Protein synthesis inhibitors enhance secretagogue sensitivity of in vitro rat pancreatic acini

Abstract

Isolated rat pancreatic acini were treated with cycloheximide and amylase release was measured. This agent increased the sensitivity to both synthetic octapeptide of cholecystokinin (CCK8) and carbamylcholine, the major secretagogues known to utilize Ca2+ as a second messenger. The mechanism of the cycloheximide effect was via inhibition of protein synthesis, as indicated by the following: 1) the concentration of cycloheximide used inhibited leucine incorporation by greater than 90%; 2) this effect was not instantaneous but increased up to a 2-h pretreatment; and 3) a similar effect was obtained with puromycin, a chemically different inhibitor of protein synthesis. Cycloheximide acted on the steps by which secretagogues mobilize cellular Ca2+ because the dose-response curve for 45Ca2+ efflux was shifted to the same extent as that for amylase release, whereas the dose-response curve for amylase release induced by the Ca2+ ionophore A23187 was not altered. The results suggest, therefore, that a rapidly turning-over protein present in pancreatic acinar cells exerts an inhibitory influence on Ca2+ mobilization by secretagogues.

Effect of Hyperthermia on Premature Intracellular Trypsinogen Activation in the Exocrine Pancreas

“…For experiments in which we studied whether the effect of hyperthermia on zymogen activation in living acini depended on the synthesis or upregulation of heat shock proteins we completely blocked protein synthesis using the inhibitor cycloheximide (300 M) for 30 min prior to the experiments, as well as throughout the incubation period, and as previously described (12). After exposure to temperatures ranging from 37 to 41°C and to either Caerulein (10 nM) or NaCl (controls) as indicated above acini were lysed in 250 l Ripabuffer at pH 8.0 containing Tris-HCl (100 mM), NaCl (300 mM), 0.2% SDS, 2% Triton-X-100, 2% Na-deoxycholate, and a protease inhibitor cocktail (1 ml/mg tissue) composed of Aprotinin (10 g/ml), Leupeptin (10 g/ml), sodiumpyrophosphate (0.01 M), sodiumfluoride (0.1 M), dihydrogenperoxide (1 mM), L-phenyl-methyl-sulfonylfluoride (PMSF, 1 mM), and 0.02% soybean-trypsin-inhibitor.…”