In accordance towards the results on acinar stimulus secretion coupling (Figure ?(Figure1),1), both providers failed to alter the increase of serum amylase and lipase levels following pancreatitis induction (Figure ?(Number3A3A and B). evaluation, freshly removed pancreata were formalin (4%) fixed, ethanol dehydrated and, inlayed in paraffin. Six micrometer slices were then stained with H&E and subjected to standard light microscopy. Serum amylase and lipase measurements Measurement of serum amylase and lipase activity was performed using commercially available packages (Boehringer Mannheim) following a manufacturers instructions. Measurement of trypsin activity After incubation with or without cerulein, acini were pelleted (10000 test, < 0.05 was considered significant. RESULTS Effects of CEP1347 and SB203580 on acinar TEPP-46 stimulus secretion coupling and trypsin activity We have previously demonstrated that JNK inhibition does not influence acinar stimulus secretion coupling. We, consequently, investigated effects p38 and/or JNK inhibition on cerulein-induced acinar amylase launch (Number ?(Figure1A).1A). Cerulein induced a dose dependent secretory response including the standard biphasic TEPP-46 inhibition with hyperstimulatory amounts (). Neither JNK nor p38 inhibition apparently modified the secretory dose response to cerulein. Therefore, treatment with either 20 M CEP1347 (), 50 M SB203580 () or both providers given simultaneously () did not alter maximal amylase launch not the biphasic dose response. We also measured the effects of stress kinase inhibition on cerulein-induced acinar trypsin activation (Number ?(Figure1B).1B). Active trypsin could be found actually in unstimulated acini, but treatment with cerulein led to a dose dependent increase of acinar trypsin activity (). Interestingly, using CEP1347 (), we observed a inclination towards reduced trypsin activation while SB203580 () appeared to increase trypsin activation. However, in isolated acini, this effect was not statistically significant. Open in a separate window Number 1 Effects of CEP1347 and SB203580 on acinar stimulus secretion coupling and trypsin activation. Acutely isolated acini were incubated with the indicated amounts of cerulein for 30 min (). Acini were also treated with 20 M CEP1347 (), 50 M SB203580 () or both providers simultaneously (). A: Amylase launch into the supernatant was identified and indicated as % of total content material. Stimulus secretion coupling was not significantly modified by stress kinase inhibitors. B: Active trypsin was measured in acinar homogenates and indicated as pg/mg protein. Although SB203580 treated acini experienced a inclination towards higher amounts of active trypsin, this effect was not statistically significant. SB203580 inhibits cerulein-induced p38 kinase activation in vivo In order to compare the effects of JNK and p38 inhibition inhibition of pancreatic TEPP-46 p38 activity could be accomplished. In contrast to JNK[9,10], p38 is definitely constitutively active in the pancreas (Number ?(Number2,2, lane 1). Treatment with 30 mg/kg cerulein resulted in strong p38 activation (lane 2). Treatment with 3 mg/kg SB203580 reduced cerulein-induced p38 activation by half while 30 mg/ kg SB203580 suppressed cerulein-induced p38 activation almost completely (Number ?(Number2,2, lanes 3 and 4). However, even the highest SB203580 dose could not reduce pancreatic p38 kinase activity below basal levels (Number ?(Number2,2, lane 4). SB203580 experienced no apparent effect on cerulein-induced activation of JNK or ERK (not shown). Open in a separate window Number 2 SB203580 inhibits cerulein-induced p38 kinase activation phosphorylation of HSP27 following Esm1 immunoprecipitation of MAPKAPK2 was used as read-out of p38 activity. 30 mg SB203580 almost completely abolished cerulein-induced pancreatic p38 activation. Effects of CEP1347 and SB203580 on biochemical guidelines of pancreatitis Relating to our earlier results, we used 30mg of either CEP1347 and/or SB203580 (Number ?(Number2)2) given sc 4h prior to cerulein. In accordance to the results on acinar stimulus secretion coupling (Number ?(Figure1),1), both providers failed to alter the increase of serum amylase and lipase levels following.