Ca2+-, phorbol ester-, and cAMP-stimulated enzyme secretion from permeabilized rat pancreatic acini

Abstract

Enzyme secretion from the exocrine pancreas is stimulated by receptor-activated breakdown of phosphatidylinositol 4,5-bisphosphate and consequent rise of both inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which leads to Ca2+ release and to activation of protein kinase C, respectively. Another way involves receptor-mediated stimulation of adenylate cyclase and consequent rise of cAMP and activation of protein kinase A. In the present work we have studied direct stimulation, inhibition, and mutual interaction of these pathways on enzyme secretion from isolated rat pancreatic acini that had been permeabilized by treatment with saponin or digitonin. The data were compared with those obtained in isolated intact acini. The data show that with increasing free Ca2+ concentrations greater than 10(-6) M protein release increases in "leaky" but not in "intact" cells and is maximal at approximately 10(-3) M, increasing about twofold compared with that in the absence of Ca2+. In the presence of the acetylcholine analogue carbachol, this effect of Ca2+ is enhanced by about threefold in leaky cells and is also present in intact cells to a similar extent. cAMP and its analogues, dibutyryl cAMP (dbcAMP) and 8-bromo-cAMP stimulate protein release by about twofold in the presence of Ca2+ in leaky cells. In intact acini cAMP has no effect, and cAMP analogues stimulate enzyme secretion by about twofold in some but not all experiments. Similarly, forskolin, an activator of adenylate cyclases and inhibitors of cyclic nucleotide-dependent phosphodiesterases, such as 3-isobutyl-1-methylxanthine (IBMX) and R0 201724, stimulate protein release in permeabilized acini. The Ca2+-binding protein calmodulin has no effect on enzyme secretion, whereas the calmodulin antagonist trifluoperazine dihydrochloride stimulates protein release in leaky but not in intact acini. The activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates protein release in a Ca2+-dependent manner and enhances cAMP-induced secretion. The effects of carbachol, TPA, cAMP, and a combination of both TPA and cAMP are inhibited by the polyamine spermine in permeabilized cells. Spermine has no effect on carbachol-induced enzyme secretion in intact cells. The data suggest that enzyme secretion from pancreatic acinar cells is mediated by cAMP protein kinase A and by Ca2+ phospholipid protein kinase C in a Ca2+-dependent way and that interaction occurs between both pathways.

Pervanadate Stimulates Amylase Release and Protein Tyrosine Phosphorylation of Paxillin and p125FAK in Differentiated AR4-2J Pancreatic Acinar Cells

“…Both the Ca 2ϩ -and the protein kinase C-induced pathways act synergistically. Artificial activation of these pathways by elevation of cytosolic Ca 2ϩ concentration in the presence of stimulators of protein kinase C, such as phorbol esters, could mimic the effect of secretagogues on enzyme secretion (29,31,32). Although different proteins have been shown to be phosphorylated in response to secretagogues of enzyme secretion, key targets in the Ca 2ϩ -and diacylglycerol-dependent cascade of stimulatory events have not yet been identified.…”

Stimulatory effects of bilirubin on amylase release from isolated rat pancreatic acini

“…TPA may exert its effect on enzyme secretion through PKC activation while not affecting Ca 2ϩ or cyclic nucleotide metabolism (8). TPA also exerts sustained amylase release in pancreatic acini (20,31). Ca 2ϩ and DAG also have been shown (8,35) to work synergistically on amylase release from pancreatic acini.…”

Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity.

“…In recent reports polyamines were assigned an inhibitory role in phospholipase C-catalyzed polyphosphoinositide hydrolysis (45,47). This feature may reflect that polyamines, by virtue oftheir polycationic nature, compete with Ca2+ for binding sites on phosphatidylinositol 4,5-bisphosphate, which is the major phospholipase C substrate.…”

“…On day 4 of culture, media were removed and the cultures were washed once with phosphate-buffered saline. They were then preincubated at 37°C for 45 min in Krebs-Ringer bicarbonate buffer ( 18) at pH 7.4 containing 2 mg/ml bovine serum albumin, 10 mM Hepes, and 2.8 mM glucose. The preincubation media were then discarded, the cultures were washed once with similar media and subsequently exposed for 30 min to different secretagogues in similar incubation buffer with 2.8 mM glucose.…”