H 7 inhibits PKC more potently in in vitro assays and is mainly used as a rather nonspecific inhibitor of protein kinase activity. Mecamylamine is a nonselective and noncompetitive antag onist of the nicotinic acetylcholine receptors and it blocks the effect of nicotine. Material and methods Reagents All reagents used in the experiment are of analytical nevertheless grade. Nicotine was purchased from Sigma and it was obtained in liquid form. Nicotine was dissolved initially with a few drops of ethanol and fur ther diluted to the required concentration with saline, pH adjusted to 7. 4 by sodium hydroxide. For con trol samples, medium containing the same amount of ethanol was used as was done for dissolution of nicotine with saline, the pH adjusted to 7. 4. Cholecystokinin was purchased from Bachem, Philadelphia, Inhibitors,Modulators,Libraries PA.

For inhibitor studies, MAPK inhibitor, UO126, jun kinase inhibitor and p 38 kinase inhibitors were pur chased from. 2 Aminoethoxydiphenyl borate, a reliable Inhibitors,Modulators,Libraries blocker of store operated Ca2 entry and H 7, a broad based, cell permeable serinethreonine kinase inhibitor, were purchased from Calbiochem. Mecamylamine, a nicotinic acetylcholine receptor antag onist, was purchased from Sigma Life Sciences. conotoxin, an N type voltage dependent calcium channels inhibitor was purchased from Peptide Inter national. Isolation of primary pancreatic acinar cells Adult male Sprague Dawley rats were used for the study. The animals Inhibitors,Modulators,Libraries were procured through a protocol approved by the Institutional Animal Care and Use Committee. The animals were acclimatized for a week under con trolled laboratory conditions prior to the study.

After an 18 hour fast, the animals were sacrificed, the pancreas removed quickly and Inhibitors,Modulators,Libraries freed from fat and lymph nodes. Pancreatic acini were isolated by enzymatic digestion according to methods reported previously. Briefly, Krebs Henseleit bicarbonate buffer, pH 7. 4, containing the minimum Eagles Medium supple ment, 67 Uml collagenase, 2 mgml bovine serum albumin, and 0. 1 mgml soybean trypsin inhibitor, was injected into the pancreatic tissue intersti tium. The injected pancreatic tissue was incubated at 37 C in a shaking water bath at a frequency of 120 timesmin for 40 minutes, followed by mechanical disruption of the tissue with gentle suction through pipettes of decreasing orifice sizes.

Acini were then purified by filtration through 150 uM polyethylene mesh and by density gradient centri Inhibitors,Modulators,Libraries fugation with KHB containing 4% BSA. Acini were prein cubated for 30 minutes in HEPES buffered Ringers solution, pH 7. 4. The HR used was the same as KHB, except that it contained 10 mmolL Hepes and 0. 5% BSA. Prior to use, inhibitor Ixazomib the buffer was gassed with 100% O2. After pre incubation, acini were washed and resuspended in fresh HR at a density of 0. 3 0. 4 mgml of acinar protein.