COMPARISON OF PRE- AND POSTSYNAPTIC -ADRENOCEPTOR BLOCKING EFFECTSƒ¿ OF E-643

IN THE ISOLATED VAS DEFERENS OF THE RAT

Tadao SHOJI Kawashima Branch-Laboratory, Laboratories of Pharmacology, Eisai Co. Ltd., Kawashimacho, Hajimagun, Gifu 483, Japan

Accepted January 18, 1981

Abstract-Effectiveness of E-643, a newly developed ƒ¿-blocker, and four -antagonists in blocking pre- and postsynaptic ƒ¿-adrenoceptors wereƒ¿

compared in the isolated rat vas deferens. The inhibitory effect of

on the field-stimulated twitch response was antagonized in the presence of

the ƒ¿-antagonists. The order of affinity (pA2) for presynaptic ƒ¿-receptors,

as assessed from parallel shift of the dose-response curve to clonidine, was:

>yohimbine>tolazoline>E-643•†. At concentrations from 10-8 to 10-6 M, neither E-643 nor prazosin had any effect on the twitch

which had been depressed by the treatment with clonidine, whereas phentol

amine, and tolazoline partially reversed it. Contractile effects of

cumulative concentrations of noradrenaline were also antagonized by

antagonists. The order of affinity (pA2) for postsynaptic ƒ¿-receptors ƒ¿-was:

E-643•†prazosin> phentolamine>yohimbine>tolazoline. Selectivity for

pre- versus postsynaptic ƒ¿-receptors was assessed by comparing KB values for pre- and postsynaptic ƒ¿-receptors. The order of selectivity for the pre

synaptic ƒ¿-receptors was : yohimbine>tolazoline>phentolamine>>prazosin

E-643. It is concluded that•† E-643 is a potent and highly selective post

synaptic ƒ¿-blocker.

E-643, 2-[4-(n-butyryl)-homopiperazine the possibility that E-643 might selectively 1-yl]-4-amino-6,7-dimethoxyquinazoline, is block the postsynaptic a-adrenoceptors with a newly developed a-blocker with potent out significant interference with presynaptic antihypertensive activity (1-4). In a previous a-receptors (5). The purpose of the present study using the isolated rabbit aorta, we study was to compare the blocking activity showed that E-643, like phentolamine, was a of E-643 on the pre and postsynaptic a specific and competitive blocker of nor adrenoceptors. (5). On the other hand, a differ Experiments were conducted using the ence was noted concerning hypotensive isolated rat vas deferens, an organ in which effects of these two drugs; that is, in both motor transmission is thought to be modulated anesthetized and unanesthetized rats, hypo by an inhibitory presynaptic a-receptors (6 tensive effects of E-643 were more remarkable 9). The results were compared with those of than those of phentolamine on the basis of yohimbine, tolazoline and phentolamine, equipotent a-receptor blockade (2, 5). E-643 compounds which are known to block had no direct relaxing action on isolated rabbit presynaptic a-adrenoceptors (10, 11) and aortic strips (5). These findings suggested with prazosin, a drug which is thought to preferentially block postsynaptic a by acting on the presynaptic a-adrenoceptors adrenoceptors (12-14). (8. 10, 12, 15). Two different methods were used to study MATERIALSAND METHODS interference by a-antagonists of the twitch Preparation and recording: The vas inhibiting effect of clonidine. One of these deferens was removed from Sprague-Dawley was to assess blocking activity of an rats weighing 280-350 g and dissected free antagonist from the shift of the dose-response from the adhering connective tissues. A curve to clonidine. Control dose-response section of 2 cm, including mainly the central curves and those in the presence of portion of the vas deferens was excised and antagonists were obtained in separate pre suspended in a 20-m1 organ bath containing parations as a full recovery from maximally Krebs solution of the following composition effective concentrations of clonidine could (mM) : NaCI, 118.4; KCI, 4.7; CaC12, 2.52; not be obtained by washout. After the twitch MgSO4.7H2O, 1.2; KH2PO4, 1.2; NaHCO3, tension to field stimulation became constant, 25.0 and glucose, 11.1. Magnesium sulfate the antagonists were added to the bath and was reduced to 0.6 mM when the twitch allowed to contact with the vas deferens for response to field stimulation was studied. 15 min prior to initiation of the cumulative The solution was gassed with a mixture of addition of clonidine. Control preparations 95% 02 and 5% C02. Temperature of the were treated with 20 PI of distilled water bathing solution was maintained at 37°C for instead of the antagonists. study of the twitch response and it was Another method was to assess the antago reduced to 30°C in order to decrease the nistic potency of an antagonist from reversal spontaneous contractions when nor of the twitch tension which had been adrenaline-induced contractions were studied potentially depressed by the treatment of Contractions of the isolated preparations clonidine. The twitch response was first were measured isometrically under a tension depressed below 20% of the original peak of 0.5-1.0 g with a force-displacement tension by 10-3 M of clonidine. Increasing transducer (Nihonkohden, SB-1T) connected concentrations (10-3 to 10-5 M) of the to a carrier amplifier (Nihonkohden, RP-5). antagonists were then added to the bath and Recordings were made on a pen-writing reversal of the twitch was tested (see an oscillograph (Nihonkohden, RJG-4000). example in Fig. 1). The tissue was exposed Field stimulation: A pair of Ag-AgCI to each successive concentration of an ring-electrodes were placed near the top antagonist until the response had reached an and bottom of the vas deferens. Field stimu equilibrium. lation was carried out at 0.2 Hz using square Effects on the postsynaptic a-adreno pulses of 3 msec duration at submaximum ceptors: Blocking activity of the a-adreno voltage (about 10 V). This stimulation ceptor antagonists on the postsynaptic a condition was fixed throughout the experi adrenoceptors was assessed by the ment. antagonism with /-noradrenaline-induced Effects on the presynaptic a-adrenoceptors: contractions of the vas deferens in the Blocking activity of the a-antagonists on the presence of cocaine (5X10-° M) and pro presynaptic a-adrenoceptors was assessed pranolol (5X10-s M). Noradrenaline was by the antagonism with an a2-adrenoceptor added to the bath in increasing concen , clonidine. Clonidine inhibits twitch trations (3x10-' to 3x10-1 M), so that a responses of the field-stimulated vas deferens :umulative dose-response curve was pro duced. After two successive dose-response antagonists were dissolved in distilled water curves of equal size had been obtained, and added to the bath in a volume of 20 iii. antagonists were added and thereby exposed /-Noradrenaline was dissolved with an equi to the tissue for 30 min. The addition of molar amount of /-ascorbic acid. When increasing concentrations of noradrenaline cocaine and were used, they were was repeated, and dose-response curves in included in the bathing solution throughout the presence of antagonists were obtained. the experiment. Effects on the twitch response: When the Statistics: pA2 values were calculated twitch tension of the vas deferens to field according to the method of van Rossum (16). stimulation became constant, a-antagonists Statistical evaluation of the data was made were added to the bath in a cumulative by Student's t-test. Differences with P values fashion (10-e to 10-5 M) and an alteration in less than 0.05 were considered to be the twitch tension was observed. The tissue significant. was allowed to contact with each successive concentration of the antagonists until the RESULTS response had reached an equilibrium. Effects on the dose-response curve to Drugs: Drugs used were clonidine hydro clonidine: Field stimulation of the vas chloride (Boehringer-Ingelheim), /-nor deferens at 0.2 Hz produced rapid twitch adrenaline hydrochloride (Fluka), yohimbine contractions. Peak tension of the twitch hydrochloride (Sigma), tolazoline hydro varied among preparations and ranged from chloride (CI BA-Geigy), phentolamine hydro 0.4 to 1.5 g. As shown in a typical trace in chloride (CIBA-Geigy), cocaine hydro Fig. 1, clonidine produced a dose-dependent chloride (Takeda) and d/-propranolol hydro inhibition of the twitch response in a low chloride (Sigma). E-643 hydrochloride and concentration range. prazosin hydrochloride were synthesized in All of the a-antagonists caused a parallel the Chemical Synthetic Laboratories of Eisai shift of the dose-response curve to clonidine, Co. Ltd. for the present study. without reduction of the maximum inhibitory The a-adrenoceptor and response, thereby suggesting a competitive

Fig. 1. Typical traces of twitch inhibition caused by increasing concentrations of clonidine (upper record) and reversal by phentolamine of the twitch which had been depressed by 10-8 M of clonidine (lower record). Clonidine and phentolamine were added to the bath at the point indicated by the arrows. Horizontal and vertical calibrations are 1 min and 1 g, respectively. type of antagonism (Fig. 2). pA2 values ence between pA2 values for E-643 and calculated from these dose-response curves prazosin. The affinity of E-643 for the are given in Table 1. The decreasing order of presynaptic a-receptors was calculated to be affinity was : phentolamine>yohimbine> one sixty-eighth of phentolamine. tolazoline> E-643>prazosin. The pA2 values Reversal of the clonidine-induced in for E-643 were significantly less (P<0.001) hibition of the twitch: The results with the than those of phentolamine, yohimbine and five a-antagonists are shown in Fig. 3. tolazoline. There was no significant differ Under conditions in which the twitch re sponses were inhibited below 20% of the original size by the treatment of clonidine (10-8 M), subsequent addition of phentol amine antagonized and completely reversed the depressed twitch in a concentration dependent manner (10' to 10-5 M) (see a typical trace in Fig. 1). In contrast, E-643 and prazosin had no effect at concentrations ranging from 10-e to 10-6 M. At a higher concentration of 10-5 M, both drugs caused partial antagonism. Almost full antagonism was obtained with tolazoline at 10 M, but Fig. 2. Effects of a-antagonists on the dose only partial reversal of the twitch was response curve to clonidine. The ordinate obtained at a lower concentration of 10-6 M. indicates per cent of twitch contraction. Twitch Yohimbine produced a biphasic effect, a tension of each vas deferens to field-stimulation before the addition of clonidine was taken as dose-dependent antagonism occurring at 100%. The abscissa represents concentrations concentrations up to 10-6 M followed by an of clonidine (3x10-10 to 10-6 M). 0------•: inhibition at 10-5 M. Control; O: E-643 (10-6 M); 0-0: Effects on the postsynaptic a-adreno prazosin (10 6 M); A: phentolamine (10-' M and 10-6 M); A: tolazoline ceptors: All of the a-antagonists used in this (10-6 M); ~: yohimbine (10 and study caused a parallel displacement of the 10-6 M). Number of experiments was 28 for dose-response curve to noradrenaline to the controls and five to six for the other studies. the right (Fig. 4). pA2 values calculated Standard errors (vertical bars) are given only on the control curve. from these dose-response curves are given

Table 1. Blocking activities of a-antagonists on the pre and postsynaptic a-adrenoceptors.

'B' i n the abscissa applies only for the lower curves and denotes the level before addition of the antagonists. Each point with a vertical bar indicates the mean with standard error of five experiments.

Fig. 3. Effects of a-antagonists on the twitch response (upper curves: O) and on the twitch which had been depressed by the treatment with clonidine (lower curves: 0). The ordinate indicates per cent of twitch contraction. Twitch tension of each vas deferens to field stimulation prior to the addition of the antagonists (upper curves) or treatment with 10_8 M of clonidine was taken as 100% and indicated by a dotted line. The abscissa represents concentrations of the antagonists (10-8 to 10-5 M).

in Table 1. The decreasing order of affinity return towards the original level. Phentol for the postsynaptic a-receptors was amine produced a biphasic effect, inhibition E-643>prazosin> phentolamine>yohimbine of the twitch at lower concentrations (up to >tolazoline. The pA2 value for E-643 was 10-6 M) followed by marked enhancement at significantly greater (P<0.001) than the a higher concentration (10-5 M). Prazosin values obtained for phentolamine, yohimbine and yohimbine produced a dose-dependent and tolazoline. There was no statistically reduction of the twitch at concentrations significant difference between the values for higher than 10-' M. Tolazoline did not E-643 and prazosin. The affinity of E-643 for significantly affect the twitch contractions at the postsynaptic a-receptors was calculated concentrations up to 10-' M, but did markedly to be 13, 676 and 1290 times greater than enhance the twitch at 10-5 M. those of phentolamine, yohimbine and tolazoline, respectively. DISCUSSION Effects on the twitch response: The results The vas deferens of the rat has been are shown in Fig. 3. E-643 slightly reduced repeatedly used for the study of presynaptic the twitch peak tension at the concentrations a-adrenoceptors (6-9, 12, 15, 17-19). Field of 10' and 10-6 M. At the concentration of stimulation of the epididymal portion of the 10-5 M, E-643 potentiated the twitch to a vas deferens with single pulses elicits a slight extent and the response tended to biphasic response consisting of an initial fast inhibition of the twitch at low concentrations similar to those reported by other workers (8, 10, 12, 19). This inhibitory action was antagonized by the a-antagonists. pA2 values showed a distinct variation in affinity for the presynaptic a-receptors. Thus, E-643, as well as prazosin, had a much lesser affinity than those of phentolamine and yohimbine. Contractile effects of cumulative concen trations of noradrenaline were also anta gonized by a-antagonists. pA2 values Fig. 4. Effects of a-antagonists on the dose revealed a difference in affinity for the response curve to noradrenaline. The ordinate indicates per cent of maximum contraction. postsynaptic a-receptors among the an The maximum attainable tension of each vas tagonists. E-643, as well as prazosin, had deferens to cumulatively applied noradrenaline much greater affinity than either yohimbine or was taken as 100%. The abscissa represents tolazoline. The pA2 values for E-643 and concentrations of noradrenaline (3x10-8 to 3 x 10-' M). •------0: Control; O: E-643 phentolamine were much the same as those (10-8 and 10-7 M); 0-0: prazosin (10-8 and we found in preparations of the rabbit aorta 10-7 M); 0 A: phentolamine (10-7 and (5). 10-s M); A: tolazoline (10-5 M); C7 0: Selectivity of each a-antagonist for pre yohimbine (10-5 M). Number of experiments versus postsynaptic a-adrenoceptors was was 40 for the controls and five for the other studies. Standard errors (vertical bars) are assessed by the ratio of KB values derived given only on the control curve. from pA2 values against noradrenaline and clonidine, respectively (KB-post/KB-pre) component (twitch) followed by a slower (Table 1). The decreasing order of selectivity component, whereas in the prostatic portion, for the presynaptic a-adrenoceptors was : the response consists principally of a single yohimbine >tolazoline > phentolamine>>pra fast component (20-23). The second slower zosinb E-643. Thus, it is evident that E-643, component is thought to be and like prazosin, can be classified into a category the initial fast component is considered to be of a-antagonists which preferentially block of nonadrenergic origin (20-22). Both the postsynaptic a-adrenoceptors. Yohimbine fast and slow components are inhibited by and tolazoline, on the other hand, are drugs low concentrations of clonidine (22). which preferentially block presynaptic a Preparations used in the present study adrenoceptors. Phentolamine is an a consisted mainly of a central portion of the antagonist which has fairly equal affinities for vas deferens. Field stimulation (0.2 Hz, both pre and postsynaptic a-receptors. 3 msec, submaximal voltage) produced only This spectrum of selectivity concerning a rapid twitch response. Thus, it seems that prazosin, phentolamine, tolazoline and the contractions elicited by the stimulation yohimbine is in good agreement with data on consisted mainly of the fast component. the rat vas deferens (8, 10, 12, 15, 19) and This assumption may be partly supported by other tissues (see ref. 11). It should be noted, the modest inhibition of the twitch caused by however, that the difference between E-643 the highest concentration of E-643 and and tolazoline was mainly due to variation in prazosin. postsynaptic potency; the presynaptic affinity Clonidine produced a dose-dependent of tolazoline was only 2.9 times greater than that of E-643 while postsynaptic affinity of T. Igarashi for comments on the manuscript. the former drug was as little as 1 /1290 of the latter. REFERENCES

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