Characterization of subtype of α1-adrenoceptor mediating vasoconstriction in perfused rat hind limb

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Abstract

The subtype of α1-adrenoceptor mediating the exogenous noradrenaline-induced vasopressor response in perfused rat hind limb was determined by functional measurements and radioligand binding assays. The potencies (pA2 values) of α1A-adrenoceptor-selective antagonists, RS-17053 (N-[2-(2-cyclopropylmethoxy-phenoxy) ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethanamine hydrochloride), WB 4101 (2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4 benzodioxane), 5-methyl-urapidil, and the α1D-adrenoceptor-selective antagonist, BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspirol[4.5]decane-7,9-dione), to inhibit the noradrenaline-induced vasopressor response determined by Schild plot were 9.47±0.21, 9.48±0.19, 8.10±0.27 and 6.66±0.14, respectively, with no slope significantly different from unity. The affinities (Ki values) of these antagonists were determined by displacement of 125I-BE 2254 (2-β(4-hydroxyphenyl)-ethylaminomethyl)-tetralone) binding from the cloned α1a-, α1b-, α1d-adrenoceptor, stably expressed in human embryonic kidney (HEK) 293 cells. The pA2 values of the above antagonists correlated well with the binding Ki values only for α1A-adrenoceptors (r=0.93), but not for α1B-adrenoceptors (r=0.51) and α1D-adrenoceptors (r=0.13). The concentration-vasopressor response curve for noradrenaline was not significantly affected by pretreatment with 50 μM chloroethylclonidine for 30 min. The results suggest that only α1A-adrenoceptors mediate the noradrenaline-induced vasopressor response in perfused rat hind limb.

Introduction

α1-Adrenoceptors can be found at numerous end organs of the autonomic nervous system, especially vascular smooth muscle. The clinical potencies of prazosin and other α1-adrenoceptor antagonists as antihypertensive drugs are evidence of the important physiological role of peripheral α1-adrenoceptors in maintaining arterial pressure in animals and humans.

α1-Adrenoceptors have been classified on the basis of pharmacological evidence into three subtypes termed α1A-, α1B-, and α1D-adrenoceptor subtypes mainly because of differential affinities to subtype-selective antagonists. Selective α1A-adrenoceptor antagonists include WB 4101 (Morrow et al., 1985; Morrow and Creese, 1986), 5-methyl-urapidil (Gross et al., 1988; Hanft and Gross, 1989), (+)-niguldipine (Boer et al., 1989), and RS-17053 (Ford et al., 1996). BMY 7378 appears to preferentially antagonize α1D-adrenoceptors (Goetz et al., 1995). Chloroethylclonidine is an alkylating antagonist that irreversibly inactivates the α1B- and α1D-adrenoceptor, but not the α1A-adrenoceptor subtype (Perez et al., 1991; Forray et al., 1994; Han et al., 1987; Minneman et al., 1988). The heterogeneity of α1-adrenoceptors is further supported by results of receptor gene cloning studies which show that three molecular subtypes (α1a-, α1b-, α1d-adrenoceptor) exist and correspond directly to the receptors expressed in intact tissues (Ford et al., 1994). One of the most useful approaches for characterizing subtype distribution in a tissue is to determine correlations between potencies of antagonists obtained in functional experiments with the tissue and affinities obtained from radioligand binding assays with cloned α1-adrenoceptor subtypes.

Investigations employing functional, radioligand binding and molecular methods have demonstrated the existence of multiple α1-adrenoceptor subtypes in vascular smooth muscle of isolated rat, rabbit, dog, and human blood vessels, such as aorta (Buckner et al., 1996), renal artery (Han et al., 1990; Piascik et al., 1994), and mesenteric artery (Han et al., 1990; Piascik et al., 1994), etc. These arteries essentially function as conduit vessels which direct blood flow into organs; the contractile properties of these vessels have a minor influence on the regulation of vascular resistance. It is more important to determine the distribution and function of α1-adrenoceptor subtypes in a resistance vascular bed. In the rat perfused mesenteric arterial bed, noradrenaline-induced increases of perfusion pressure are mainly mediated by the activation of α1A-adrenoceptors based on the high affinities for 5-methyl-urapidil (pA2 9.0–9.2) and WB 4101 (pA2 9.6, Kong et al., 1994; Cunningham et al., 1994; Williams and Clarke, 1995) and the lack of affinity for chloroethylclonidine (Williams and Clarke, 1995). The α1-adrenoceptor in the rat kidney vascular bed has also been characterized as an α1A-adrenoceptor subtype (Eltze et al., 1991; Blue et al., 1991, Blue et al., 1992, Blue et al., 1995). A consistent observation in isolated perfused kidney studies is the weak inhibitory effect of chloroethylclonidine on the vasopressor response to α1-adrenoceptor stimulation. In addition, the potencies of several subtype-selective antagonists on the noradrenaline-induced vasocontractile response in the isolated rat perfused kidney correlated significantly with their binding affinity in the cloned α1a-adrenoceptor (r=0.85). In contrast, correlation with binding affinity for the cloned α1b-adrenoceptor (r=0.02) or α1d-adrenoceptor (r=0.12) is poor (Blue et al., 1995). Those results confirm that the α1A-adrenoceptor is the major subtype in renal resistance arterioles. Other reports show that, in isolated perfused rat mesentery or kidney, sympathetic nerve stimulation provokes vasoconstriction which is extremely sensitive to low concentrations of α1A-adrenoceptor antagonists and non-sensitive to chloroethylclonidine (Kong et al., 1994; Blue et al., 1991, Blue et al., 1992). Therefore, α1A-adrenoceptors may be located at the neurovascular synapses where they receive direct sympathetic nerve innervation (Kong et al., 1994; Williams and Clarke, 1995; Eltze et al., 1991; Blue et al., 1992).

The distribution of α1-adrenoceptor subtypes in other resistance vasculature has not been reported yet. Skeletal muscle is the largest organ in the body, comprising an estimated 35–45% of body weight in non-obese individuals. The muscle resistance vessels have a key role in regulating the total systemic vascular resistance and hence arterial blood pressure. In the present study, we characterized subtypes of the α1-adrenoceptor distributed in the resistance vasculature of skeletal muscle by determining functional potencies of α1-adrenoceptor subtype-selective antagonists and comparing them with binding affinities at cloned α1-adrenoceptor subtypes.

Section snippets

Preparation of isolated perfused rat hind limb

The animals were anesthetized with pentobarbital sodium (60 mg/kg, i.p.). Surgery was performed as described by Colquhoun et al. (1990). Flow was restricted to two hind limbs by cannulation with PE-50 polyethylene to two lateral common iliac arteries. Perfusion was performed in a temperature-controlled cabinet, and the arterial perfusion medium was temperature-equilibrated by passage through a heat exchanger. The perfusion Krebs solution contained the following (mM): NaCl, 118; KCl, 4.7; CaCl2,

pA2 values of α1-adrenoceptor subtype-selective antagonists

Prazosin (3–30 nM in the absence of yohimbine) or yohimbine (1–10 μM in the absence of prazosin) competitively antagonized noradrenaline-induced vasoconstriction (Fig. 1), with pA2 values of 9.04±0.12 and 6.35±0.14, and slope in the Schild plot of 0.85±0.13 and 0.86±0.06, respectively. In the presence of 0.1 μM yohimbine to block α2-adrenoceptors, prazosin (1–10 nM), RS-17053 (1–10 nM), WB 4101 (1–10 nM), 5-methyl-urapidil (10–100 nM), and BMY 7378 (0.3–3 μM) produced parallel shifts to the

Discussion

The vasopressor responses induced by exogenous noradrenaline or by sympathetic activity were mediated by both α1-adrenoceptor and α2-adrenoceptor in cat and dog hind limb vasculature (Gardiner and Peters, 1982; Karasawa and Koss, 1993). Medgett and Ruffolo (1988)reported that α1- and α2-adrenoceptors exist in the femoral vascular bed of rat hind limb and that both mediate exogenous noradrenaline-induced vasoconstriction. However, only the α1-adrenoceptor was involved in neurogenic

Acknowledgements

This study is supported by grants from the National Natural Science Foundation of China, and a grant from the China Medical Board of New York Inc., No. 93-951.

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