5 x 1 0 8 M (zx), 8 . 8 x 1 0 -8 M (t3) sulpiride. Abscissa: negative log of drug molar concentration. Ordinate: % of inhibition. Each point is a mean of 6 experiments.
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Fig. 6. Concentration-response curve of inhibitory effects of clonidine on the field-stimulation-evoked contractions of the mouse vas deferens. (0) control; (Lx) in the presence of 7.4 x 10 -7 M SCH 23390; ([3) in the presence of 8 . 8 x 1 0 - s M sulpiride. Abscissa: negative log of drug molar concentration. Ordinate: % of inhibition. Each point is a mean of 6 experiments.
225
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- 0 . 8 0 + 0 . 0 2 5 . Yohimbine failed to modify the concentration-response curve to LY 171555; only the S K & F 38393 control concentration-response curve was slightly impaired, but a Schild plot could not be constructed for yohimbine since the effects of this compound could only be observed at a single concentration (1.4 × 10 -4 M) on the maximum inhibitory response; lower concentrations of yohimbine were ineffective. Pretreatment with H 1- and H2-histamine receptor antagonists (2.4 X 10 -4 M chlorpheniramine and 7.4 × 1 0 - 4 M ranitidine), respectively, and with antiserotoninergic (2.2 × 10 -5 M methysergide) did not modify the concentration-response curves to the dopaminergic agonists used. Both LY 171555 and S K & F 38393 were tested on the resting tone of the unstimulated vas deferens; in no case did we observe modifications of the resting tone even at concentrations higher than those required to inhibit the field-stimulation-induced contractions. Both LY 171555 (from 1.9 X 1 0 - 9 M to 1.9 x 1 0 - 6 M ) and S K & F 38393 (from 1.7 x 10 -7 M to 1.7 × 10 -5 M) were tested on the exogenous noradrenaline; the contractile responses evoked by noradrenaline were never modified in the presence of both dopaminergic agonists (Fig. 8A, B).
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Fig. 7. Concentration-response curve of inhibitory effects of dopamine (e) on the field-stimulation-evoked contractions of the mouse vas deferens and its antagonism by ( 0 ) 4.3 x 10 - 9 M, (A) 8.6 x 10 -9 M, (r-q) 1.4× 10 -8 M yohimbine. Abscissa: negative log of drug molar concentration. Ordinate: % of inhibition. Each point is a mean of 6 experiments.
rightward in a parallel manner in the presence of the selective a2-adrenoceptor antagonist, yohimbine (Fig. 5); the calculated pA 2 value was 8.33 + 0.07 (n = 6); a plot of log (concentration ratio - 1) against log (antagonist concentrations) (4.3 x 1 0 - 9 M, 8.6 x 1 0 - 9 M, 1.4 X 10 -8) gave a linear regression with a slope of - 1 . 0 4 + 0.007. The control concentration-response curve to clonidine was not shifted by pretreatment with both sulpiride and SCH 23390, at least at those concentrations modifying the concentration-response curves to the dopaminergic agonists (Fig. 6). The control concentration-response curve to dopamine was shifted to the right in a parallel manner in the presence of yohimbine (Fig. 7); the calculated p A 2 value was 8.4 + 0.04 (n = 6); a plot of log (concentration ratio - 1 ) against log (antagonist concentrations) (4.3 × 1 0 - 9 M , 8 . 6 X 1 0 - 9 M , 1.4 X 10 -8 M) gave a linear regression with a slope
Discussion
Authors have denied over the past years the existence of dopamine receptors in the rat vas deferens [21,27]. By using the relatively specific
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Fig. 8. Concentration-response curve of contractile responses of the m o u s e vas deferens to exogenous noradrenaline. A: (e) control; ( 0 ) 1.9 X 10 - 8 M, (zx) 1.9 × 10 - 7 M, ([3) 1.9 x 10 - 6 M LY 171555. B: (e) control; ( 0 ) 1.7 x 10 -8 M, (A) 1.7 × 10 - 7 M, (D) 1.7 × 10 -6 M S K & F 38393. Abscissa: negative log of drug molar concentration. Ordinate: % of m a x i m u m contractile response. Each point is a mean of 5 experiments.
226
drugs for each category of peripheral dopamine receptors, we have observed in both cases an inhibition of field-stimulation-induced twitches in this tissue. Two main questions arose: (1) whether the inhibition was adrenergic or dopaminergic in nature; the effectivity or ineffectivity of the antagonists could answer this first question; (2) whether the inhibition was due to an interaction with pre- or postjunctional sites; experiments performed on the baseline tone of the unstimulated tissue and on the contractile responses to exogenous noradrenaline could answer this second question. All the agonists used produced an inhibition of field-stimulated vas deferens; by comparing the estimated p D 2 values, which can reflect differences in affinities or intrinsic efficacies, the rank order of agonist potencies was LY 171555 > clonidine > S K & F 38393 > dopamine. The relatively specific DA 1 antagonist, SCH 23390, and the relatively specific DA 2 antagonist, sulpiride, produced parallel, concentration-dependent shift to the right of the S K & F 38393 and LY 171555 concentration-response curves, respectively; in both cases the antagonism may be defined as competitive in type and it was surmountable by higher concentrations of agonists. The pA 2 estimates for SCH 23390 against S K & F 38393 was found to be 6.89 and the p A 2 estimates for sulpiride against LY 171555 was found to be 8.80; the two mean slopes of the regression lines were near the theoretical value of 1. In no case was the activity of LY 171555 influenced by SCH 23390, nor that of S K & F 38393 by sulpiride; the p A 2 values of both SCH 23390 and sulpiride against dopamine could not be calculated; only very high concentrations of both antagonists slightly counteracted the m a x i m u m inhibitory effects of dopamine. Both SCH 23390 and sulpiride were ineffective on the inhibitory responses produced by clonidine. The inhibition produced by dopamine was antagonized by yohimbine; the p A 2 value of yohimbine against dopamine was 8.40; however it was obvious why a-adrenergic blocking agents antagonized the inhibitory activity of dopamine, namely this amine has also an amimetic activity. The strongest evidence that both LY 171555 and S K & F 38393 do not act via
adrenergic receptors, was provided by the lack of effects of yohimbine on the LY 171555 and SK& F 38393 concentration-response curves. Only the S K & F 38393 concentration-response curve was slightly modified, but a Schild plot could not be constructed since the effects of yohimbine could only be observed at a single concentration (1.4 × 10 `-4 M) on the m a x i m u m inhibitory response. Interference of both LY 171555 and S K & F 38393 with other receptors was also studied: neither the antagonists for histamine receptors nor the antagonists for serotonin receptors were effective. Both LY 171555 and S K & F 38393 seem to be selective agonists for one type of receptor which could be defined as dopaminergic in type. Arguments can be also provided in favour of the prejunctional localization of both LY 171555- and S K & F 38393-activated sites; both compounds never modified the baseline tone of the tissue nor the contractile responses to exogenous noradrenaline. Therefore both LY 171555- and S K & F 38393-mediated effects seem to be due to an interaction with prejunctional sites. The discrepancy between our results and those of other authors [21,27] could be due in part to species differences and in part to the lack, until recently, of relatively selective d o p a m i n e r e c e p t o r agonists and antagonists. In addition to the physiological function of the prejunctional c~2-adrenoceptors on sympathetic nerve endings in the negative feedback regulation of noradrenaline release, a possible physiological function for the prejunctional dopamine receptors can be envisaged. It has been suggested that d o p a m i n e and prejunctional dopamine receptors participate in the negative feed-back in situations of prolonged nerve activity; under these conditions dopamine is released from noradrenergic nerves and acts on prejunctional DA receptors to decrease the noradrenaline release in order to save transmitter [13]. Another point which deserves to be discussed concerns with the hypothesis that noradrenaline and ATP are co-transmitters in the sympathetic nerves of rodent vasa [2]; a possible interference of dopamine or dopaminergic agonists with the purinergic component was not analyzed since the protocol of stimulation evoked a monophasic response completely inhibited by c~-adrenoceptor antagonists
227
(phentolamine or prazosin); whether dopamine or dopaminergic agonists may cause a prejunctional inhibition of ATP-release is open to further investigations, taking into account the notion that variation of the parameters of stimulation is useful to detect subtle drug effects and that the supposed purinergic component dominates during stimulation of a given frequency and duration [17].
Acknowledgements This work was supported by a grant from the Italian National Research Council (CNR No CT 85.497.07). We are grateful to Prof. G. Di Chiara and Dr. E. Ongini for the gift of LY 171555 and SCH 23390.
10
11
12
13
14
15 16
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