Epidermal growth factor induces the differential release of GP2 and amylase from AR4-2J cells.

Epidermal growth factor (EGF) stimulates secretion of glycoprotein 2 (GP2) in a time-and concentration-dependent manner from the AR4-2J pancreatoma cell line. Cell differentiation induced by dexamethasone treatment for 3 d, however, did not significantly alter either basal or EGF-stimulated GP2 release. Basal and EGF-stimulated GP2 release were similarly unaffected by caerulein, which promotes amylase secretion by a regulated route. A brief exposure to cycloheximide profoundly blocked EGF-evoked GP2 secretion. Furthermore, EGF-stimulated GP2 release was not accompanied by significant alterations in intracellular ionic calcium levels, in contrast to the stimulatory actions of caerulein. We conclude that EGF-stimulated release of GP2 occurs via a novel secretory pathway that is neither regulated nor constitutive as currently defined.

Modulation of phospholipase A2 activity in zymogen granule membranes by GTP[S]; evidence for GTP-binding protein regulation.

In membranes associated with purified pancreatic zymogen granules, GTP[S] elicited a concentration-dependent activation of phospholipase A2 (PLA2), which was converted to inhibition in the presence of added Ca2+. The GTP-binding protein inhibitor GDP[S] blocked both the stimulatory and inhibitory actions of GTP[S]. We conclude that in zymogen granule membranes GTP-binding proteins exert a dual regulation of PLA2 activity.

Regulation of phosphatidylinositol 4-kinase activity in rat pancreatic acini.

Previous data showed that carbachol (CCh) elicits a concentration-dependent increase in phosphatidylinositol (PtdIns) 4-kinase activity in homogenates derived from agonist-stimulated pancreatic acini. In the present study CCh elicited a time-dependent increase in PtdIns 4-kinase activity, which was blocked by n-methylscopolamine and mimicked by muscarine. A membrane-associated PtdIns 4-kinase activity was identified that displayed maximal activity in the pH range of 5.5-8.5. The zymogen granule fraction possessed relatively high specific enzyme activity, which was sensitive to CCh stimulation. The enzyme had apparent Km values for PtdIns and ATP of 4 and 60 microM, respectively. CCh caused no discernible change in the Km for either PtdIns or ATP but increased Vmax. Dioctanoylglycerol and oleoyl acetylglycerol augmented PtdIns 4-kinase activity, which was blocked by staurosporine, thus suggesting a possible role for protein kinase C in the regulation of PtdIns 4-kinase. These collective findings demonstrate a muscarinic receptor-mediated regulation of PtdIns 4-kinase activity in exocrine pancreas, involving a protein kinase C-dependent process.

Identification and characterization of carboxyl ester hydrolase as a phospholipid hydrolyzing enzyme of zymogen granule membranes from rat exocrine pancreas.

Salt-washed (0.6 m NaCl) zymogen granule membranes (ZGM) of rat pancreatic acinar cells were utilized to identify and characterize membrane protein(s) responsible for phospholipase and lysophospholipase activities. Five major bands were identified in salt-washed ZGM by Coomassie Brilliant Blue. A 70-kDa protein with enzymatic activity was retained in significant quantities after several washes with 0.6 M NaCl but could be displaced from ZGM by 2 m NaCl or by 100 mg/ml heparin. By contrast, GP2, an integral membrane protein, was not displaced under these conditions. These findings suggest that the enzyme is a peripheral membrane protein of ZGM. Renaturation of ZGM proteins following electrophoresis revealed that the 70-kDa protein possessed phospholipase activity. Identification of the 70-kDa protein as a membrane-associated carboxyl ester hydrolase was based upon: (a) the use of a specific polyclonal antiserum, (b) N-terminal sequence, (c) two-dimensional gel analysis, (d) enzymatic characterization, and (e) co-localization to an area of a non-reducing gel containing significant phospholipase activity. Other ZGM proteins, namely GP2 and GP3, could not be demonstrated to possess phospholipase activity under the experimental conditions employed. Our finding that carboxyl ester hydrolase from ZGM exhibits PLA1 and lysophospholipase activities represents the first identification and characterization of a protein responsible for phospholipase activity in secretory granule membranes.

Glycoprotein 2 of zymogen granule membranes shares immunological cross-reactivity and sequence similarity with phospholipase A2.

Glycoprotein 2 (GP2), the major protein of rat pancreatic zymogen granule membranes (ZGMs), cross-reacted with an antiserum against porcine secretory phospholipase A2 (sPLA2). Amino acid sequence comparison showed 45% similarity and 23% identity between porcine PLA2 and the C-terminal portion of GP2. An antiserum to intestinal brush border Ca(2+)-independent PLA2 (bbPLA2) also recognized GP2. The antigenic and sequence similarities between GP2, sPLA2, and bbPLA2 imply that the role of GP2 in cellular function is associated with phospholipid binding and/or hydrolysis.