Identification and characterization of a hormonally regulated form of phospholipase A2 in rat renal mesangial cells.
The activity of phospholipase A2 (PLA2), the regulatory enzyme in arachidonic acid release and prostaglandin synthesis, was measured in cell-free extracts of rat renal mesangial cells. Arginine vasopressin (AVP) and phorbol myristate acetate (PMA) both stimulated PLA2 activity as assayed by the release of free arachidonic acid from exogenously added [14C]arachidonyl-phosphatidylcholine. This represents a direct in vitro demonstration of hormone-induced changes in PLA2 activity. The stimulated activity was recovered following fractionation by DEAE-cellulose anion exchange and FPLC Superose 12 gel filtration. Stimulated activity from AVP- and PMA-treated cells comigrated as a single peak, suggesting that these agents are stimulating a single form of the enzyme. The molecular weight of this hormonally regulated form of PLA2 is approximately 60,000. The enzyme has an obligate requirement for Ca2+, having no activity in the presence of EGTA, and has a pH optimum in the alkaline range. Following cell disruption in the presence of chelators, the enzyme is recovered in a high speed supernatant. However, it appears that it can bind to a crude membrane fraction in a Ca2+-dependent manner, similar to other Ca2+-phospholipid binding proteins. The observed stable modification in PLA2 activity by AVP and PMA suggests a phosphorylation of PLA2 or PLA2 modulators by protein kinase C.
D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002413:Cations, Divalent; D001127:Arginine Vasopressin; D004533:Egtazic Acid; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate