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Functional characterization of muscarinic receptors in rat duodenum
1038
Abstracts
Vol. 56, No.s 11112, 1995
73 FUNCTIONAL CHABACTEBIZATION RABBIT ANOCOCCYGEUS MUSCLE
OF
NEUBONAL
MUSCAIUNIC
BECEPTOBS
IN
J. Gross, M. Waelbroeck*, E. Mutschler, and G. Lambrecht. Dept. of Pharmacology, Univ. of D-60439 Frankfurt Main, Germany, *Dept. of Biochemistry and Nutrition, Medical School, Free Univ. of B-1070 Brussels, Belgium. The rabbit anococcygeus muscle (RAM) is innervated by excitatory sympathetic and inhibitory non-adrenergic, noncholinergic (?WNC) nerves (Graham and Sneddon, EJP 237: 93, 1993). The aim of the present study was to characterize the muscarinic receptor subtypes mediating NANC-relaxation (Creed et al., J. Physiol. 273: 121, 1979) and to investigate whether the noradrenaline (NA)-induced neurogenic contractions can be affected by muscarinic agonists and subtype-prefering antagonists. a) NANC-relaxations: In preparations with histamine- (2 pM) raised tone the cumulative addition of the muscarinic agonist (+)-muscarine (EC59 = 6.7 pM) elicited relaxations (isometric recording). These relaxations were sensitive to tetrodotoxin (TTX; 1 pM) and to the NO-synthase inhibitor Nw-nitroL-arginine (NO-Arg; 100 pM) indicating that NO plays an important role as the inhibitory NANC-transmitter. b) Muscarinic modulation of noradrenergic tt&mission: Electrical field stimulation (1 Hz, 0.5 ms, 35 V, for 45-240 s; applied every 15 min) induced TTX- (1 pM) and prazosin- (IC59 = 2.8 nM) sensitive contractions (isotonic recording). These effects were dose-dependently inhibited by the muscarinic agonist arecaidine propargyl ester (EC50 = 22.4 t&I). NO-Arg (300 pM) was present throughout the experiments to isolate NA-induced neurouenic contractions. These results demonstrate the existence of prejunctional inhibitory muscarinic receptors on sympathetic nerves in RAM. Muscarinic 1 pA2 values In both experimental protocols [a) and b)], all antagonists displayed a antagonist a) W simple competitive antagonism, e.g. Schild-plots were linear and the Pirenzepine ( 7.14 1 6.46 slopes were not significantly different from unity (P > 0.05). A comparison of the pA2-values obtained in RAM (see table) with the binding affinities at Ml - M4 receptors (see F%ffet al., this volume) suggests that the functional muscarinic receptors mediating NANC-relaxation and heteroinhibition of noradrenaline release in RAM are of the M4 and M2 subtypes, respectively.
74 FUNCTIONAL
CELARACTERIZ A TION OF MUSCAIUNIC
RECEPTORS
IN BAT DUODENUM
0. Pfaff, J. Gross, M. Waelbroeck*, E. Mutschler and G. Lambrecht. Dept. of Pharmacology, Univ. of D-60439 Frankt%/Main, Germany; *Dept. of Biochemistry and Nutrition, Medical School, Free Univ. of B-1070 Brussels, Belgium It has been reported that muscarinic receptors mediating relaxations in rat duodenum via activation of non-adrenergic, non-cholinergic (NANC) neurons are of the Ml or M4 subtype (Micheletti et al., BJP 100: 395, 1990). Based on the results obtained in binding studies, Liebmann et al. (NSAF 345: 7, 1992) suggested that the rat duodenal smooth muscle contains a heterogeneous population of Ml, M2 and M4, but not of M3 muscarinic receptors. However, the postjunctional muscarinic receptor mediating contraction of rat duodenum has not been characterized so far. Thus, the aim of the present study was to characterize the receptor subtypes responsible for the above mentioned functional responses by the use of a battery of subtype-prefering antagonists. Tetrodotoxin-sensitive (3pM) relaxations of intact rat duodenum segments to single doses of the Ml-selective agonist 4-(4-fluorophenylcarbamoyloxy)-2-butynyl-N-methyl-pyrrolidinium tosylate (4-F-PyMc#; EC59: 790 nM) were measured isotonically. Tetrodotoxin-insensitive (3pM) contractile responses to cumulative addition of arecaidine propargyl ester (APE; EC59: 74 nM) were studied on longitudinal muscle strips. The calculated pA2-values of the antagonists investigated (evaluation by Schild analysis; slopes not different from unitv) and the pK;-values obtained in binding studies at Ml (NB-OK 1 cells), M2 (rat heart), M3 (rat pancreas) and M4 receptors (rat &iatum) are listed in the table below. A comparison of the functional Muscarinic pA2-values PKi-values antagonists relaxation contraction Ml pA2 values obtained in rat M2 Jvl3 M4 Pirenzqine 7.90 1 6.89 8.3 6.5 6.8 7.1 duodenum with the binding AQ-R.4741 7.40 7.26 7.8 8.5 6.6 8.2 affinities at Ml - M4 receptors p-F-HHSiD 7.37 7.83 7.8 6.5 7.8 7.8 clearly demonstrates that the Himbacine 6.77 7.33 7.1 8.0 6.5 8.1 postjunctional receptor mediaGuanylpirenzqine 7.31 7.6 5.4 6.0 6.2 ting contraction in rat duodenum is of the M3 subtype (r = 0.96) g;:= 8.47 6.20 6.1 8.3 5.8 7.0 5.9 8.1 5.9 7.9 whereas the receptor mediating Atropine 9.24 9.5 9.2 8.7 9.5 NANC relaxation is of the Ml (S>Dimethindene 6.36 7.1 7.8 6.7 7.0 subtype (r = 0.99). (R)-Dimethindene 5.49 6.0 6.3 5.6 6.0 Tripitramine 8.33 7.60 8.801 9.571 7.42j 8.19l Supp. by DFG. ‘Maggie et al., Eur. J. Pharmacol. - Mol. Pharmacol. Sect. 268: 3, 1994
Abstracts
Vol. 56, No.s 11112, 1995
73 FUNCTIONAL CHABACTEBIZATION RABBIT ANOCOCCYGEUS MUSCLE
OF
NEUBONAL
MUSCAIUNIC
BECEPTOBS
IN
J. Gross, M. Waelbroeck*, E. Mutschler, and G. Lambrecht. Dept. of Pharmacology, Univ. of D-60439 Frankfurt Main, Germany, *Dept. of Biochemistry and Nutrition, Medical School, Free Univ. of B-1070 Brussels, Belgium. The rabbit anococcygeus muscle (RAM) is innervated by excitatory sympathetic and inhibitory non-adrenergic, noncholinergic (?WNC) nerves (Graham and Sneddon, EJP 237: 93, 1993). The aim of the present study was to characterize the muscarinic receptor subtypes mediating NANC-relaxation (Creed et al., J. Physiol. 273: 121, 1979) and to investigate whether the noradrenaline (NA)-induced neurogenic contractions can be affected by muscarinic agonists and subtype-prefering antagonists. a) NANC-relaxations: In preparations with histamine- (2 pM) raised tone the cumulative addition of the muscarinic agonist (+)-muscarine (EC59 = 6.7 pM) elicited relaxations (isometric recording). These relaxations were sensitive to tetrodotoxin (TTX; 1 pM) and to the NO-synthase inhibitor Nw-nitroL-arginine (NO-Arg; 100 pM) indicating that NO plays an important role as the inhibitory NANC-transmitter. b) Muscarinic modulation of noradrenergic tt&mission: Electrical field stimulation (1 Hz, 0.5 ms, 35 V, for 45-240 s; applied every 15 min) induced TTX- (1 pM) and prazosin- (IC59 = 2.8 nM) sensitive contractions (isotonic recording). These effects were dose-dependently inhibited by the muscarinic agonist arecaidine propargyl ester (EC50 = 22.4 t&I). NO-Arg (300 pM) was present throughout the experiments to isolate NA-induced neurouenic contractions. These results demonstrate the existence of prejunctional inhibitory muscarinic receptors on sympathetic nerves in RAM. Muscarinic 1 pA2 values In both experimental protocols [a) and b)], all antagonists displayed a antagonist a) W simple competitive antagonism, e.g. Schild-plots were linear and the Pirenzepine ( 7.14 1 6.46 slopes were not significantly different from unity (P > 0.05). A comparison of the pA2-values obtained in RAM (see table) with the binding affinities at Ml - M4 receptors (see F%ffet al., this volume) suggests that the functional muscarinic receptors mediating NANC-relaxation and heteroinhibition of noradrenaline release in RAM are of the M4 and M2 subtypes, respectively.
74 FUNCTIONAL
CELARACTERIZ A TION OF MUSCAIUNIC
RECEPTORS
IN BAT DUODENUM
0. Pfaff, J. Gross, M. Waelbroeck*, E. Mutschler and G. Lambrecht. Dept. of Pharmacology, Univ. of D-60439 Frankt%/Main, Germany; *Dept. of Biochemistry and Nutrition, Medical School, Free Univ. of B-1070 Brussels, Belgium It has been reported that muscarinic receptors mediating relaxations in rat duodenum via activation of non-adrenergic, non-cholinergic (NANC) neurons are of the Ml or M4 subtype (Micheletti et al., BJP 100: 395, 1990). Based on the results obtained in binding studies, Liebmann et al. (NSAF 345: 7, 1992) suggested that the rat duodenal smooth muscle contains a heterogeneous population of Ml, M2 and M4, but not of M3 muscarinic receptors. However, the postjunctional muscarinic receptor mediating contraction of rat duodenum has not been characterized so far. Thus, the aim of the present study was to characterize the receptor subtypes responsible for the above mentioned functional responses by the use of a battery of subtype-prefering antagonists. Tetrodotoxin-sensitive (3pM) relaxations of intact rat duodenum segments to single doses of the Ml-selective agonist 4-(4-fluorophenylcarbamoyloxy)-2-butynyl-N-methyl-pyrrolidinium tosylate (4-F-PyMc#; EC59: 790 nM) were measured isotonically. Tetrodotoxin-insensitive (3pM) contractile responses to cumulative addition of arecaidine propargyl ester (APE; EC59: 74 nM) were studied on longitudinal muscle strips. The calculated pA2-values of the antagonists investigated (evaluation by Schild analysis; slopes not different from unitv) and the pK;-values obtained in binding studies at Ml (NB-OK 1 cells), M2 (rat heart), M3 (rat pancreas) and M4 receptors (rat &iatum) are listed in the table below. A comparison of the functional Muscarinic pA2-values PKi-values antagonists relaxation contraction Ml pA2 values obtained in rat M2 Jvl3 M4 Pirenzqine 7.90 1 6.89 8.3 6.5 6.8 7.1 duodenum with the binding AQ-R.4741 7.40 7.26 7.8 8.5 6.6 8.2 affinities at Ml - M4 receptors p-F-HHSiD 7.37 7.83 7.8 6.5 7.8 7.8 clearly demonstrates that the Himbacine 6.77 7.33 7.1 8.0 6.5 8.1 postjunctional receptor mediaGuanylpirenzqine 7.31 7.6 5.4 6.0 6.2 ting contraction in rat duodenum is of the M3 subtype (r = 0.96) g;:= 8.47 6.20 6.1 8.3 5.8 7.0 5.9 8.1 5.9 7.9 whereas the receptor mediating Atropine 9.24 9.5 9.2 8.7 9.5 NANC relaxation is of the Ml (S>Dimethindene 6.36 7.1 7.8 6.7 7.0 subtype (r = 0.99). (R)-Dimethindene 5.49 6.0 6.3 5.6 6.0 Tripitramine 8.33 7.60 8.801 9.571 7.42j 8.19l Supp. by DFG. ‘Maggie et al., Eur. J. Pharmacol. - Mol. Pharmacol. Sect. 268: 3, 1994