Functional characterization of phosphoinositide-specific phospholipase C-beta 1 and -beta 3 in intestinal smooth muscle.
Soluble and membrane phosphoinositide-specific phospholipases obtained separately from dispersed circular and longitudinal intestinal muscle cells were characterized for substrate specificity and G protein dependence using selective antibodies to various isoforms of phospholipase C (PLC) and G protein subunits. Western blot analysis disclosed the presence of the main PLC isozymes, PLC-gamma 1, PLC-delta 1, and PLC-beta 1. Soluble PLC from circular and longitudinal muscle was stimulated by guanosine 5'-O-(3-thiophosphate) and inhibited by PLC-beta 1 antibody (80-90%) and PLC-beta 3 antibody (approximately 25%) but not by G protein antibodies. Membrane PLC from circular and longitudinal muscle was stimulated by cholecystokinin octapeptide (CCK-8) and inhibited selectively by PLC-beta 1 antibody (85%), PLC-beta 3 antibody (15%), and G alpha q/11 antibody (90%). CCK-8-induced contraction in permeabilized circular muscle cells was also selectively inhibited by PLC-beta 1 antibody (76%), PLC-beta 3 antibody (24%), and G alpha q/11 antibody (86%). The combined effects of PLC-beta 1 and PLC-beta 3 antibodies on PLC activity and muscle contraction were additive, causing complete inhibition. Soluble and membrane PLC from circular and longitudinal muscle were immunologically similar but functionally different. The enzymes from circular muscle preferentially hydrolyzed endogenous and exogenous phosphatidylinositol 4,5-biphosphate (PIP2), confirming previous findings of preferential hydrolysis of PIP2 in dispersed intestinal circular muscle cells.
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology; D005769:Gastrointestinal Motility / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006168:Guinea Pigs; D007295:Inositol Phosphates / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D007527:Isoenzymes / Q000378:metabolism*; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D012844:Sincalide / Q000494:pharmacology; D012995:Solubility; D010738:Type C Phospholipases / Q000378:metabolism* / Q000502:physiology
D000911:Antibodies, Monoclonal; D007295:Inositol Phosphates; D007527:Isoenzymes; D010716:Phosphatidylinositols; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D012844:Sincalide