Phospholipase C, but not InsP3 or DAG, -dependent activation of the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle cells

In visceral smooth muscles, both M2 and M3 muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCβ activation. Thus, we studied their relevance for muscarinic cationic current (mICAT) 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 μM EGTA or 50–100 μM fluo-3 for confocal fluorescence imaging) or with [Ca2+]i ‘clamped’ at 100 nM using 10 mM BAPTA/CaCl2 mixture. Using a cAMP-elevating agent (1 μM isoproterenol) or a membrane-permeable cAMP analog (10 μM 8-Br-cAMP), we found no evidence for mICAT modulation through a cAMP/PKA pathway. With low [Ca2+]i buffering, the PLC blocker U-73122 at 2.5 μM almost abolished mICAT, in some cases without any significant effect on [Ca2+]i. When [Ca2+]i was buffered at 100 nM, U-73122 reduced both carbachol- and GTPγS-induced mICAT maximal conductances (IC50=0.5–0.6 μM) and shifted their activation curves positively. U-73343, a weak PLC blocker, had no effect on GTPγS-induced mICAT, but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mM. Aristolochic acid and D-609, which inhibit PLA2 and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mICAT, suggesting that these enzymes were not involved. InsP3 (1 μM) in the pipette or OAG (20 μM) applied externally had no effect on mICAT 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 mICAT in response to carbachol when [Ca2+]i was strongly buffered to 100 nM. It is concluded that phosphatidylinositol-specific PLC modulates mICATvia Ca2+ release, but also does so independently of InsP3, DAG, Ca2+ store depletion or a rise of [Ca2+]i. Our present results explain the previously established ‘permissive’ role of the M3 receptor subtype in mICAT generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve. British Journal of Pharmacology (2004) 141, 23–36. doi:10.1038/sj.bjp.0705584