Comparison of Agonistic and Antagonistic Actions of Guanabenz and Guanfacin on A, and A2-Adrenoceptors in Isolated Smooth Muscles

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Comparison of Agonistic and Antagonistic Actions of Guanabenz and Guanfacin on a, and a2-Adrenoceptors in Isolated Smooth Muscles

Kouichi TAKEUCHI, Miki KOGURE and Takao HASHIMOTO Departmentof Pharmacology,Meiji College of Pharmacy, 1-35-23, Nozawa,Setagaya-ku, Tokyo 154, Japan Accepted November22, 1986

Abstract-The effects of guanabenz, guanfacine and clonidine on a-adrenoceptors were investigated in isolated rat vas deferens and rabbit aortic strip. All 3 drugs at low concentrations (10-9-10-8 M) caused inhibition of twitch responses of the rat vas deferens induced by nerve stimulation. These effects were competitively antagonized by yohimbine. Guanfacine and clonidine at relatively high concen trations (10-°-10-4 M) produced contractions of the rat vas deferens which were antagonized by prazosin. These contractile responses were not much affected by denervation. Prazosin-sensitive contractions by guanfacine and clonidine were also observed in the rabbit aortic strip, which were not affected by reserpine pre treatment. In both tissues, the intrinsic activity of guanfacine was almost identical to that of norepinephrine, whereas that of clonidine was less than one half. Guanabenz and clonidine showed a competitive antagonistic effect against nor epinephrine and phenylephrine in both the rat vas deferens and rabbit aorta, the antagonisms being similar in potency. The results indicate that all 3 drugs are potent agonists on the presynaptic a2-adrenoceptor. In contrast, on the postsynaptic ai -adrenoceptor, guanfacine and guanabenz showed only agonistic and antagonistic actions, respectively, whereas clonidine exhibited partial agonistic characteristics.

Guanabenz and guanfacine are newly adrenoceptors in peripheral tissues, the introduced antihypertensive drugs possessing effects of guanabenz, guanfacine and a guanidine residue in their structural formula clonidine were comparatively investigated in (1-5). In order to elucidate the mechanisms the rat vas deferens a preparation which has of action, many experiments have been been shown to be convenient and useful for carried out in a number of animals under assessing the actions of adrenergic drugs and various conditions. At present, the hypo has provided much important information tensive actions of these drugs are thought to (14). To compare the effects with those on be exerted through a stimulation of a the vascular smooth muscle, the rabbit aortic adrenoceptors in the central nervous system strip was also used. as well as a direct inhibition of the release of neurotransmitter from peripheral sympa Materials and Methods thetic nerves (6-9). The mode of action of The animals used in this study were male these drugs thus appears to be analogous to Wistar-strain rats (200-300 g in body weight) that of clonidine (10-13). Nevertheless, the and Albino rabbits (3.0-4.0 kg in body precise mechanisms of these drugs including weight). Dissected smooth muscle pre clonidine are still to be determined. parations were placed in an organ bath of 20 In the present study, in an attempt to ml capacity containing a modified Krebs clarify the actions of sympathomimetic hypo Ringer solution of the following com tensive drugs on the pre and postsynaptic a position (mM): NaCI, 120; KCI, 4.7; MgCl2, 1.2; CaCl2, 2.2; NaHCO3, 25; KH2PO4, 1.2 examined againstthe action of norepinephrine and glucose, 14. The bathing medium was or phenylephrine, and their potencies were maintained at 37±1 °C and aerated with a assessed by determining pA2 values. mixture of 95% 02 and 5% C02 Similar experiments were carried out on The rat vas deferens the surgically denervated tissues. Action on a2-adrenoceptors: The rat was Surgical denervation: The animals were sacrificed by a blow on the head, and both anesthetized with sodium pentobarbital (30 right and left vasa deferentia were dissected mg/kg, i.p.). Postganglionic denervation of out and set up. After equilibration for at least the left vas deferens was performed under a 60 min, agonistic actions of guanabenz, dissecting microscope by the method of guanfacine or clonidine on presynaptic a Kasuya et al. (15), which involves cutting adrenoceptors were determined by examining the nerve fibers emanating from the hypo the effects of these drugs on the twitch gastric plexus, leaving the deferential artery responses of the rat vas deferens induced by intact. The right vas deferens served as the transmural nerve stimulation. For this purpose, control. The tissues were used 1 week after square wave pulses (1 msec duration, supra the operation. maximal voltage) generated by a stimulator The rabbit aortic strip (Nihon Kohden, SEN-7103) at 0.05 Hz The rabbits were sacrificed by a blow on were passed through platinum bar electrodes the head, and the thoracic aorta was quickly which were placed in parallel with the excised. The aortic strip, 3 mm in width and preparation at a distance of 0.5 cm. The 25-30 mm in length, was prepared by isometric contraction of the vas deferens was helically cutting the tissue according to the recorded on an ink-writing oscillograph method of Furchgott et al. (16). The strip was (Nihon Kohden, RM-85) through a force set up in an organ bath containing modified displacement transducer (Nihon Kohden, TB Krebs-Ringer's solution maintained at 37± 611 T). 1 °C. A resting tension of 4.0 g was ap When the twitch responses attained a plied to the tissue, and 4 hr were allowed steady state, guanabenz, guanfacine or for equilibration. The contraction and relax clonidine was cumulatively added at an ation of the aortic smooth muscle were interval of about 5 min, and the inhibiting isotonically recorded on an electric kymo effects on the twitch responses were graph. investigated. Antagonistic action of The direct stimulating effects of guanabenz, yohimbine was assessed by examining the guanfacine or clonidine on the aortic strip effects on the twitch-inhibiting action of were examined first. Dose-response curves guanabenz, guanfacine and clonidine. The for these compounds were determined, and extent of a2-antagonistic action of yohimbine an antagonism by prazosin was also in was assessed by determining pA2 values vestigated. Subsequently, the antagonistic from rightward displacement of the dose effects of guanabenz, guanfacine and response curves for guanabenz, guanfacine clonidine against norepinephrine or phenyle or clonidine. phrine were examined, and pA2 values were Action on al-adrenoceptor: In this series determined. Similar experiments were also of experiments, isotonic contractions of the performed on the tissue deprived of its rat vas deferens were recorded on an electric sympathetic nervous influence. Because kymograph. One gram was applied to the surgical denervation of the rabbit aorta was tissue as a resting tension. Direct stimulating technically impossible, the tissues obtained actions of guanabenz, guanfacine or from the rabbits pretreated with reserpine 48 clonidine on the smooth muscle of the rat vas hr (1.5 mg/kg, i.p.) and 24 hr (1.0 mg/kg, i.p.) deferens were examined. The dose-response before sacrifice were used in these experi studies were performed, and the antagonism ments. by prazosin was also investigated. Sub Materials sequently, the a, -antagonistic actions of In the present study, the following drugs guanabenz, guanfacine and clonidine were were used: guanabenz acetate (Nippon the twitch response at doses over 1 x 10-9 M (Fig. 2). At the maximum concentration (1 x 10-7 M), depending on the preparation, some of the drugs induced small contractions presumably due to a direct activation of the postsynaptic a-adrenoceptor (vide infra). From the dose-response relationship, their pD2 values were determined (Table 1). The rank order of potency of the twitch inhibiting action was as follows: clonidine> guanfacine>guanabenz. Yohimbine competitively antagonized against the twitch-inhibiting action of either guanabenz, guanfacine or clonidine. The Fig. 1. Structural formulas of guanabenz, guan antagonistic effect of yohimbine (3 x 10-8, facine and clonidine.

Shoji), guanfacine HCI (Sandoz), clonidine HCI (Boehringer Ingelheim), yohimbine HCI (Wako), prazosin HCI (Pfizer), L-norepine phrine bitartrate (Wako), L-phenylephrine (Tokyo Kasei), reserpine (Apoplon®, Daiichi Seiyaku). The structural formulas of guanabenz, guanfacine and clonidine are shown in Fig. 1. Statistical methods The pA2 and pD2 values were determined according to the method of van Rossum (17). Statistical evaluation of the results was made by Student's t-test. Differences with P values less than 0.01 were considered to be sig nificant. Results Actions on nerve stimulation -induced Fig. 2. Typical records showing the influence of twitch responses of the rat vas deferens: guanabenz, guanfacine and clonidine on the twitch Transmural nerve stimulation at 0.05 Hz response. The doses of each of the drugs, 3x`10-9, produced twitch responses of the rat vas 10-8, 3x10-8 and 10-' M cumulatively added, are deferens. Guanabenz, guanfacine and clo indicated by an arrow. The right bar indicates 0.5 g nidine caused dose-dependent inhibition of in tension.

Table 1. Comparison of a-agonistic actions (pD2) of guanabenz, guanfacine and clonidine on the presynaptic a2-adrenoceptor in isolated rat vas deferens and antagonism by yohimbine (pA2) 10-7 M) against each drug was assessed by a competitive manner, whereas guanfacine calculating its pA2 values from the rightward (up to 10-6 M) did not exert antagonistic displacement of the dose-response curves for action. The effect of clonidine on the dose these agonists (Table 1). response curves to norepinephrine was Postsynaptic actions on the rat vas determined in the normal (Fig. 4a) and deferens: Guanfacine and clonidine produced denervated tissues (Fig. 4c). From the contractile responses of the rat vas deferens rightward displacement of the dose-response at relatively high concentrations (10-6-10-4 curve to norepinephrine or phenylephrine, M). The maximum contractile effect of the potency of the antagonistic action (pA2 guanfacine was almost identical to that of value) of guanabenz and clonidine (10-6 norepinephrine; i.e., the intrinsic activity of 10-5 M) was assessed (Table 2). guanfacine was 0.96 (Fig. 3a, Table 2). The In order to check whether the effects of contractile action of clonidine was half and these drugs were exerted directly or indirectly, its intrinsic activity was 0.50 (Fig. 3a, Table similar experiments were performed in the rat 2). On the other hand, guanabenz did not vas deferens that underwent surgical sympa produce appreciable contractile responses thectomy 1 week ago, and the results are even at high concentrations (up to 10-4 M). summarized in Table 2. The intrinsic activity The effect of guanfacine was antagonized of guanfacine was decreased and pA2 values by prazosin (10-8, 10-7 M); i.e., the dose of guanabenz and clonidine as the antagonists response curve for guanfacine was shifted to against norepinephrine and phenylephrine the right and the maximum response was were slightly increased following denervation. decreased (Fig. 3b). Yohimbine at higher The other parameters for each drug were not concentration (10-6 M) produced similar influenced by denervation. antagonism against guanfacine (data not Actions on the rabbit aortic strip: In order shown). The contractile effect of clonidine to further analyze the effects of these drugs on was also antagonized by prazosin (data not the postsynaptic a-adrenoceptors, the rabbit shown). aortic strips were utilized. Guanfacine and The influences of guanabenz, guanfacine clonidine produced contractions of the rabbit and clonidine on the contractile responses aorta dose-dependently from 10-6 M to 10-4 to norepinephrine or phenylephrine were M, the effect of guanfacine being more potent subsequently investigated. Guanabenz (Fig. 5a). In contrast, guanabenz caused only and clonidine showed antagonistic actions a small contraction at 10-4 M (Fig. 5a). The against norepinephrine and phenylephrine in maximum contractile effect of guanfacine was

Fig. 3. Dose-response curves for a) norepinephrine, guanabenz, guanfacine and clonidine, and b) displacement by prazosin of the dose-response curves for guanfacine in isolated rat vas deferens, nor epinephrine (0), guanabenz (/), guanfacine (0), clonidine (0), prazosin (10-8 M A, 10-7 M A). Ordinate: % of the maximum contraction to norepinephrine a) and guanfacine b). Abscissa: negative log dose (M) of the compounds. Each value is presented as a mean with S.E.M. (bar) of 10 experiments. a, and a2-Adrenergic Actions Fig. 4. Effect of clonidine on the dose-response curves to norepinephrine in isolated rat vas deferens (a: normal and c: denervated tissues) and rabbit aortic strips (b: normal and d: reserpine-treated tissues). Ordinate: % of the maximum contraction to norepinephrine. The abscissa shows the negative log dose (M) of norepinephrine: norepinephrine alone (:D) and in the presence of 10-' M (10), 10-6 M (0) and 10-5 M (/) clonidine. High concentrations (10-6, 10-5 M) of clonidine caused partial contractions of the tissues, but superimposed responses to norepinephrine exhibited a clear parallelism. Each value is presented as a mean with S.E.M. (bar) of 6-7 experiments.

Fig. 5. Dose-response curves for a) norepinephrine, guanabenz, guanfacine and clonidine, and b) displacement by prazosin of the dose-response curves for guanfacine in isolated rabbit aortic strips, norepinephrine (0), guanabenz (/), guanfacine (0), clonidine (LI), prazosin (10-8 M, A 10' M A). Ordinate: % of the maximum contraction to norepinephrine a) and guanfacine b). Abscissa: negative log dose (M) of the compounds. Each value is presented as a mean with S.E.M. (bar) of 10 experiments. almost identical to that of norepinephrine order of potency for agonists on presynaptic (0.97 in intrinsic activity), whereas those of a2-adrenoceptors was found to be as follows: clonidine and guanabenz were much smaller clonidine>guanfacine>guanabenz, the dif (Table 3). The contractile activities of these ferences among them were very small, and drugs were antagonized by prazosin, an both guanfacine and guanabenz can be example with guanfacine being presented in assumed to be potent presynaptic a2 Fig. 4b. adrenoceptor agonists as well as clonidine. Guanabenz and clonidine (10-6-10-5 M) The view that guanfacine and guanabenz caused competitive antagonism against nor are potent a2-adrenoceptor agonists is in epinephrine and phenylephrine. The effect agreement with other published observations, of clonidine on the dose-response curves to where these drugs as well as clonidine have norepinephrine was determined in the normal been demonstrated to decrease the magnitude (Fig. 4b) and reserpine-treated tissues (Fig. of the responses to sympathetic nerve stimu 4d). From the rightward displacement of the lation in several organs (2, 18-20). Moreover, dose-response curves for norepinephrine or guanfacine and guanabenz have been phenylephrine, pA2 values of guanabenz reported to decrease 3H-norepinephrine and clonidine were calculated (Table 3). overflow in response to postganglionic Guanfacine (up to 10-6 M) did not affect the sympathetic nerve stimulation in isolated dose-response relationship of norepinephrine. mouse atria (18), rat cortex slices (18) and The results of similar experiments on the isolated rabbit heart (19, 20). In the study of aortic strips from reserpine-pretreated rabbits the release of 3H-norepinephrine in the rabbit are collectively presented in Table 3. The pulmonary artery, clonidine and guanabenz agonistic and antagonistic actions of clonidine have been shown to suppress preferentially were slightly increased and decreased, the release of norepinephrine induced by respectively, following reserpine treatment. electrical stimulation at relatively low The effects of guanfacine and guanabenz frequencies (21, 22). were essentially identical to those on the In the rabbit atrial and ileal preparations control tissues. with intact nerves, it has been demonstrated that guanabenz but not clonidine showed Discussion dual inhibitory actions on the adrenergic In the present study, it was demonstrated neurotransmission, i.e., a presynaptic a2 that either guanabenz, guanfacine or clonidine adrenoceptor-stimulating action and a caused dose-dependent inhibition of the guanethidine-like neuron-blocking action twitch response of the rat vas deferens (23). Therefore, in addition to a selective induced by transmural nerve stimulation. stimulation of the presynaptic a2-adreno This inhibition of the twitch response by ceptor, the possibility cannot be excluded these drugs was unlikely to be mediated that guanabenz might attenuate the twitch through an antagonism against released response of the rat vas deferens through a norepinephrine at postsynaptic a-adreno guanethidine-like neuron blockade most ceptors, because twitch-inhibiting actions probably at high concentrations. of these drugs were exerted at far lower At relatively high concentrations, concentrations compared with their blocking guanfacine and clonidine produced con action on postsynaptic a-adrenoceptors (cf., tractile responses of the rat vas deferens dose pD2 values in Table 1 vs. pA2 values in Table dependently. Guanfacine was almost a full 2). Furtheremore, the twitch-inhibiting agonist, whereas clonidine was a partial actions of these drugs were competitively agonist, the intrinsic activity of which was antagonized by yohimbine, a selective a2 0.5. The effects were antagonized by adrenocepter antagonist. It can be con prazosin, a selective a, -adrenoceptor sidered, therefore, that these drugs produced antagonist. Almost similar results were attenuation of the twitch responses of the obtained in isolated rabbit aorta, except for rat vas deferens through a stimulation of the a very little contractile activity of guanabenz presynaptic a2-adrenoceptor. Although the and a little smaller value in the intrinsic activity of clonidine compared with those in In anesthetized rats, guanfacine has been the rat vas deferens. Smooth muscle shown to produce a biphasic response in the contracting actions of guanfacine and blood pressure, i.e., an increase followed by clonidine and an antagonism by prazosin a decrease in blood pressure, and the initial were qualitatively similarly detected even hypertension was considered to be due to a after denervation of the rat vas deferens or direct stimulation of a-adrenoceptors in reserpine pretreatment of the rabbit aorta. blood vessels (26). The transient elevation of Thus, these results indicate that guanfacine blood pressure following clonidine adminis and clonidine caused contractions of the tration has also been demonstrated to be rat vas deferens and rabbit aorta by directly due to a direct stimulation of at -adrenoceptor acting on the smooth muscles. Because these (27). As far as the peripheral actions are tissues exhibit exclusively postsynaptic a, concerned, therefore, guanabenz appears to adrenoceptors with scarce a2-adrenoceptors be the most favorable hypotensive drug since (24, 25), their action appears to be exerted it exhibits almost agonistic and antagonistic most probably through stimulation of postsy actions on the presynaptic a2 and post naptic at -adrenoceptors. synaptic ai -adrenoceptors, respectively, Denervation of the rat vas deferens usually In summary, guanabenz, guanfacine and brings about supersensitivity as well as a clonidine exerted agonistic action on the large increase in the maximum response to presynaptic a2-adrenoceptor in the rat vas norepinephrine (15). In the control and deferens at a very low concentration range denervated tissues, the contractile response (10-9-10-7 M). Guanfacine and clonidine to guanfacine and clonidine was plotted as showed agonistic action on the postsynaptic the percentage of the maximum response to a1 -adrenoceptor in the rat vas deferens and norepinephrine in each preparation, and rabbit aorta at relatively high concentrations various parameters were determined. There (10-6-10-4 M), guanfacine being almost a fore, even if the values were quantitatively full agonist. Guanabenz and clonidine ex different to some extent between the control hibited a competitive a, -adrenoceptor and denervated preparations, the tissue antagonistic action, the potencies of which exhibited denervation supersensitivity to were nearly identical to each other. It remains either of the drugs. A similar arguement can to be clarified as to what extent do these be applied to the case of reserpine pretreat peripheral effects of the drugs contribute to ment of the rabbit aorta. their hypotensive effects which have been Guanabenz and clonidine showed compe thought to be mostly exerted through acting titive antagonistic actions against norepine on the central nervous system. phrine in the rat vas deferens and rabbit aortic preparation. Norepinephrine has been References demonstrated to produce contractions of the 1 Baum, T., Eckfeld,D.K., Metz, N., Dinish,J.L., rat vas deferens and rabbit aorta through Rowles, G., Van Pelt, R., Shropshire, A.T., stimulating postsynaptic a, -adrenoceptors Fernandez, S.P., Gluckman, M.I. and Bruce, (24, 25). It is thus assumed that guanabenz W.F.: 2,6-Dichlorobenzylideneamino guanidine possesses a competitive blocking action on acetate (Wy-8678). 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