Abstract

We have reexamined the muscarinic receptor subtype mediating carbachol-induced contraction of rat urinary bladder and investigated the role of phospholipase (PL)C, D, and A₂ and of intra- and extracellular Ca²⁺ sources in this effect. Based on the nonsubtype-selective tolterodine, the highly M₂ receptor-selective (R)-4-{2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl}-piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M₃ receptor-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M₃ receptors. Carbachol stimulated inositol phosphate formation in rat bladder slices, and this was abolished by the phospholipase C inhibitor 1-(6-[([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122; 10 μM). Nevertheless, U 73,122 (1–10 μM) did not significantly affect carbachol-stimulated bladder contraction. Carbachol had only little effect on PLD activity in bladder slices, but the PLD inhibitor butan-1-ol, relative to its negative control butan-2-ol (0.3% each), caused detectable inhibition of carbachol-induced bladder contraction. The cytosolic PLA₂ inhibitor arachidonyltrifluoromethyl ketone weakly inhibited carbachol-induced contraction at a concentration of 300 μM, but the cyclooxygenase inhibitor indomethacin (1–10 μM) remained without effect. The Ca²⁺ entry blocker nifedipine (10–100 nM) almost completely inhibited carbachol-induced bladder contraction. In contrast, 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96,365; 10 μM), an inhibitor of store-operated Ca²⁺ channels, caused little inhibition. We conclude that carbachol-induced contraction of rat bladder largely depends on Ca²⁺ entry through nifedipine-sensitive channels and, perhaps, PLD, PLA₂, and store-operated Ca²⁺ channels, whereas cyclooxygenase and, surprisingly, also PLC are not involved to a relevant extent.