Characterization of polyphosphoinositide-specific phospholipase C in rat parotid gland membranes

Abstract

Hydrolysis of exogenously added, [ 3H]inositol-labeled, phosphatidylinositol 4,5-bisphosphate (PIP 2) by rat parotid membranes was increased, dose-dependently, by the muscarinic cholinergic agonist carbamylcholine (carbachol) in the presence of guanosine 5′- O-thiotriphosphate (GTPγS). The stimulation was inhibited by atropine and guanosine 5′- O-thiodiphosphate (GDPβS). GTPγS alone stimulated PIP 2 hydrolysis, with halfmaximal activation at 0.1 μ m. This was inhibited by GDPβS but not by atropine. Agonist stimulation of PIP 2 hydrolysis was dependent on the presence of lipids (phosphatidylserine:phosphatidylethanolamine:PIP 2 = 1:1:1). When PIP 2 was added as micelles with detergent (sodium deoxycholate) only, basal hydrolysis was elevated, thus decreasing the relative stimulation by GTPγS and carbachol. The water-soluble hydrolysis products formed under either condition were 1,4,5-inositol trisphosphate, 1,4-inositol bisphosphate, and cyclic inositol trisphosphate. Hydrolysis of exogenous phosphatidylinositol (PI) was also stimulated by carbachol in the presence of GTPγS but the extent of PI hydrolysis was 44-fold lower than PIP 2 hydrolysis. When [Ca 2+] in the medium was increased from 100 n m to 1 μ m, basal hydrolysis of both PI and PIP 2 increased (9.3- and 19.2-fold, respectively). However, levels of basal and stimulated PIP 2 hydrolysis were higher (37.9- and 29.6-fold, respectively) than those of PI hydrolysis. Antibodies (both polyclonal and monoclonal) raised against phospholipase C (PLC β 1) from bovine brain did not react with any component in either rat parotid membranes or cytosol, although a reactivity was detected in rat brain membranes. A monoclonal antibody against bovine brain PLC γ 1 detected a ~ 150-kDa protein only in the parotid cytosol, while antisera against bovine brain PLC δ 1 enzyme showed no reactivity with parotid membranes or cytosol. Together, these observations suggest that while there appears to be a protein similar to bovine brain PLC γ 1 in parotid gland cytosol, the PLC which mediates PIP 2 hydrolysis in rat parotid membranes and can be regulated by the muscarinic receptor via a G-protein is distinct from the well-characterized PLC enzymes γ 1, δ 1, and β 1.