a-Adrenoceptors in Canine Mesenteric Artery Are Predominantly 1A Subtype: Pharmacological and Immunochemical Evidence

We wanted to determine which a-adrenoceptor subtypes mediate phenylephrine (PE) contraction of dog mesenteric artery in vitro. We studied antagonisms in response to prazosin, 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane, 5-methylurapidil, N-[2(2-cyclopropyl methoxy phenoxy)ethyl]5-chloro-a,a-dimethyl-1Hindole-3-ethanamine HCl (RS 17053), 8–3-[4-(2-methoxyphenyl)1-piperazinyl]propylcarbamoyl)-3-methyl-4-oxo-22-phenyl-4H-1benzopyran 2HCl [SB216469 (Rec 15/2739)], BMY 7378, 8-[2(1,4-benzodioxan-2-ylmethylamino)ethyl]8-azaspirol[4,5]decane7,9-dione HCl, MDL 72832, and 7-chloro-2-bromo-3,4,5,6tetrahydro-4-methylfurol[4,3,2-ef]3-benzapine. pKB values for prazosin, 5-methylurapidil, MDL 72832, and RS-17053 were consistent with action on a1A-adrenoceptors but decreased with concentration. pKB values (9.6) for Rec 15/2739 (a1L/1A-adrenoceptor selective) were constant. Antagonism by BMY 7378, 7-chloro-2-bromo3,4,5,6-tetrahydro-4-methylfurol[4,3,2-ef]3-benzapine, and 8-[2-(1,4benzodioxan-2-ylmethylamino)ethyl]8-azaspirol[4,5]decane-7,9dione HCl gave pKB values between those expected for a1Aand a1D-adrenoceptors. Chloroethylclonidine (100 mM) shifted EC50 values for PE rightward and decreased Emax values but left large residual responses. After 100 mM chloroethylclonidine, either BMY 7378 (100 nM) or RS-17053 (300 nM) increased EC50 values for PE contractions with pKB values like those of controls. At 6 nM, phenoxybenzamine increased the EC50 values and reduced Emax values; prior Rec 15/2739, but not prior BMY 7378, protected receptors against inactivation. An antibody against the a1Badrenoceptors immunostained muscle of aorta but not mesenteric artery. We conclude that dog mesenteric artery contains a1Aadrenoceptors. Discrepancies among responses expected if only these receptors are present may result from pleiotropic functional effects at this receptor and the presence of a1L-adrenoceptors. Shi et al. (1989a,b, 1990) showed that receptors of dog mesenteric and saphenous veins (DMVs and DSVs, respectively) had similar KD (dissociation constant in saturation ligand binding studies) values for and densities of [H]prazosin (PR) binding sites. However, in dog mesenteric arteries (DMAs), the KD value for prazosin binding was lower at a similar receptor density. Only DMVs and DSVs, which had higher densities of [H]rauwolscine binding sites than DMAs, responded by contraction to a2-adrenoceptor agonists. All three vessels responded to phenylephrine (PE) with similar pD2 values and similar efficacies. Responses to norepinephrine were effected only through a1-adrenoceptors in DMAs, but a2-adrenoceptor activation also contributed to the norepinephrine-induced contraction of DMVs and DSVs. Later, Shimamoto et al. (1992) showed that responses of DMAs to UK 14304, an a2-adrenoceptor-selective agonist, were promoted by agents that produced threshold contractile stimulation by enhanced Ca entry, but a1-adrenoceptors as well as a2-adrenoceptors mediated these responses. Received for publication February 9, 1999. 1 This work was supported by a grant-in-aid and by a Career Investigatorship Award (C.Y.K.) from the Ontario Heart and Stroke Foundation and by a National Institutes of Health Grant GM41470 to R.D.B. This work was also aided by a Martin Wills student scholarship (to A.D.) from the Heart and Stroke Foundation of Ontario. Portions were presented in abstract form (Kwan CY, Low AM, Lu-Chao H and Daniel EE (1997) Characterization of a-adrenoceptor subtypes in dog mesenteric artery. Canadian Federation of Biological Sciences, annual meeting, London, Ontario, Canada. ABBREVIATIONS: DMV, dog mesenteric vein; C-E, concentration-effect; CEC, chloroethylclonidine; DMA, dog mesenteric artery; DMSO, dimethyl sulfoxide; DSV, dog saphenous vein; Emax, maximum response to phenylephrine; KB, calculated antagonist dissociation constant in functional studies; KD, dissociation constant in saturation ligand binding studies; MDL 72832, 8-[4-(1,4-benzodioxan-2-ylmethylamino)butyl]8azaspirol[4,5]decane-7,9-dione HCl; MDL 73005EF, 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]8-azaspirol[4,5]decane-7,9-dione HCl; PE, phenylephrine; PBZ, phenoxybenzamine; Rec 15/2739, (SB216469; 8–3-[4-(2-methoxyphenyl)-1-piperazinyl]propylcarbamoyl)-3-methyl-4-oxo22-phenyl-4H-1-benzopyran 2HCl; RS-17053, N-[2-(2-cyclopropyl methoxy phenoxy)ethyl]5-chloro-a,a-dimethyl-1H-indole-3-ethanamine HCl; SK&F 105854, 7-chloro-2-bromo-3,4,5,6-tetrahydro-4-methylfurol[4,3,2-ef]3-benzapine; WB 4101, 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl1,4-benzodioxane; 5-MU, 5-methylurapidil. 0022-3565/99/2912-0671$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 291, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 291:671–679, 1999 671 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from However, the question of which of the various subtypes of a1-adrenoceptors mediate contraction in DMAs remains unanswered. After the subclassification of a1-adrenoceptors into a1A and a1B subtypes based on greater sensitivity of the latter to inactivation by chloroethylclonidine (CEC; Han et al., 1987a,b), molecular biological studies have defined three subtypes, now known as a1A, a1B, and a1D, all with high affinity for prazosin (Lomasney et al., 1991a,b). The a1Badrenoceptor defined pharmacologically proved to be similar to the cloned a1b-adrenoreceptor, with a lower affinity for 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane (WB 4101) and 5-methylurapidil (5-MU) than the a1Aadrenoreceptor, which had high affinity for these antagonists, as reviewed by Ford et al. (1994). The a1D-adrenoceptor is now recognized to be a distinct subtype (Schwinn and Lomasney, 1992; Perez et al., 1994; see reviews in Ford et al., 1994; Hieble et al., 1995a), distinguished from the a1A-adrenoceptor by a low affinity for 5-MU and a high affinity for recently described agents such as BMY 7378, 8-[2-(1,4benzodioxan-2-ylmethylamino)ethyl]8-azaspirol[4,5]decane-7,9dione HCl (MDL 73005EF; Saussy et al., 1994; Goetz et al., 1995), and 7-chloro-2-bromo-3,4,5,6-tetrahydro-4-methylfurol[4,3,2-ef]3benzapine (SK&F 105854; Hieble et al., 1995b) in cloned and expressed rat and human receptors. All these subclasses of receptors have high binding affinity (pKi . 9) for prazosin when expressed in cell lines. Naturally occurring receptors have been found to have lower affinities (pKB or pKi , 9) in some tissues (Muramatsu et al., 1990; Ohmura et al., 1992) and have been classified as a1L-adrenoceptors in contrast to the high-affinity types, a1H-adrenoceptors. Receptors with low affinity for prazosin have not been cloned, but some antagonists [e.g., SB216469; 8–3-[4(2-methoxyphenyl)-1-piperazinyl]propylcarbamoyl)-3-methyl-4-oxo-22-phenyl-4H-1-benzopyran 2HCl (Rec 15/2739)] with high affinity for a1A-adrenoceptors have been reported to distinguish between them and other a1(perhaps a1L-) adrenoceptor subtypes (Testa et al., 1996, 1997; Leonardi et al., 1997). Recently, Ford et al. (1997) showed that the a1A-adrenoceptor expressed in CHO-K1 cells demonstrated binding properties [high affinity to prazosin, 5-MU, N-[2-(2-cyclopropyl methoxy phenoxy)ethyl]5-chloro-a,adimethyl-1H-indole-3-ethanamine HCl (RS-17053), Rec 15/ 2739, WB 4101, and (1)-niguldipine] expected of a1A-adrenoceptors. However, when a functional property, inhibition of production of inositol phosphates, was evaluated, many antagonists gave lower affinity interactions, as expected for a1Ladrenoceptors. Rec 15/2739 showed the same functional as binding affinity for the a1A-adrenoceptor. The authors suggested that the a1L-adrenoceptor was a pleiotropic expression of this receptor. The goal of this study was to characterize the a1-adrenoceptor subtypes of DMAs by using functional interactions as well as immunostaining studies. The results can be compared with those from other canine blood vessels that have different a-adrenoceptors (Daniel et al., 1996, 1997; Low et al., 1998). Materials and Methods Animal and Tissue Preparation. Mongrel dogs of either sex, weighing 10 to 25 kg, were kept under standard conditions in our animal quarters, fasted 24 h before use, and euthanized with an i.v. overdose (100 mg/kg) of pentobarbital. These procedures were approved by the University Animal Care Committee following the guidelines of the Canadian Council on Animal Care. Segments to be used for functional studies were placed in Krebs-Ringer solution (see below for composition). Functional Studies. Rings of DMAs from the first or, occasionally, the second branch (3 mm wide) were mounted individually in 10-ml organ baths filled with modified Krebs’ solution composed of 115.5 mM NaC1, 4.6 mM KC1, 1.16 mM MgSO4, 1.16 mM NaH2PO4, 2.5 mM CaC12, 21.9 mM NaHCO3, and 11.1 mM glucose, pH 7.4; gassed with a 95% O2/5% CO2 gas mixture; and kept at 37°C. The endothelium was removed by rubbing with forceps, confirmed by showing that carbachol could not relax a contraction induced by PE (3 mM) or 60 mM KCl. The tissues were subjected to a 3g preload tension, which gave the maximum contractile response, and allowed to equilibrate for 2 h. Rings were subjected to repeated exposures to 100 mM KCl, followed by washing, until contractions were regular. Cumulative dose-response curves were constructed before and 30 min after incubation with increasing concentrations of antagonists (except prazosin incubated for 45 min) were added. The concentration of agonist in the bath was increased approximately 3-fold at each step after the response to the previous dose had plateaued. Data were discarded if a 2-fold shift or more in the EC50 value for PE occurred in concomitant time controls. When CEC was used as an antagonist, it was not feasible to carry out successive exposures to different PE concentrations. In these studies, of eight arterial rings, two were time controls, and two each were exposed to different concentrations of CEC. Data are expressed