Characterization of polyphosphoinositide-specific phospholipase C in rat parotid gland membranes.
Hydrolysis of exogenously added, [3H]inositol-labeled, phosphatidylinositol 4,5-bisphosphate (PIP2) by rat parotid membranes was increased, dose-dependently, by the muscarinic cholinergic agonist carbamylcholine (carbachol) in the presence of guanosine 5'-O-thiotriphosphate (GTP gamma S). The stimulation was inhibited by atropine and guanosine 5'-O-thiodiphosphate (GDP beta S). GTP gamma S alone stimulated PIP2 hydrolysis, with half-maximal activation at 0.1 microM. This was inhibited by GDP beta S but not by atropine. Agonist stimulation of PIP2 hydrolysis was dependent on the presence of lipids (phosphatidylserine:phosphatidylethanolamine:PIP2 = 1:1:1). When PIP2 was added as micelles with detergent (sodium deoxycholate) only, basal hydrolysis was elevated, thus decreasing the relative stimulation by GTP gamma S 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 GTP gamma S but the extent of PI hydrolysis was 44-fold lower than PIP2 hydrolysis. When [Ca2+] in the medium was increased from 100 nM to 1 microM, basal hydrolysis of both PI and PIP2 increased (9.3- and 19.2-fold, respectively). However, levels of basal and stimulated PIP2 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 beta 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 gamma 1 detected a approximately 150-kDa protein only in the parotid cytosol, while antisera against bovine brain PLC delta 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 gamma 1 in parotid gland cytosol, the PLC which mediates PIP2 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 gamma 1, delta 1, and beta 1.
Hiramatsu
Y
Y
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Horn
V J
VJ
Baum
B J
BJ
Ambudkar
I S
IS
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010306:Parotid Gland / Q000201:enzymology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013873:Thionucleotides / Q000494:pharmacology
D007295:Inositol Phosphates; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D002217:Carbachol; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium