title: Phospholipase C, but not InsP3 or DAG, -dependent activation of the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle cells.
abstract: 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 [Ca(2+)](i) (either using 50 microm EGTA or 50-100 microm fluo-3 for confocal fluorescence imaging) or with [Ca(2+)](i) 'clamped' at 100 nm using 10 mm BAPTA/CaCl(2) mixture. 2. Using a cAMP-elevating agent (1 microm isoproterenol) or a membrane-permeable cAMP analog (10 microm 8-Br-cAMP), we found no evidence for mI(CAT) modulation through a cAMP/PKA pathway. 3. With low [Ca(2+)](i) buffering, the PLC blocker U-73122 at 2.5 microm almost abolished mI(CAT), in some cases without any significant effect on [Ca(2+)](i). When [Ca(2+)](i) was buffered at 100 nm, U-73122 reduced both carbachol- and GTPgammaS-induced mI(CAT) maximal conductances (IC(50)=0.5-0.6 microm) 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 1 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 microm) in the pipette or OAG (20 microm) applied externally had no effect on mI(CAT) or its inhibition by U-73122. Ca(2+) store depletion (evoked by InsP(3), 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 [Ca(2+)](i) was strongly buffered to 100 nm. 6. It is concluded that phosphatidylinositol-specific PLC modulates mI(CAT) via Ca(2+) release, but also does so independently of InsP(3), DAG, Ca(2+) store depletion or a rise of [Ca(2+)](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.
authors.affiliation: Department of Nerve-Muscle Physiology, A.A. Bogomoletz Institute of Physiology, Kiev 01024, Ukraine. zholosa@sghms.ac.uk
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Zholos
authors.forename: Yaroslav D
authors.initials: YD
authors.lastname: Tsytsyura
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Gordienko
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Tsvilovskyy
authors.forename: Tom B
authors.initials: TB
authors.lastname: Bolton
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D034341:Aristolochic Acids / Q000494:pharmacology; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism* / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism; D006168:Guinea Pigs; D007082:Ileum / Q000166:cytology / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000166:cytology* / Q000502:physiology; D009636:Norbornanes; D018408:Patch-Clamp Techniques / Q000379:methods; D054799:Phospholipase C beta; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D011760:Pyrrolidinones / Q000494:pharmacology; D043585:Receptor, Muscarinic M2 / Q000187:drug effects / Q000502:physiology; D043587:Receptor, Muscarinic M3 / Q000187:drug effects / Q000502:physiology*; D012433:Ryanodine / Q000494:pharmacology; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C011439:1,2-diacylglycerol; D000067956:Adenylyl Cyclase Inhibitors; D034341:Aristolochic Acids; D001952:Bridged-Ring Compounds; D004075:Diglycerides; D004963:Estrenes; D007211:Indoles; D007295:Inositol Phosphates; D007527:Isoenzymes; D009636:Norbornanes; D011760:Pyrrolidinones; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D013859:Thiocarbamates; D013871:Thiones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; D012433:Ryanodine; D015124:8-Bromo Cyclic Adenosine Monophosphate; D002110:Caffeine; C046498:tricyclodecane-9-yl-xanthogenate; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; C038237:1-oleoyl-2-acetylglycerol; D002217:Carbachol; C000228:aristolochic acid I; D010741:Phospholipases A; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D007545:Isoproterenol; D002118:Calcium; C000543:cyclopiazonic acid