Roles of M2 and M3 muscarinic receptors in the modulation of β-adrenoceptor-mediated relaxation of the rat urinary bladder

Lambertus P.W. Witte1, Noach de Haas1, Mathai Mammen2, Eric L. Stangeland2, Jayashree Aiyar2, Martin C. Michel1

1AMC, Amsterdam, Netherlands, 2Theravance, South San Francisco, USA

β-Adrenoceptor stimulation is the most important mechanism to induce bladder smooth muscle relaxation. β-Adrenoceptor-mediated bladder relaxation is less potent and/or effective if tone had been induced by muscarinic receptor stimulation (Longhurst and Levendusky, 1995). The bladder expresses more M2- than M3-receptors, but bladder contraction occurs predominantly if not exclusively via M3-receptors. Therefore, we have explored the roles of M2- and M3- receptors in the impairment of β-adrenergic bladder relaxation and the signalling mechanisms involved in such interaction.

Isoprenaline concentration-response curves for relaxation of isolated bladder strips from male Wister rats (weight 300 ± 22 g, strip length 19.9 ± 3.6 mm, strip weight 9.5 ± 2.7 mg) were generated against passive tension (10 mN) or tone induced by the following conditions: KCl (50 mM), carbachol (1 μM), M3-selective stimulation (carbachol in presence of the M2-selective antagonist THRX-182,087 [N-(3-{(R)-1-[1-(1H-Imidazole-4-carbonyl)-piperidin-4-ylmethyl]-piperidin-2-ylmethyl}-phenyl)-4-methoxy-benz-amide]), M2-selective stimulation (M3-sparing agonist THRX-199,874 [4-(2-Oxo-2,3-dihydro-benzimidazol-1-yl)-1,4’-bipiperidinyl-1’-carboxylic acid ethyl ester]). M3-selective stimulation was also tested in the presence of phospholipase C inhibition (10 μM U 73,122 [1-(6-[(17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione]) and protein kinase C inhibition (1 μM chelerythrine), M2-selective stimulation in the presence of an adenylyl cyclase inhibitor (1 μM SQ 22,536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine]). Data are mean ± S.E.M. of at least 6 paired strips, and a p < 0.05 (t-test or ANOVA) was considered significant.

Carbachol significantly reduced the efficacy (but not potency) of isoprenaline relaxation as compared to KCl-induced tension (Emax 50±2% vs. 31±2%, p<0.05). While selective M2 stimulation did not significantly enhance basal tension, it attenuated the potency of isoprenaline to induce relaxation (pEC50 7.7±0.1 vs. 8.1±0.1, p<0.05). Selective M3-stimulation caused similar tension as carbachol alone, but less attenuation of the isoprenaline response (Emax 51±3% vs. 40±3%, pEC50 7.4±0.1 vs. 7.6±0.1, both p<0.05).

During M3-stimulation, phospholipase C inhibition partly reversed the attenuation of the isoprenaline response whereas protein kinase C inhibition did not alter the isoprenaline response. In the presence of adenylyl cyclase inhibition, M2 stimulation reduced the potency but concomitantly enhanced the efficacy of the isoprenaline response (possibly related to a small increase in starting tension).

We conclude that rat bladder contraction mostly occurs via M3 receptors, whereas both M2 and M3 receptors contribute to attenuation of the isoprenaline response. While phospholipase C stimulation does not contribute to the contraction response, it does contribute to the attenuation of isoprenaline-induced relaxation. The role of adenylyl cyclase inhibition in the M2 receptor component is difficult to interpret based on our data. These data show that receptor subtypes and signalling pathways involved in attenuation of relaxation partly differ from those involved in causing rat bladder contraction by muscarinic agonists.

Longhurst PA and Levendusky M (1995) Br. J. Pharmacol. 127: 1744-1750