Identification of phosphoinositide-specific phospholipase C-beta 1 and GTP-binding protein, Gq alpha, in bovine iris sphincter membranes: characteristics of the phospholipase and its coupling to cholinergic muscarinic receptors.
Previously, we have established that treatment of iris sphincter smooth muscle with carbachol (CCh) results in increased phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) and in muscle contraction. To throw more light on the mechanism of muscarinic stimulation of PLC in this tissue we have investigated the properties of this enzyme and its regulation by GTP analogs and protein phosphorylation in bovine iris sphincter membranes. The data obtained can be summarized as follows: (1) the presence of PLC-beta 1 and a GTP-binding protein, Gq alpha, was detected in the microsomal (membrane) fraction by anti-PLC beta 1 and anti-Gq alpha antibodies, respectively. The membrane PLC hydrolysed exogenously added PIP2 and this hydrolysis was increased dose-dependently by Ca2+ (1-10 microM) but the enzyme activity was inhibited by Mg2+. (2) Addition of guanosine 5'-O-thiotriphosphate (GTP gamma S, 0.1 microM) to the membrane fraction increased PIP2 hydrolysis by 30%, whereas addition of CCh (10 microM) was without effect. However, when added together, CCh and GTP gamma S increased PIP2 hydrolysis by 46%. This effect was significantly inhibited by atropine and by the anti-PLC-beta 1 and anti-Gq alpha antibodies. (3) Removal of PLC-beta 1 from the membranes with 2 M KCl resulted in a significant reduction of the CCh-induced PIP2 hydrolysis, and this effect of the muscarinic agonist was restored when the membrane fraction was supplemented with PLC-beta 1 purified from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
C J
CJ
Zhou
R A
RA
Akhtar
A A
AA
Abdel-Latif
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000032:analysis*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D007498:Iris / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D009130:Muscle, Smooth / Q000201:enzymology; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010738:Type C Phospholipases / Q000032:analysis* / Q000037:antagonists & inhibitors
D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002217:Carbachol; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D008274:Magnesium; D002118:Calcium