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).