Phospholipase C, but not InsP(3) or DAG, -dependent activation of the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle cells

1 In visceral smooth muscles, both M-2 and M-3 muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCbeta activation. Thus, we studied their relevance for muscarinic cationic current (mI(CAT)) generation, which underlies cholinergic excitation. Experiments were performed on single guinea-pig ileal cells using patch-clamp recording techniques under conditions of weakly buffered [Ca2+](i) (either using 50 muM EGTA or 50-100 muM fluo-3 for confocal fluorescence imaging) or with [Ca2+](i) 'clamped' at 100 nM using 10 mM BAPTA/CaCl2 mixture. 2 Using a cAMP-elevating agent (I pm isoproterenol) or a membrane-permeable cAMP analog (10 muM 8-Br-cAMP), we found no evidence for m(ICA)T modulation through a cAMP/PKA pathway. 3 With low [Ca2+](i) buffering, the PLC blocker U-73122 at 2.5 muM almost abolished mI(CAT), in some cases without any significant effect on [Ca2+](i). When [Ca2+](i) was buffered at 100 rim, U-73122 reduced both carbachol- and GTPgammaS-induced mI(CAT) maximal conductances (IC50=0.5-0.6 muM) and shifted their activation curves positively. 4 U-73343, a weak PLC blocker, had no effect on GTPgammaS-induced mI(CAT), but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to I mm. Aristolochic acid and D-609, which inhibit PLA(2) and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mI(CAT), suggesting that these enzymes were not involved. 5 InsP(3) (1 muM) in the pipette or OAG (20 muM) applied externally had no effect on mI(CAT) or its inhibition by U-73122. Ca2+ store depletion (evoked by InSP3, or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mI(CAT) in response to carbachol when [Ca2+](i) was strongly buffered to 100 nM. 6 It is concluded that phosphatidylinositol-specific PLC modulates mI(CAT) via Ca2+ release, but also does so independently of InsP(3), DAG, Ca2+ store depletion or a rise of [Ca2+](i). Our present results explain the previously established 'permissive' role of the M-3 receptor subtype in mI(CAT) generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.