inositol-1-4-5-trisphosphate and Pancreatitis

Fine-tuning of the activity of inositol 1,4,5-trisphosphate receptors (IP

Topics: Acute Disease; Animals; Calcium Signaling; Disease Models, Animal; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Ion Channel Gating; Male; Mice; Mice, Inbred C57BL; Pancreatitis; Proteolysis; Rats; Rats, Wistar

Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH(2) terminus to expose a tethered ligand. We previously demonstrated that PAR-2 activates ion channels in pancreatic duct epithelial cells (PDEC). Using real-time optical fluorescent probes, cyan fluorescence protein-Epac1-yellow fluorescence protein for cAMP, PH(PLC-delta1)-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cgamma (PKCgamma)-C1-yellow fluorescence protein for diacylglycerol, we now define the signaling pathways mediating PAR-2 effect in dog PDEC. Although PAR-2 activation does not stimulate a cAMP increase, it induces phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Intracellular Ca(2+) mobilization from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and a subsequent Ca(2+) influx through store-operated Ca(2+) channels cause a biphasic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca(2+)](i) in turn causes a biphasic increase in exocytosis. A protein kinase assay revealed that trypsin also activates PKC isozymes to stimulate additional exocytosis. Paralleling the increased exocytosis, mucin secretion from PDEC was also induced by trypsin or the PAR-2 activating peptide. Consistent with the serosal localization of PAR-2, 1 microm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 microm trypsin at 37 degrees C damaged the epithelial barrier sufficiently so that it could reach and activate the serosal PAR-2 to stimulate exocytosis. Thus, in PDEC, PAR-2 activation increases [Ca(2+)](i) and activates PKC to stimulate exocytosis and mucin secretion. These functions may mediate the reported protective role of PAR-2 in different models of pancreatitis.

Topics: Animals; Calcium; Calcium Channels; Calcium Signaling; Cells, Cultured; Cyclic AMP; Disease Models, Animal; Dogs; Epithelial Cells; Exocytosis; Inositol 1,4,5-Trisphosphate; Ion Channels; Mucins; Pancreatic Ducts; Pancreatitis; Phosphatidylinositol 4,5-Diphosphate; Protein Kinase C; Receptor, PAR-2; Trypsin; Type C Phospholipases

The pancreatic acinar unit is a classical example of a polarized tissue. Even in isolation, these cells retain their polarity, and this has made them particularly useful for Ca2+ signaling studies. In 1990, we discovered that this cell has the capability of producing both local cytosolic and global Ca2+ signals. The mechanisms underlying this signal generation have now been established. Furthermore, it has become clear that the local signals are sufficient for the control of both fluid and enzyme secretion, whereas prolonged global signals are dangerous and give rise to acute pancreatitis, a disease where the pancreas digests itself.

Topics: Calcium; Calcium Signaling; Cell Communication; Cell Compartmentation; Cell Membrane; Cell Polarity; Cytosol; Endoplasmic Reticulum; Humans; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Pancreas, Exocrine; Pancreatitis

Young female mice fed a choline-deficient, ethionine-supplemented (CDE) diet rapidly develop acute hemorrhagic pancreatitis. We have observed that pancreatic acini prepared from these mice are unable to secrete amylase in response to addition of the cholinergic agonist carbachol, although they retain the ability to secrete amylase in response to the Ca2+ ionophore A23187. The CDE diet does not alter the binding characteristics (Kd or the maximal number of binding sites) for muscarinic cholinergic receptors as tested using the antagonist [3H]N-methylscopolamine nor the competition for this binding by carbachol. Addition of carbachol to acini prepared from mice fed the CDE diet does not result in as marked an increase in cytosolic free Ca2+ levels as that noted in control samples (evaluated using quin2 fluorescence). These observations indicate that the CDE diet interferes with stimulus-secretion coupling in mouse pancreatic acini at a step subsequent to hormone-receptor binding and prior to Ca2+ release. This conclusion is confirmed by our finding that the hormone-stimulated generation of [3H]inositol phosphates (inositol trisphosphate, inositol bisphosphate, and inositol monophosphate) from acini labeled with [3H]myoinositol is markedly reduced in acini prepared from mice fed the CDE diet. This reduction is not due to a decrease in phosphatidylinositol-4,5-bisphosphate. This communication represents the first report of a system in which a blockade of inositol phosphate generation can be related to a physiologic defect and pathologic lesion.

Topics: Aminoquinolines; Amylases; Animals; Calcimycin; Calcium; Carbachol; Choline Deficiency; Diglycerides; Enzyme Activation; Ethionine; Female; Fluorescence; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mice; N-Methylscopolamine; Pancreas; Pancreatitis; Protein Kinase C; Scopolamine Derivatives; Sugar Phosphates; Tritium; Type C Phospholipases