Protease-activated receptor-2 (PAR-2) is a widely expressed tethered ligand receptor that can be activated by trypsin and other trypsin-like serine proteases. In the exocrine pancreas, PAR-2 activation modulates acinar cell secretion of digestive enzymes and duct cell ion channel function. During acute pancreatitis, digestive enzyme zymogens, including trypsinogen, are activated within the pancreas. We hypothesized that trypsin, acting via PAR-2, might regulate the severity of that disease, and to test this hypothesis, we examined the effect of either genetically deleting or pharmacologically activating PAR-2 on the severity of secretagogue-induced experimental pancreatitis. We found that experimental acute pancreatitis is more severe in PAR-2(-/-) than in wild-type mice and that in vivo activation of PAR-2, achieved by parenteral administration of the PAR-2-activating peptide SLIGRL-NH2, reduces the severity of pancreatitis. In the pancreas during the early stages of pancreatitis, the MAPK ERK1/2 is activated and translocated to the nucleus, but nuclear translocation is reduced by activation of PAR-2. Our findings indicate that PAR-2 exerts a protective effect on pancreatitis and that activation of PAR-2 ameliorates pancreatitis, possibly by inhibiting ERK1/2 translocation to the nucleus. Our observations suggest that PAR-2 activation may be of therapeutic value in the treatment and/or prevention of severe clinical pancreatitis, and they lead us to speculate that, from a teleological standpoint, PAR-2 may have evolved in the pancreas as a protective mechanism designed to dampen the injurious effects of intrapancreatic trypsinogen activation.

The initiation of insulin release from rat islet ␤ cells relies, in large part, on calcium influx through dihydropyridine-sensitive (␣ 1D ) voltage-gated calcium channels. Components of calcium-dependent insulin secretion and whole cell calcium current, however, are resistant to L-type channel blockade, as well as to -conotoxin GVIA, a potent inhibitor of ␣ 1B channels, suggesting the expression of additional exocytotic calcium channels in the islet. We used a reverse transcription-polymerase chain reaction-based strategy to ascertain at the molecular level whether the ␣ 1A calcium channel isoform was also present. Results revealed two new variants of the rat brain ␣ 1A channel in the islet with divergence in a putative extracellular domain and in the carboxyl terminus. Using antibodies and cRNA probes specific for ␣ 1A channels, we found that the majority of cells in rat pancreatic islets were labeled, indicating expression of the ␣ 1A channels in ␤ cells, the predominant islet cell type. Electrophysiologic recording from isolated islet cells demonstrated that the dihydropyridine-resistant current was sensitive to the ␣ 1A channel blocker, -agatoxin IVA. This toxin also inhibited the dihydropyridineresistant component of glucose-stimulated insulin secretion, suggesting functional overlap among calcium channel classes. These findings confirm the presence of multiple high voltage-activated calcium channels in the rat islet and implicate a physiologic role for ␣ 1A channels in excitation-secretion coupling in ␤ cells.The metabolism of glucose in ␤ cells is linked to membrane excitation through increases in the components that influence the ATP/ADP ratio (1, 2). A local increase in ATP relative to ADP inhibits the ATP-sensitive K ϩ (K ATP ) channel, giving rise to oscillations in K ATP permeability and consequent fluctuations in membrane potential (3, 4). ATP-induced depolarizations evoke insulin release through the activation of voltagedependent calcium channels, promoting calcium influx (3-6). If extracellular calcium is eliminated, glucose-stimulated insulin secretion is abolished, highlighting the important role of calcium channels in insulin homeostasis (7-11).Of the six genes (A-E, S) encoding the pore-forming ␣ 1 subunits of high voltage-activated calcium channels (12), all are capable of coupling to exocytotic machinery, although differences in tissue distribution and efficacy with which they stimulate secretion vary among the channel classes (for review, see Ref. 13). Pharmacological and heterologous expression studies have identified selective antagonists for certain of these: -agatoxin IV blocks ␣ 1A , -conotoxin GVIA blocks ␣ 1B , and 1,4-dihydropyridines block ␣ 1C , ␣ 1D , and ␣ 1S (13). No selective antagonist has been identified to date for ␣ 1E calcium channels, although they are sensitive to nonselective calcium channel antagonists (e.g. -grammotoxin SIA and cadmium).Previous work on pancreatic ␤ cells demonstrated that calcium influx and depolarization-evoked insulin release are blocked 60-80% by...

Glucagon-like peptide 1 (GLP-1) is a physiological stimulus of pancreatic beta-cell function. This enteroendocrine hormone is produced by intestinal L cells, and is delivered via the bloodstream to GLP-1 receptors (GLP-1Rs) on pancreatic beta-cells. In addition, there is evidence that beta-cell GLP-1Rs maintain sustained basal activity even in the absence of intestinal peptide, an observation that has raised the question whether these receptors have some degree of ligand-independent function. Here, we provide an alternative explanation for basal receptor activity based on our finding that biologically relevant amounts of fully processed GLP-1 are locally generated by insulinoma cell lines, as well as by alpha-cells of isolated rat islets in primary culture. Presence of GLP-1 was established by immunocytochemistry, as well as by selective ELISAs and bioassays of cell supernatants. A GLP-1R antagonist significantly reduced insulin secretion/production in beta-TC-6 insulinoma cells and isolated rat islets, suggesting a functionally important loop between locally produced GLP-1 and its cognate receptor. Treatment with this antagonist also inhibited the growth of beta-TC-6 cells. These observations provide novel insight into the function of insulin-producing cell lines and native beta-cells during in vitro culture, and they support the idea that locally produced GLP-1 may play a role in intra-islet regulation.

Truncated forms of glucagon-like peptide-1 are the most potent endogenous stimuli of insulin secretion and have powerful antidiabetogenic effects. To determine the structure and coupling mechanisms of the human GLP-1 receptor we have isolated two pancreatic islet cDNAs, encoding the 463 amino acid receptor and differing mainly in their 3' untranslated regions. The deduced amino acid sequence is 90% homologous with the rat GLP-1 receptor. Northern blot analysis shows expression of a single 2.7 kb transcript in pancreatic tissue. When expressed in COS-7 cells the recombinant receptor conferred specific, high affinity GLP-1(7-37) binding. GLP-1(7-37) increased intracellular cAMP in a concentration dependent manner and caused an increase in the free cytosolic calcium ([Ca2+]i) from an intracellular pool, characteristic of phospholipase C (PLC) activation. Thus, like the structurally related glucagon and parathyroid hormone receptors, the human GLP-1 receptor can activate multiple intracellular signaling pathways including adenylyl cyclase and PLC. Knowledge of the GLP-1 receptor structure will facilitate the development of receptor agonists and elucidation of the important role of GLP-1 in normal physiology and disease states.