Br. J. Pharmacol. (1994), 112, 519-524 '." © MamlaMacmillan PresPress LtdLtd,1-994-1994 Mediation by M3-muscarinic receptors of both endothelium-dependent contraction and relaxation to acetylcholine in the aorta of the spontaneously hypertensive rat 'Chantal M. Boulanger, Keith J. Morrison & Paul M. Vanhoutte Center for Experimental Therapeutics, Baylor College of Medicine, Houston, Texas 77030, U.S.A. 1 Experiments were designed to characterize the subtype(s) of endothelial muscarinic receptor that mediate(s) endothelium-dependent relaxation and contraction in the aorta of spontaneously hypertensive rats (SHR). 2 Rings of SHR aorta with endothelium were suspended in organ baths for the measurement of isometric force. Ecothiopate (an inhibitor of acetylcholinesterase) was present throughout the experiments. Endothelium-dependent contraction to acetylcholine was studied in quiescent aortic rings in the presence of N0-nitro-L-arginine (to prevent the formation of nitric oxide). Endothelium-dependent relaxation to acetylcholine was obtained during contraction to phenylephrine and in the presence of indomethacin (to inhibit cyclo-oxygenase activity). Responses to acetylcholine were assessed against the non-preferential muscarinic receptor antagonist, atropine, and the preferential antagonists pirenzepine (M,), methoctramine (M2) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3). 3 The potency of acetylcholine in inducing endothelium-dependent contraction was 6.54 ± 0.07 (EC5,,). Atropine, pirenzepine, methoctramine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent contraction to acetylcholine. The pA2 values for these muscarinic receptor antagonists were estimated from Arunlakshana-Schild plots to be (- logM) 9.48 ± 0.07, 6.74 ± 0.22, 6.30 ± 0.20 and 9.39 ± 0.22 respectively. The potency of acetylcholine in inducing endothelium- dependent relaxation was 7.82 ± 0.09 (ICW). Atropine, pirenzepine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent relaxation to acetylcholine but methoctramine had no effect. The pA2 values for atropine and 4-DAMP for the relaxation to acetylcholine were estimated from Arunlakshana-Schild plots to be (- log M) 9.15 ± 0.23 and 9.63 ± 0.28, respectively. These results suggest that the muscarinic M3 receptor subtype mediates both endothelium-dependent relaxation and contraction to acetylcholine in SHR aorta. Keywords: Endothelium-derived relaxing factor; endothelium-derived contracting factor; muscarinic receptors; spontaneously hypertensive rats (SHR) Introduction Endothelial cells contribute to the local regulation of thelium-dependent contractions to acetylcholine are not vasomotor tone by releasing dilator and constrictor sub- observed in the aorta of normotensive rats (Luscher & Van- stances. The relaxing substances which are generated by the houtte, 1986). In the SHR, they are abolished in the presence endothelium include endothelium-derived relaxing (EDRF; of inhibitors of cyclo-oxygenase; the EDCF involved may be identified as nitric oxide or a related nitrogen-containing an endoperoxide (Luscher & Vanhoutte, 1986; Auch-Schwelk compound) and hyperpolarizing (EDHF) factors (e.g. Furch- et al., 1990; Ito et al., 1991). Thus, acetylcholine elicits both gott & Zawadzki, 1980; Furchgott & Vanhoutte, 1989; endothelium-dependent contraction and relaxation in the Luscher & Vanhoutte, 1990; Moncada et al., 1991). Can- aorta of SHRs (Luscher & Vanhoutte, 1986). didates for endothelium-derived contracting substances in- In blood vessels, the release of EDRF evoked by acetyl- clude endoperoxides, thromboxane A2, superoxide anions choline is mediated via the activation of different muscarinic and possibly endothelins (e.g. Furchgott & Vanhoutte, 1989; receptors. The Ml subtype mediates endothelium-dependent Yanagisawa & Masaki, 1989; Luscher & Vanhoutte, 1990; relaxations in the canine femoral artery (Rubanyi et al., Bassenge & Heusch, 1990). In the aorta of the normotensive 1987), the bovine pial artery (Garcia-Villalon et al., 1991), rat, acetylcholine causes an endothelium-dependent relaxa- the rabbit saphenous artery (Komori & Suzuki, 1987) and tion, which is mediated via both the activation of a con- the rabbit pulmonary artery (Orphanos & Catravas, 1989). stitutive, calmodulin-dependent nitric oxide synthase enzyme Activation of the M2 subtype causes the release of EDRF in and the subsequent production of nitric oxide (Luscher & the bovine coronary artery (Duckles, 1988), the rabbit ear Vanhoutte, 1986; Schini & Vanhoutte, 1992), and the release artery (Hynes et al., 1986), the porcine cerebral artery (Van of EDHF (Chen et al., 1988). In the aorta of the adult Charldorp & Van Zwieten, 1989) and the canine femoral spontaneously hypertensive rat (SHR), low concentrations of artery (Rubanyi et al., 1987). The M3 muscarinic receptors acetylcholine (10-9 to 3 x 10-0M) induce an endothelium- mediate endothelium-dependent relaxation in pial arterioles dependent relaxation which is comparable to that observed in of mice (Shimizu et al., 1983), the rabbit ear artery (Duckles the aorta from normotensive control rats (Luscher & Van- & Garcia-Villalon, 1990), the rat pulmonary artery (McCor- houtte, 1986). However, relaxation to higher concentrations mack et al., 1988), the bovine coronary artery (Brunner et al., of acetylcholine (3 x 10-7 to 3 x I0- M) is attenuated in the 1991), the rabbit aorta (Jaiswal et al., 1991), the rabbit pial hypertensive strain, due to the concomitant release of an artery (Garcia-Villalon et al., 1991) and the cat cerebral endothelium-derived contracting factor (EDCF). Endo- artery (Dauphin & Hamel, 1990; Alonso et al., 1991). In addition, activation of different muscarinic receptor subtypes in the same preparation can cause the release of different ' Author for correspondence. endothelial vasoactive factors; indeed, in the rabbit 520 C.M. BOULANGER et al. saphenous artery, the release of EDRF and that of and to optimize the endothelium-dependent contractile res- endothelium-derived hyperpolarizing factor are mediated by ponse (Ito et al., 1991; Auch-Schwelk et al., 1992). The activation of M2 and Ml subtypes, respectively (Komori & response to acetylcholine was investigated under control con- Suzuki, 1987). ditions and in the presence of increasing concentrations of In the SHR aorta, the endothelium-dependent contraction the muscarinic antagonists. Only one concentration of to acetylcholine is blocked by the non-preferential muscarinic antagonist was studied per tissue. antagonist, atropine, but not by the nicotinic receptor Endothelium-dependent relaxation to acetylcholine was antagonist, hexamethonium (LUscher & Vanhoutte, 1986). studied in aortic rings, with endothelium, in the presence of Furthermore, the release of EDCF occurs in response to indomethacin (10-5M; 40 min; to prevent the formation of higher concentrations of acetylcholine than are required to vasoactive prostanoids). Responses to acetylcholine were elicit the release of EDRF; this suggests that different sub- measured in tissues which were contracted with phenyle- types of muscarinic receptors may mediate the two responses. phrine (3 x 10-8 M to 3 x 10-7 M; to match the level of con- The present series of experiments used preferential mus- traction between preparations) under control conditions and carinic receptor antagonists to characterize the muscarinic in the presence of increasing concentrations of the muscarinic receptor subtype(s) that mediate(s) the endothelium- antagonists. Only one concentration of antagonist was dependent contraction and relaxation in response to acetyl- studied per tissue. choline in the aorta from SHR. Drugs Methods The following drugs were used: acetylcholine HCl, atropine sulphate, indomethacin, phenylephrine, thrombin (Sigma Organ bath experiments Chemical, St. Louis, MO, U.S.A.), N0-nitro-L-arginine (Ald- rich Chemical Company, Milwaukee, WI, U.S.A.); eco- Experiments were performed on thoracic aortae from male thiopate iodide (as phospholine iodide, 1.8% ophthalmic SHR (8-10 months old; weight 350-412 g; Harlan Sprague solution, St Lukes Hospital Pharmacy, Houston, TX, Dawley, Indianapolis, IN, U.S.A). All procedures using U.S.A.); pirenzepine, methoctramine and 4-diphenylacetoxy- animals were in accordance with the guidelines of the Animal N-methylpiperidine methiodide (4-DAMP) (Research Protocol Review Committee of Baylor College of Medicine. Biochemical Inc., Natick, MA, U.S.A.). Drug concentrations Systolic arterial blood pressure was measured by the tail cuff are expressed as final molar concentrations in the bath solu- method, and averaged 213 ± 6 mmHg (n = 41). The rats were tion. Drugs were prepared in distilled water, except anaesthetized with pentobarbitone sodium (50 mg kg-', indomethacin which was dissolved in distilled water contain- intraperitoneally). The thoracic aorta was dissected free, ing NaCO3 (3 x 10-5 M) and sonicated before use. A stock excised, and placed in cold modified Krebs-Ringer bicar- solution of ecothiopate was prepared in sterile diluent (com- bonate solution of the following composition (mM): NaCl position: chlorobutanol, 0.55%; mannitol, 1.2%; boric acid, 118.3, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaC12 2.5, NaHCO3 0.06% and sodium phosphate, 0.026%). Subsequent dilutions 25.0, calcium disodium edetate (EDTA) 0.026, and glucose were made in control solution. 11.1 (control solution). The blood vessels were cleaned of adherent connective tissue and cut into rings (4-5 mm long).