Porcine pancreatic phospholipase A(2) stimulates secretin release from secretin-producing cells

We have isolated, from canine pancreatic juice, two 14-kDa proteins with se cretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A(2) (PLA(2)). In this study we have obtained evi dence that secretin-releasing activity is an intrinsic property of pancreat ic PLA(2). Porcine pancreatic PLA(2) from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid ch romatography, They were tested for stimulation of secretin release from mur ine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enri ched mucosal cell preparations isolated from rat upper small intestine. Eac h enzyme preparation was found to contain several components of secretin-re leasing activity, Each bioactive fraction was purified to homogeneity by re chromatography and then subjected to mass spectral analysis and assays of P LA(2) and secretin-releasing activities. It was found that the fraction wit h highest enzymatic activity also had the highest secretin-releasing activi ty and the same M-r as porcine pancreatic PLA(2). Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA(2), suggesting that it was identical to the enzy me. Purified porcine pancreatic PLA(2) also stimulated secretin release con centration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum . Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory eff ect of purified pancreatic PLA(2) on secretin secretion from STC-1 cells wa s inhibited by an L-type Ca2+ channel blocker, by down-regulation of protei n kinase C or by pretreatment of the cell with pertussis toxin, It is concl uded that porcine pancreatic PLA(2) possesses an intrinsic secretin-releasi ng activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through t he L-type channel and activation of protein kinase C.