NFATc3 regulates trypsinogen activation, neutrophil recruitment, and tissue damage in acute pancreatitis in mice.

BACKGROUND & AIMS: The signaling mechanisms that regulate trypsinogen activation and inflammation in acute pancreatitis (AP) are unclear. We explored the involvement of the calcium- and calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in development of AP in mice. METHODS: We measured levels of myeloperoxidase and macrophage inflammatory protein 2 (CXCL2), trypsinogen activation, and tissue damage in the pancreas 24 hours after induction of AP by retrograde infusion of taurocholate into the pancreatic ducts of wild-type, NFAT luciferase reporter (NFAT-luc), and NFATc3-deficient mice. We isolated acinar cells and measured NFAT nuclear accumulation, trypsin activity, and expression of NFAT-regulated genes. RESULTS: Infusion of taurocholate increased the transcriptional activity of NFAT in the pancreas, aorta, lung, and spleen of NFAT-luc mice. Inhibition of NFAT with A-285222 blocked taurocholate-induced activation of NFAT in all organs. A-285222 also reduced taurocholate-induced increases in levels of amylase, myeloperoxidase, and CXCL2; activation of trypsinogen; necrosis of acinar cells; edema; leukocyte infiltration; and hemorrhage in the pancreas. NFATc3-deficient mice were protected from these effects of taurocholate. Similar results were obtained using an l-arginine-induced model of AP. Reverse-transcription polymerase chain reaction and confocal immunofluorescence analyses showed that NFATc3 is expressed by acinar cells. NFATc3 expression was activated by stimuli that increase intracellular calcium levels, and activation was prevented by the calcineurin blocker cyclosporin A or A-285222. Activation of trypsinogen by secretagogues in acinar cells was prevented by pharmacologic inhibition of NFAT signaling or lack of NFATc3. A-285222 also reduced expression of inflammatory cytokines such as CXCL2 in acinar cells. CONCLUSIONS: NFATc3 regulates trypsinogen activation, inflammation, and pancreatic tissue damage during development of AP in mice and might be a therapeutic target.

Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons.

We have studied the Ca(2+) leak pathways in the endoplasmic reticulum of pancreatic acinar cells by directly measuring Ca(2+) in the endoplasmic reticulum ([Ca(2+)](ER)). Cytosolic Ca(2+) ([Ca(2+)](C)) was clamped to the resting level by a BAPTA-Ca(2+) mixture. Administration of cholecystokinin within the physiological concentration range caused a graded decrease of [Ca(2+)](ER), and the rate of Ca(2+) release generated by 10 pm cholecystokinin is at least 3x as fast as the basal Ca(2+) leak revealed by inhibition of the endoplasmic reticulum Ca(2+)-ATPase. Acetylcholine also evokes a dose-dependent decrease of [Ca(2+)](ER), with an EC(50) of 0.98 +/- 0.06 microm. Inhibition of receptors for inositol 1,4,5-trisphosphate (IP(3)) by heparin or flunarizine blocks the effect of acetylcholine but only partly blocks the effect of cholecystokinin. 8-NH(2) cyclic ADP-ribose (20 microm) inhibits the action of cholecystokinin, but not of acetylcholine(.) The basal Ca(2+) leak from the endoplasmic reticulum is not blocked by antagonists of the IP(3) receptor, the ryanodine receptor, or the receptor for nicotinic acid adenine dinucleotide phosphate. However, treatment with puromycin (0.1-1 mm) to remove nascent polypeptides from ribosomes increases Ca(2+) leak from the endoplasmic reticulum by a mechanism independent of the endoplasmic reticulum Ca(2+) pumps and of the receptors for IP(3) or ryanodine.