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The pathological activation of digestive zymogens within the pancreatic acinar cell probably plays a central role in initiating many forms of pancreatitis. To examine the relationship between zymogen activation and acinar cell injury, we investigated the effects of secretagogue treatment on isolated pancreatic acini. Immunofluorescence studies using antibodies to the trypsinogen-activation peptide demonstrated that both CCK (10(-7) M) hyperstimulation and bombesin (10(-5) M) stimulation of isolated acini resulted in trypsinogen processing to trypsin. These treatments also induced the proteolytic processing of procarboxypeptidase A1 to carboxypeptidase A1 (CA1). After CCK hyperstimulation, most CA1 remained in the acinar cell. In contrast, the CA1 generated by bombesin was released from the acinar cell. CCK hyperstimulation of acini was associated with cellular injury, whereas bombesin treatment did not induce injury. These studies suggest that 1) proteolytic zymogen processing occurs within the pancreatic acinar cell and 2) both zymogen activation and the retention of enzymes within the acinar cell may be required to induce injury.

The pathological activation of zymogens within the pancreatic acinar cell plays a role in acute pancreatitis. To identify the processing site where activation occurs, antibodies to the trypsinogen activation peptide (TAP) were used in immunofluorescence studies using frozen sections from rat pancreas. Saline controls or animals receiving caerulein in amounts producing physiological levels of pancreatic stimulation demonstrated little or no TAP immunoreactivity. However, after caerulein hyperstimulation (5 micrograms. kg-1. h-1) for 30 min and the induction of pancreatitis, TAP immunoreactivity appeared in a vesicular, supranuclear compartment that demonstrated no overlap with zymogen granules. The number of vesicles and their size increased with time. After 60 min of hyperstimulation with caerulein, most of the TAP reactivity was localized within vacuoles >/=1 micrometer that demonstrated immunoreactivity for the granule membrane protein GRAMP-92, a marker for lysosomes and recycling endosomes. Pretreatment with the protease inhibitor FUT-175 blocked the appearance of TAP after hyperstimulation. These studies provide evidence that caerulein hyperstimulation stimulates trypsinogen processing to trypsin in distinct acinar cell compartments in a time-dependent manner.

To identify the muscarinic subtype present on the rat pancreatic acinar cell, we examined the effects of different muscarinic receptor antagonists on amylase secretion and proteolytic zymogen processing in isolated rat pancreatic acini. Maximal zymogen processing required a concentration of carbachol 10- to 100-fold greater (10(-3) M) than that required for maximal amylase secretion (10(-5) M). Although both secretion and conversion were inhibited by the M3 antagonist 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) (50% inhibition approximately 6 x 10(-7) M and 1 x 10(-8) M, respectively), the most potent inhibitor was the M1 antagonist telenzepine (50% inhibition approximately 5 x 10(-10) M and 1 x 10(-11) M, respectively). Pirenzepine, another M1 antagonist, and the M2 antagonist methoctramine did not reduce amylase secretion or zymogen processing in concentrations up to 1 x 10(-5) M. Analysis of acinar cell muscarinic receptor by PCR revealed expression of both m1 and m3 subtypes. The pancreatic acinar cell has a distinct pattern of muscarinic antagonist sensitivity (telenzepine >> 4-DAMP > pirenzepine) with respect to both amylase secretion and zymogen conversion.

Exocrine pancreatic zymogens are proteolytically processed to active forms after they are secreted into the small intestine. However, intracellular conversion of zymogens to active forms can be stimulated by treating pancreatic acinar cells with high doses of cholecystokinin (0.1 microM) or carbamylcholine (0.1 mM). The high doses of cholecystokinin are unlikely to be achieved physiologically. The ability of ethanol to sensitize the acinar cell to zymogen conversion Induced by cholecystokinin or carbamylcholine was examined. Ethanol (10-200 mM) had no effect alone or when combined with carbamylcholine. However, ethanol (25 mM) added with low-dose cholecystokinin (0.1 nM) generated zymogen conversion that was 1) sixfold higher than cholecystokinin alone and 2) equivalent to that generated by highdose cholecystokinin (10 microM). The ability of ethanol to enhance cholecystokinin-induced zymogen conversion was dependent on the dose of ethanol and the duration of ethanol treatment. The cholecystokinin receptor antagonist, L-364,718, blocked the conversion stimulated by the addition of ethanol with cholecystokinin. This effect of ethanol did not change the affinity or number of cholecystokinin receptors, suggesting an effect more distal in the stimulus-activation cascade. These findings demonstrate that ethanol selectively sensitizes the pancreatic acinar cell to cholecystokinin-stimulated zymogen proteolysis.

We recently reported that elevations in the intracellular Ca2+ concentration ([Ca2+]i) enhance low-voltage-activated, T-type, Ca2+ channel activity via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Here, we document CaMKII activity in bovine adrenal glomerulosa (AG) cells and assess the importance of CaMKII in depolarization-induced Ca2+ signaling. AG cell extracts exhibited kinase activity toward a CaMKII-selective peptide substrate that was dependent on both Ca2+ [half-maximal concentration for Ca2+ activation (K0.5) = 1.5 microM] and calmodulin (K0.5 = 46 nM) and was sensitive to a calmodulin antagonist and a CaMKII peptide inhibitor. On cell treatment with elevated extracellular potassium (10-60 mM) or angiotensin II, Ca(2+)-independent CaMKII activity increased to 133-205% of basal activity. Ca(2+)-independent kinase activity in agonist-stimulated extracts was inhibited by the CaMKII inhibitor peptide, 1(-)[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a cell-permeable inhibitor of CaMKII, reduced the agonist-induced stimulation of Ca(2+)-independent CaMKII activity. KN-62 also diminished depolarization-induced increases in [Ca2+]i without affecting the membrane potential. These observations suggest that CaMKII is activated in situ by aldosterone secretagogues and augments Ca2+ signaling through voltage-gated Ca2+ channels.

Ca2+/calmodulin (CaM)-dependent protein kinase II is a major effector of the Ca2+ signaling pathway. It has a wide tissue distribution and phosphorylates multiple substrates. Villus enterocytes from rat ileum contain a Ca2+/CaM-dependent kinase activity that phosphorylates the exogenous neural substrate synapsin I. This phosphorylation is blocked by a specific peptide inhibitor. Antibodies made to rat brain Ca2+/CaM-dependent protein kinase II label a single band with a relative molecular mass of approximately 50 kDa in isolated rat enterocytes by immunoblot. Almost one-half of this immunoreactive protein is preferentially found in a particulate compared with a soluble subcellular fraction of the enterocytes. Virtually all of the 50-kDa band in the particulate fraction is insoluble in nonionic detergent, suggesting that the kinase is associated with the enterocyte cytoskeleton. Antibodies to Ca2+/CaM-dependent protein kinase II immunocytochemically detect fibrillar structures concentrated in the terminal web region of intestinal epithelial cells that colocalized with myosin II. This enzyme may have a role in regulating the intestinal epithelial cytoskeleton.

An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with [32P]orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.

To implicate the Ca2+-calmodulin-dependent protein kinase (CDPK) in the secretory response of pancreatic acinar cells to postnatal neurohumoral stimulation, we examined enzymatic protein kinase activity during pancreatic development. CDPK was investigated by measuring the phosphorylation of endogenous proteins and an exogenous substrate, synapsin I. Ca2+-calmodulin (CaM)-stimulated phosphorylation of endogenous proteins as well as synapsin I progressively increased in pancreatic supernatants from rats of increasing age. When compared with embryonic pancreas (1 day before birth), Ca2+-CaM-stimulated protein kinase activity increased two- and sixfold in neonatal (1 day after birth) and adult pancreas (60 days after birth), respectively, when normalized to protein content. To further characterize CDPK activity, soluble fractions prepared from embryonic, neonatal, and adult pancreas were separated by gel filtration chromatography. CDPK activities from embryonic and neonatal pancreas were contained in fractions of relative molecular weights (Mr) less than 200,000. In contrast, kinase activity from adult pancreas was predominantly comprised of a Mr approximately 550,000 species that coeluted with a type II CDPK. Increasing amounts of a Mr = 51,000 CaM-binding protein as a function of increasing age were consistent with the developmental appearance of the CaM-binding subunit of type II Ca2+-CaM-stimulated protein kinase. CaM levels increased in parallel with total Ca2+-CaM-stimulated protein kinase activity. Coordinate enhancement of type II CDPK activity and secretagogue responsiveness during pancreatic maturation may suggest a role for this enzyme in the secretory process.