Signal transduction underlying carbachol-induced contraction of rat urinary bladder.: I.: Phospholipases and Ca2+ sources

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 A(2) and of intra-and extracellular Ca2+ sources in this effect. Based on the nonsubtype-selective tolterodine, the highly M-2 receptor-selective (R)-4-{2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl}-piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M-3 receptor-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M-3 receptors. Carbachol stimulated inositol phosphate formation in rat bladder slices, and this was abolished by the phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122; 10 muM). Nevertheless, U 73,122 (1-10 muM) 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 PLA(2) inhibitor arachidonyltrifluoromethyl ketone weakly inhibited carbachol-induced contraction at a concentration of 300 muM, but the cyclooxygenase inhibitor indomethacin ( 1- 10 muM) remained without effect. The Ca2+ entry blocker nifedipine (10-100 nM) almost completely inhibited carbachol-induced bladder contraction. In contrast,1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96,365; 10 muM), 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, PLA(2), and store-operated Ca2+ channels, whereas cyclooxygenase and, surprisingly, also PLC are not involved to a relevant extent.