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 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, , and the preferential antagonists (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 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 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). Statistical analysis In some preparations, the endothelium was removed by gently rubbing the intimal surface with a small forceps. In Experiments with each muscarinic antagonist were performed the remaining rings, care was taken not to touch the inner on rings from the same animals studied in parallel. surface of the blood vessel. The presence or absence of In the experiments in which concentration-contraction functional endothelial cells was confirmed by the presence or curves to acetylcholine were determined in quiescent prepara- absence of relaxation in response to thrombin (1 u ml-'), tions (endothelium-dependent contraction), the results of respectively (Luscher & Vanhoutte, 1986). each experiment are expressed as percentage of the maximal The rings were suspended horizontally between two response to acetylcholine (10-5 M) observed in control stainless steel wires in organ baths, which contained 25 ml of preparations. EC50 represents the negative logarithm of the control solution (37°C) aerated with 95% 02 5% CO2. The concentration of acetylcholine that elicits fifty percent (50%) preparations were connected to force transducers (Statham of its maximal response (measured at 10- M). Universal UC2 or Grass FT 03C, Quincy, MA, U.S.A.) for In the experiments in which endothelium-dependent relaxa- recording of changes in isometric tension. Prior to tion to acetylcholine was studied, aortic rings were con- experimentation, the preparations were stretched progres- tracted with equi-effective concentrations of phenylephrine sively and exposed to phenylephrine (3 x 1O-7M) at each (3 x 10-8 to 3 x 10-7 M) [mean contractile response level of force until the optimal point of the length-active 1.6 ± 0.1 g; n = 24]. For each preparation, the relaxation is force relationship was reached. After the procedure the rings expressed as percentage inhibition of the contraction to were allowed to equilibrate for 40 min. All rings were then phenylephrine. ICm represents the negative logarithm of the exposed to phenylephrine (3 X 10- M) to determine maximal concentration of acetylcholine that induces fifty percent responsiveness. Experiments were performed on parallel rings (50%) inhibition of the contraction to phenylephrine with endothelium, in the presence of ecothiopate (3 x 10-6 M) (3 x 10-8 to 3 x 10-7 M). to prevent degradation of acetylcholine by acetyl- pA2 values (estimates of the equilibrium dissociation con- . Ecothiopate did not affect the endothelium- stants) for the muscarinic receptor antagonists were deter- dependent responses to acetylcholine in this preparation mined from graphs of log concentration-ratios minus 1 (data not shown). Responses to acetylcholine were assessed (CR - 1) versus log concentration of antagonist (Arunlak- against the non-preferential muscarinic receptor antagonist, shana & Schild, 1959). CR is defined as the concentration of atropine, and the preferential antagonists, pirenzepine (Ml), agonist required to induce 50% maximal response in the methoctramine (M2) and 4-diphenylacetoxy-N-methylpiperi- presence of antagonist divided by the agonist concentration dine methobromide (4-DAMP; M3). Unless otherwise stated, that elicits the same degree of response in the absence of the incubation period with the different antagonists was antagonist. The pA2 values were calculated if the slope of the 40 min. plot was not significantly different from unity. When endothelium-dependent contraction to acetylcholine Results are given as means ± s.e.mean. Statistical evalua- was investigated, aortic rings with endothelium were tion was done by Student's t test for paired or unpaired incubated with NG-nitro-L-arginine (10-4 M, 20 min) to pre- observations. When more than two means were compared, vent the formation of nitric oxide (Schini & Vanhoutte, 1992) two-way analysis of variance (ANOVA) was used. Means ENDOTHELIAL MUSCARINIC RECEPTORS IN SHR 521 were considered significantly different when P was less than acetylcholine (10-a M) was 12.0 ± 1% of that to phenyle- 0.05. phrine (3 X I0-5 M) (n = 7; P < 0.05). Atropine (3 X I0-9 M-3 X I0-7 M) caused a parallel right- ward displacement of the concentration-response curve to Results acetylcholine. The response to acetylcholine (10-4 M) was reduced in preparations incubated in the presence of atropine (3 x I0-8 and 3 x I0-7 M) (Figure 1). Pirenzepine (10-6 M- Contraction IO- M) (Figure 1), methoctramine (10-6 M-10-5 M) (Figure Acctylcholine induced concentration- and endothelium- 2) and 4-DAMP (10-9 M- I0- M) (Figure 2) caused parallel dependent contraction of rings of aorta from SHR treated dextral shifts in the concentration-contraction curve (at the with Nt-nitro-L-arginine (10-4 M; 20 min) (Figure 1). The linear portion of the curve) to acetylcholine, with no change EC50 value for acetycholine for contraction under control in the maximal response. conditions was 6.54 ± 0.07 (n = 12). The contraction evoked The pA2 values for atropine, pirenzepine, methoctramine by acetylcholine was 60.6 ± 4.1% of that induced by and 4-DAMP for contraction to acetylcholine were (- log M) phenylephrine (3 x 10-5 M; 4.0 ± 0.4 g, n = 7), whereas in 9.48 ± 0.07, 6.74 ± 0.22, 6.30 ± 0.20 and 9.39 ± 0.22, respec- rings denuded of endothelium, the maximal contraction to tively (Table 1). The effect of 4-DAMP on endothelium-

a a 120 - a) 120 a) c c ._ ._ 0 100 100

._ 4) a) - 0 80 C._ 80 m 0 Q 0 +o a) 0 60 - 60 4- EC.) ._c 0 40 - C._ 40 C aL) 0a) x E 20 - E 20 E 0 4 F 0

-7 -6 -5 -4 -3 Acetylcholine (log M) Acetylcholine (log M)

b b 1)f - 120 a) a)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~O C .C 100 .) 100 a) /~~~~~~t a) 80 7 .) 80 - 0

0 Q 0 60 0 60

C 0x 40 a) 0 .5 40 .75C.) E 20 a.) 20 E E

0 -7 -6 -5 -4 -3 Acetylcholine (log M) Acetylcholine (log M)

Figure 1 Effect of the non-preferential muscarinic antagonist Figure 2 Effect of the preferential M2 muscarinic antagonist muscarinic antagonist piren- methoctramine (a) and the preferential M3 muscarinic antagonist atropine (a) and the preferential Ml evoked zepine (b) on the endothelium-dependent contraction evoked by 4-DAMP (b) on the endothelium-dependent contraction by acetylcholine in the SHR rat aorta in the presence of N0-nitro-L- acetylcholine in the SHR rat aorta in the presence of N0-nitro-L- arginine (10-4 M). The experiments were performed on parallel rings arginine (10-4 M). The experiments were performed on parallel rings with endothelium under control conditions (@) and in the presence with endothelium, under control conditions (-) and in the presence x M 3 x 10- M of either atropine (a: 3 x 10-9 M (A), 3 x 10-8 M (0), 3 x 10-7 M of either methoctramine (a: 3 10-' (A), (0), M 10-9 M 10-8 M (0), 10-7 M (0)) or pirenzepine (b: 106 M (A), 3 x 10-6 M (0), 10-5 M (0)). 3 x 10-1 (0)) or 4-DAMP (b: (A), ± are as The data are given as mean ± s.e.mean and are expressed as percen- (0)). The data are given as mean s.e.mean and expressed tage of the maximal contraction to acetylcholine (10-5 M) obtained percentage of the maximal contraction to acetylcholine (10-' M) under control conditions (experiments with atropine: 2.0 ± 0.3 g, obtained under control conditions (experiments with methoctramine: ± = 4-DAMP: 2.6 ± 0.3, n = 6). n = 6; experiments with pirenzepine: 2.0 ± 0.3 g, n = 6). 2.8 0.4 g, n 7; experiments with 522 C.M. BOULANGER et al.

dependent contraction to acetylcholine was evaluated after (10-6M- 10-5 M) (Figure 3) and 4-DAMP (10-9- 10-8 M) 60 min incubation with the antagonist. Indeed, in a previous (Figure 4) caused parallel dextral shifts in the concentration- set of experiments performed at 40 min incubation, the slope relaxation curve (at the linear portion of the curve) to acetyl- of the Arunlakshana-Schild plot was significantly greater choline, with no change in the maximal response. Methoctra- than unity, suggesting non-equilibrium conditions. mine (10-6 M-10-5 M) (Figure 4) did not significantly affect the relaxation to acetylcholine. The ICm values for acetyl- choline under control conditions and in the presence of Relaxation methoctramine (10-6M, 3 x 10-6 M and 10-5 M) were 7.79 ± 0.13, 7.41 ± 0.13, 7.48 ± 0.12 and 7.28 ± 0.15, respec- Acetylcholine induced an endothelium-dependent relaxation tively. of rings of aorta from SHR treated with indomethacin The pA2 values for the muscarinic antagonists for relaxa- (10-5 M; 45 min) (Figure 3). The ICm value for acetylcholine tion to acetylcholine are shown in Table 1. A pA2 value for for relaxation under control conditions was 7.82 ± 0.09 pirenzepine was not calculated because the slope of the (n= 12). Arunlakshana-Schild plot was significantly greater than unity Atropine (3 X 10-9 M-3 x 10-7 M) (Figure 3), pirenzepine (Table 1).

a a 120 120

100 100

80 80 c c 0 0 Co 60 .F_Co x x 60 Lo 40 0- I0-0 40

20 20

0

-9 -8 -7 -6 -5 Acetylcholine (log M) Acetylcholine (log M) b

a c 0 0 '._Co co x Co x -o

0- 0I-R

Acetylcholine (log M) Acetylcholine (log M) Figure 3 Effect of the non-preferential muscarinic antagonist Figure 4 Effect of the preferential M2 muscarinic antagonist atropine (a) and the preferential Ml muscarinic antagonist methoctramine (a) and the preferential M3 muscarinic antagonist pirenzepine (b) on the endothelium-dependent relaxation evoked by 4-DAMP (b) on the endothelium-dependent relaxation evoked by acetylcholine in the SHR rat aorta in the presence of indomethacin acetylcholine in the SHR rat aorta in the presence of indomethacin (10-5M). The experiments were performed on parallel rings with (10- Nm). The experiments were performed on parallel rings with endothelium under control conditions (@) and in the presence of endothelium under control conditions (0) and in the presence of either atropine (a: 3 x 10-9 M (A), 3 x 10-8 M (0), 3 x 10-' M (0)) either methoctramine (a: 10-6 M (A), 3 x 10-6 M (0), 10-S M (0)) or pirenzepine (b: 10-6 M (A), 3 x 10-6 M (0), 10-S M (0)). The or 4-DAMP (b: 10-9M (A), 3 x 10-9 M (0), 10-8 M (0)). The data data are given as mean ± s.e.mean and are expressed as percentage are given as mean ± s.e.mean and are expressed as percentage of the inhibition of the contraction to phenylephrine (3 x 10-8 M to contraction to phenylephrine (3 X 10-8 M to 3 x 10-7 M). In the set 3 x 10-7 M). In the experiments with atropine (a), the contraction to of experiments involving methoctramine (a), the contraction induced phenylephrine averaged 1.6 ± 0.1 g (control), 1.4 ± 0.1 g (atropine by phenylephrine averaged 1.5 ± 0.2 g (control), 1.8 ± 0.3 g (meth- 3 x 10-9 M), 1.5 ± 0.1 g (atropine 3 X 10-1 M) and 1.7 ± 0.2 g octramine 10-6M), 1.4 ± 0.3 g (methoctramine 3 X 10-6M) and (atropine 3 x 10-7 M) (n = 6). In the set of experiments involving 1.4 ± 0.2 g (methoctramine 1O-S M) (n = 6). In the experiments with pirenzepine (b), the contraction evoked by phenylephrine averaged 4-DAMP (b), the contraction evoked by phenylephrine averaged 1.6 ± 0.1 g (control), 1.8 ± 0.1 g (pirenzepine 10-6 M), 1.4 ± 0.1 g 1.5 ± 0.2 g (control), 1.7 ± 0.3 g (4-DAMP 10-9 M),1.4 ± 0.2 g (4- (pirenzepine 3 x 10-6M) and 1.9 ± 0.3 g (pirenzepine 10-5 M) (n = 6). DAMP 3 X 109Mm) and 1.5±0.2g (4-DAMP 10-8m) (n=6). ENDOTHELIAL MUSCARINIC RECEPTORS IN SHR 523

Table 1 pA2 values (mean ± s.e.mean of 6 observations) ramine is lower than that reported for an homologous for muscarinic antagonist versus acetylcholine in the SHR population of M2 muscarinic receptors (about 7.9; Michel & aorta (rings with endothelium) Whiting, 1988; Duckles & Garcia-Villalon, 1990; 1993 Recep- pA2 tor Nomenclature). The conclusion that M3 muscarinic recep- Antagonist Effect (- log M) Slope tors mediate endothelium-dependent relaxation to acetyl- choline is prompted also by the fact that pirenzepine only Atropine Relaxation 9.15 ± 0.23 1.04 affects the relaxations to acetylcholine at concentrations Contraction 9.48 ± 0.07 0.99 which are at least one hundred times higher than the Pirenzepine Relaxation 1.71* reported affinity of the antagonist for Ml receptors (pA2 8.00; Contraction 6.74 ± 0.22 1.19 1993 Receptor Nomenclature). Furthermore, methoctramine, Methoctramine Relaxation a preferential antagonist of muscarinic M2 receptors, does Contraction 6.30 ± 0.20 0.83 not affect endothelium-dependent relaxation to acetylcholine, 4-DAMP Relaxation 9.63 ± 0.28 1.02 even at higher concentrations. Contraction 9.39 ± 0.22 0.99 Methoctramine displayed different effects on endothelium- These parameters were obtained from Arunlakshana-Schild dependent contraction and relaxation to acetylcholine in the plot analysis when the slope of the plot was not different SHR aorta. Indeed, endothelium-dependent contractions to from unity. The asterisk denotes a slope which is acetylcholine were reduced by methoctramine, while significantly different from unity. endothelium-dependent relaxation was not. Both the release of EDCF and the direct contractile effect of acetylcholine on smooth muscle contribute to the endothelium-dependent con- traction in the present experiments. Although the direct effect Discussion of acetycholine on preparations without endothelium is too small to allow proper pharmacological identification of the The present results confirm that acetylcholine causes both receptor subtype involved, its antagonism by methoctramine endothelium-dependent relaxation and contraction in the could be responsible for the rightward shift of the aorta from SHR. These effects are mediated by activation of concentration-dependent contraction to acetylcholine by the muscarinic receptors since atropine, a non-preferential mus- preferential M2 muscarinic antagonist, while the release of carinic antagonist, displayed an 'apparent' competitive EDCF would be mediated by activation of a M3 receptor. antagonism of the responses to acetylcholine. The muscarinic However, the present experiments do not rule out the pos- receptor subtype(s) that mediate(s) the endothelium- sibility that different subtypes of muscarinic M3 receptors dependent contraction and relaxation to acetylcholine was mediate endothelium-dependent relaxation and contraction characterized by use of preferential muscarinic receptor to acetylcholine in the SHR aorta. This possibility is further antagonists. Using functional studies, three muscarinic recep- supported by the fact that the release of the contractile factor tor subtypes have been identified on endothelial cells: the Ml occurs in response to higher concentrations of acetylcholine subtype (Komori & Suzuki, 1987; Orphanos & Catravas, than are required to elicit the release of EDRF (Luscher & 1989; Garcia-Villalon et al., 1991), the M2 subtype (Hynes et Vanhoutte, 1986). al., 1986; Duckles, 1988) and the M3 subtype (McCormack et The endothelium-dependent relaxation mediated by activa- al., 1988; Duckles & Garcia-Villalon, 1990; Dauphin & tion of muscarinic M3 receptors is well documented in mice Hamel, 1990; Brunner et al., 1991; Jaiswal et al.. 1991; and rabbit pial arteries, the rabbit aorta, the bovine coronary Garcia-Villalon et al., 1991; Alonso et al., 1991; Shimizu et artery, the rat pulmonary artery and the cat cerebral artery al., 1993). Each muscarinic receptor subtype may contribute (McCormack et al., 1988; Dauphin & Hamel, 1990; Duckles to the local control of vascular reactivity. & Garcia-Villalon, 1990; Alonso et al., 1991; Brunner et al., Endothelium-dependent contraction to acetylcholine was 1991; Garcia-Villalon et al., 1991; Jaiswal et al., 1991; obtained in the presence of N0-nitro-L-arginine to negate the Shimizu et al., 1993). The results in these as well as the SHR contribution of endothelium-derived nitric oxide (Ito et al., rat aorta are at variance with those obtained in blood vessels 1991; Schini & Vanhoutte, 1992; Auch-Schwelk et al., 1992). such as the bovine pial artery (Garcia-Villalon et al., 1991), Endothelium-dependent relaxation to acetylcholine was the rabbit saphenous artery (Komori & Suzuki, 1987), the obtained in the presence of indomethacin, to prevent the rabbit pulmonary artery (Orphanos & Catravas, 1989) and formation of vasoactive prostanoids and to abolish the effect the canine femoral artery (Rubanyi et al., 1987) where the Ml of endothelium-derived contracting factor (Luscher & Van- subtype mediates endothelium-dependent responses to acetyl- houtte, 1986). Both responses to acetylcholine appear to be choline. Other endothelium-dependent responses to acetyl- mediated by activation of the muscarinic M3 receptor sub- choline are caused by activation of the M2 subtype such as in type. This conclusion is based on the fact that 4-DAMP, a the bovine coronary artery (Duckles, 1988), the rabbit ear preferential antagonist of muscarinic M3 receptors, displays artery (Hynes et al., 1986), the porcine cerebral artery (Van competitive antagonism of the endothelium-dependent con- Charldorp & Van Zwieten, 1989) and the canine femoral traction and relaxation to acetylcholine and yields for both arteries (Rubanyi et al., 1987). This may reflect species responses a pA2 value that is consistent with activation of an heterogeneity, which is common in mammalian blood vessels homologous population of muscarinic M3 receptors (McCor- (LUscher & Vanhoutte, 1990). mack et al., 1988; Michel et al., 1989; Alonso et al., 1991; In conclusion, the present results suggest that the release of 1993 Receptor Nomenclature). Although certain endothe- both endothelium-derived contracting (EDCF) and relaxing lium-dependent responses to acetylcholine are mediated by (EDRF) factors upon stimulation with acetylcholine is activation of Ml or M2 receptors (Rubanyi et al., 1987; mediated by activation of M3 muscarinic receptors in the Komori & Suzuki, 1987; Orphanos & Catravas, 1989; SHR aorta. Garcia-Villalon et al., 1991), this does not appear to be the case in the SHR aorta. For example, the pA2 value of pirenzepine for endothelium-dependent contraction to acetyl- choline is consistent with activation of both M2 and M3 The authors wish to thank Barnabas Desta, Nusret Didzik and muscarinic receptor subtypes (range from 5.6 to 7.0; Eglen et Shawn Latta for their technical assistance and Joanna Bale for al., 1989; Fuder et al., 1989; Van Charldorp & Van Zwieten, secretarial help. This work was supported, in part, by a grant from 1989; range 6.4 to 7.6; Duckles, 1988; Van Charldorp & Van the National Institute of Health (HL 35614) and a Grant-in-Aid Zwieten, 1989; Mei et al., 1989), but the pA2 .for methoct- from the 'Institut de Recherches Internationales Servier', Paris (F). 524 C.M. BOULANGER et al.

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