Alpha-1D adrenoceptors are involved in reserpine-induced supersensitivity of rat tail artery

We examined reserpine-induced chemical denervation supersensitivity with special reference to alpha-1 adrenoceptor (AR) subtypes. Chronic treatment with reserpine for 2 weeks depleted noradrenaline in the tail artery and spleen of rats. Noradrenaline in the thoracic aorta was negligible before and after reserpine treatment. The treatment with reserpine produced supersensitivity in the contractile responses of the rat tail artery to phenylephrine, 5-HT and KCl, resulting in leftward shift of concentration–response curves (11.6-, 2.5- and 1.1-fold at EC50 value, respectively). These results suggest a predominant sensitization of the alpha-1 AR-mediated response by reserpine treatment. BMY 7378 at a concentration (30 nM) specific for blocking the alpha-1D AR subtype, but not KMD-3213 at a concentration (10 nM) selective for blocking the alpha-1A AR subtype, inhibited the supersensitivity of the phenylephrine-induced response in the reserpine-treated artery. On the other hand, the response to phenylephrine in reserpine-untreated artery was selectively inhibited by the same concentration of KMD-3213, but not by BMY 7378. Prazosin, a subtype-nonselective antagonist, blocked the responses to phenylephrine with the same potency, regardless of reserpine treatment. In the thoracic aorta and spleen, no supersensitivity was produced in the responses to phenylephrine by reserpine treatment. In a tissue segment-binding study using [3H]-prazosin, the total density and affinity of alpha-1 ARs in the rat tail artery were not changed by treatment with reserpine. However, alpha-1D AR with high affinity for BMY 7378 was significantly detected in reserpine-treated tail artery, in contrast to untreated artery. Decreases in alpha-1A AR with high affinity for KMD-3213 and alpha-1B AR with low affinities for KMD-3213 and BMY 7378 were also estimated in reserpine-treated tail artery. Alpha-1D AR mRNA in rat tail artery increased to three-folds by reserpine treatment, whereas the levels of alpha-1A and 1B mRNAs were not significantly changed. The present results suggest that chronic treatment with reserpine affects the expression of alpha-1 AR subtypes of rat tail artery and that the induction of alpha-1D ARs with high affinity for catecholamines is in part associated with reserpine-induced supersensitivity. Keywords: Supersensitivity, reserpine, chemical denervation, alpha-1 adrenoceptor (AR), intact tissue segment binding, mRNA, rat tail artery Introduction Supersensitivity is one of the classical and pharmacological phenomena and is defined as an increase in functional affinity (that is, a decreased EC50) for agonists (Fleming et al., 1973; Fleming, 1976; Westfall, 1981). Supersensitivity may be caused by a variety of procedures, including surgical and chemical denervation and chronic treatment with antagonists (Westfall, 1981; Insel, 1989). The one common feature of these treatments has been reported that they all cause chronic interruption of neuronal stimulation on target cells. Reserpine depletes catecholamine in the adrenergic nerve due to inhibition of catecholamine reuptake by binding the pre-junctional storage vesicle for catecholamine (Berkowitz et al., 1971; Stitzel, 1976; Giachetti & Shore, 1978). Chronic treatment with reserpine is known to cause supersensitivity of the adrenergic and nonadrenergic responses in sympathetically innervated tissues, which has been respectively called specific and nonspecific supersensitivities (Fleming et al., 1973). In extensive studies, several mechanisms underlying the supersensitivity have been proposed; increases in the density or affinity of postjunctional receptors (Bito & Dawson, 1970; Colucci et al., 1981), changes in the resting membrane potential (Fleming & Westfall, 1975; Abel et al., 1981; Fleming, 1987) and alterations of calcium homeostasis (Hudgins & Harris, 1970; Carrier, 1975). Recently, alpha-1 ARs have been classified into at least three subtypes, alpha-1A, -1B and -1D, in molecular and pharmacological evaluations (Minneman et al., 1994; Hieble et al., 1995; Michel et al., 1995; Piascik & Perez 2001). These subtypes distribute distinctly and play key roles in adrenergic functions in many tissues (McGrath & Wilson, 1988; Muramatsu et al., 1995; 1998; Graham et al., 1996; Daly et al., 2002). In rats, for example, the adrenergic contraction of the tail artery is predominantly mediated by the alpha-1A AR subtype (Lachnit et al., 1997; Murata et al., 1999; Gisbert et al., 2003), while the adrenergic contractions of the thoracic aorta and spleen are mainly through alpha-1D and alpha-1B AR subtypes, respectively (Burt et al., 1995; Muramatsu et al., 1995; Buckner et al., 1996; Saussy et al., 1996). Recent studies further developed various drugs specific for each subtype. Prazosin is a classical antagonist which shows no subtype selectivity (Muramatsu et al., 1995; Hancock, 1996). BMY 7378 and KMD-3213 are well characterized as highly selective antagonists for alpha-1D and alpha-1A AR subtypes, respectively (Goetz et al., 1995; Shibata et al., 1995; Saussy et al, 1996; Yamagishi et al., 1996; Murata et al., 1999). Furthermore, several agonists including phenylephrine show relatively high affinity for the alpha-1D subtype (Lomasney et al., 1991; Buckner et al., 1996; Graham et al., 1996; Piascik & Perez, 2001). Stassen et al. (1998) evaluated the relationship between the presence of adrenergic nerves and the presence of alpha-1 ARs in the arterial trees of rat, and suggested that the presence of alpha-1 AR subtypes may be closely related to the sympathetic innervation. They also demonstrated that chemical sympathectomy of rats with 6-hydroxydopamine caused a distinct reduction in alpha-1A AR subtype, concomitant with a marked reduction of noradrenaline content. We hypothesized that the supersensitivity may in part reflect the changes in expression of alpha-1 AR subtypes. In the present study, we examined a possible relationship between reserpine-induced supersensitivity and alpha-1 AR subtypes. To this end, we analyzed the concentration–contractile response curve for phenylephrine, the binding of [3H]-prazosin, the effects of the alpha-1 AR subtype-selective ligands KMD-3213 and BMY 7378, and the mRNA levels of alpha-1 AR subtypes.