a1D-Adrenoceptors Cause Endothelium-Dependent Vasodilatation in the Rat Mesenteric Vascular Bed

The vasodilator activity of a1-adrenoceptor agonists was tested in the rat mesenteric vascular bed (MVB), and the mechanism involved was investigated in cultured endothelial cells isolated from the bovine coronary vascular bed. In preparations preconstricted by U46619, noradrenaline and phenylephrine induced a slight relaxant effect at nanomolar concentrations. This effect was abolished in endothelium-denuded preparations and in preparations pretreated with 100 mM N-nitro-L-arginine methyl ester plus 3 mM indomethacin. Both the phospholipase C inhibitor U73122 and the endoplasmic reticulum Ca-ATPase inhibitor thapsigargin inhibited the vasorelaxant effect of phenylephrine. The cellular level of inositol monophosphate (IP1) in bovine endothelial cells doubled after a 15-min exposure to 0.03 to 0.1 nM phenylephrine. The activity of cNOS was significantly increased following exposure to the same concentrations of phenylephrine. Both chloroethylclonidine and the selective a1D-adrenoceptor antagonist BMY 7378 reduced, in a concentration-dependent manner, the relaxant effect induced by phenylephrine, whereas the selective a1A-adrenoceptor antagonist (1)-niguldipine was ineffective. BMY 7378 also blocked the cNOS activation induced by phenylephrine. Conversely, the increase in perfusion pressure induced by micromolar concentrations of phenylephrine was blocked by 1 nM (1)-niguldipine, but was unaffected by BMY 7378. These findings demonstrate that nanomolar concentrations of phenylephrine, which are devoid of any contractile effect, induced a slight endothelium-dependent vasorelaxation in the rat MVB through the stimulation of a1D-adrenoceptors, located on endothelial cells, which act through phospholipase C stimulation, followed by IP1 generation, and nitric-oxide synthase activation. Conversely, the increase in perfusion pressure induced by micromolar concentrations of phenylephrine is attributable to the stimulation of a1A-adrenoceptors. The identification of endothelial a2-adrenoceptors involved in a vasorelaxant response to catecholamines was made in the late 1980s (Vanhoutte and Miller, 1989). Conversely, vascular postjunctional a1-adrenoceptors have been widely considered as vasoconstrictor receptors since their stimulation by sympathomimetic amines causes contraction of resistance arteries in most vascular beds. However, two reports (Zschauer et al., 1997; Boer et al., 1999) have produced evidence for the functional presence of vasorelaxant a1-adrenoceptors in the brachial and pulmonary arteries isolated from the rabbit and rat, respectively. According to these reports, the pharmacological stimulation of a1-adrenoceptors located on endothelial cells, is able to generate nitric oxide (NO), whereas the stimulation of a2-adrenoceptors releases a relaxing prostanoid (Zschauer et al., 1997; Boer et al., 1999). According to a recent report (Nishina et al., 1999), a2-adrenoceptors are involved in a vasorelaxant response induced by noradrenaline in conduit arteries of the neonatal rat. The release of endothelium-derived relaxing factors from endothelial cells, by activation of a1or a2-adrenoceptors has also been previously demonstrated in other vascular beds (Kaneko and Sunano, 1993; Hu et al., 1994). Endothelial vasorelaxant adrenoceptors may represent a local control mechanism, which is, at least in part, involved in the modulation of the vasoconstrictor response to sympathomimetic amines. In fact, it is well known that the vascular response to sympathetic stimulation is enhanced by endothelium removal and NO synthase inhibitor administration (Greenberg et al., 1989; Bennet et al., 1992; Amerini et al., 1995; Zschauer et al., 1997; Boer et al., 1999). The aims of the present study were to test the functional presence of putative relaxant a1-adrenoceptors in a preconstricted vascular preparation in vitro and to identify the cellular mechanisms inThis study was supported by a grant from the University of Florence Ministero dell’Universitá e della Ricerca Scientifica e Tecnologica (ex 60%). ABBREVIATIONS: NO, nitric oxide; MVB, mesenteric vascular bed; L-NAME, N-nitro-L-arginine methyl ester; DMEM, Dulbecco’s modified Eagle’s medium; IP1, inositol monophosphate; ACh, acetylcholine; CVEC, coronary venular postcapillary endothelial cell; cNOS, constitutive nitric-oxide synthase; CEC, chloroethylclonidine hydrochloride; IP3, inositol trisphosphate. 0022-3565/01/2963-869–875$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 296, No. 3 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 3099/887794 JPET 296:869–875, 2001 Printed in U.S.A. 869 at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from volved in the vasorelaxant response. Moreover, since at least three subtypes of a1-adrenoceptors (a1A, a1B, and a1D) are coexpressed in many tissues (Zhong and Minneman, 1999), we also determined the subtype(s) of a1-adrenoceptors involved in the relaxant effect. For these purposes the effects of noradrenaline, of the selective a1-adrenoceptor agonist phenylephrine and of pharmacological tools able to interfere with their actions were investigated in the MVB preparation isolated from the rat. Moreover, the transduction mechanisms were tested in cultured endothelial cells isolated from the bovine coronary vascular bed. Materials and Methods Indomethacin, 9,11-dideoxy-11a, 9a-epoxymethano-prostaglandin F2a (U46619), N -nitro-L-arginine methyl ester hydrochloride (LNAME), bradykinin, Dowex 50WX8-400 resin, Dulbecco’s modified Eagle’s medium (DMEM), lithium chloride, thapsigargin, L-arginine, HEPES (sodium salt), HEPES (free acid), acetylcholine (ACh), noradrenaline hydrochloride, and phenylephrine hydrochloride were purchased from Sigma (St. Louis, MO); (6)-1-[2,3-(dihydro-7-methyl-1Hinden-4-yl)oxyl]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551 hydrochloride), (6)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino] ethoxy]benzamide methanesulfonate (CGP 20712A methanesulfonate), and [2,6-dichloro(N-b-chloroethyl-N-methyl)-4-aminomethyl] phenylimino2-imidazolidine dihydrochloride (chloroethylclonidine dihydrochloride) were purchased from Research Biochemicals International (Boston, MA); 1-(6((17b-3-methoxy-estra-1,3,5(10)-trien-17-yl)amino)hexyl)-1Hpyrrole-2,5-dione (U73122), 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien17-yl) amino)hexyl)-2,5-pyrrolidine-dione (U73343) were purchased from Biomol (Plymouth Meeting, PA); ((S)-1,4-dihydro-2,6-dimethyl-4(3-nitrophenyl)-3,5-pyridinedicarboxylic acid, 3-(4,4-diphenyl-1piperidinyl) propylmethyl ester), (S)-(1)-niguldipine hydrochloride; and (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane7,9-dione), BMY 7378 dihydrochloride were purchased from Tocris Cookson Ltd. (Bristol, UK); bovine calf serum was purchased from Hyclone (Logan, UT); [H]arginine, myo-2-[H]inositol were purchased from Amersham Life Science Ltd. (Buckinghamshire, UK); and anion exchange columns were prepared with AG-K8 (200–400 mesh, formate form) from Bio-Rad Laboratories (Richmond, CA). A stock solution of indomethacin (10 mM) was prepared in ethanol; U73122, thapsigargin, and CGP 20712A were made in dimethyl sulfoxide; chloroethylclonidine was made in methanol; and the other drugs were dissolved daily in double distilled water and further dilutions to the final concentrations were made in Krebs’ solution. Control experiments showed that the concentrations of dimethyl sulfoxide used modified neither the vasoconstrictor response to U46619 nor the relaxation induced by different