Identification of Phosphoinositide-specific Phospholipase C-β1 and GTP-binding Protein, G qα, in Bovine Iris Sphincter Membranes: Characteristics of the Phospholipase and its Coupling to Cholinergic Muscarinic Receptors

Abstract

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 (PIP 2) into 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3) 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-β 1 and a GTP-binding protein. G qα, was detected in the microsomal (membrane) fraction by anti-PLC β1 and anti-G qα antibodies, respectively. The membrane PLC hydrolysed exogenously added PIP 2 and this hydrolysis was increased dose-dependently by Ca 2+ (1-10 μM) but the enzyme activity was inhibited by Mg 2+. (2) Addition of guanosine 5'-O-thiotriphosphate (GTPγS, 0·1 μM) to the membrane fraction increased PIP 2 hydrolysis by 30%, whereas addition of CCh (10 μM) was without effect. However, when added together, CCh and GTPγS increased PIP 2 hydrolysis by 46%. This effect was significantly inhibited by atropine and by the anti-PLC-β1 and anti-G qα antibodies. (3)Removal of PLC-β1 from the membranes with 2 m KCl resulted in a significant reduction of the CCh-induced PIP 2 hydrolysis, and this effect of the muscarinic agonist was restored when the membrane fraction was supplemented with PLC-β1 purified from bovine brain. (4) CCh-stimulated PIP 2 hydrolysis was significantly reduced in membrane fractions prepared from iris sphincter pretreated with the β-adrenergic agonist, isoproterenol. Thus, it can be concluded that in bovine iris sphincter: (a) muscarinic stimulation of PLC-β1, and subsequently PIP 2 hydrolysis, is mediated through the G-protein. G qα; and (b) that phosphorylation of G qα and/or muscarinic receptor by protein kinase A could be the mechanism through which isoproterenol inhibits CCh-stimulated hydrolysis of PIP 2 and muscle contraction in this tissue.