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Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca2+ release through IP3 receptors.

ABSTRACT: Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, which is the initiating step for alcohol-induced pancreatitis. We used intact or two-photon permeabilized acinar cells isolated from wild-type mice or mice in which inositol trisphosphate receptors of type 2 or types 2 and 3 were knocked out. In permeabilized pancreatic acinar cells even a relatively low ethanol concentration elicited calcium release from intracellular stores and intracellular trypsinogen activation. The calcium sensor calmodulin (at a normal intracellular concentration) markedly reduced ethanol-induced calcium release and trypsinogen activation in permeabilized cells, effects prevented by the calmodulin inhibitor peptide. A calmodulin activator virtually abolished the modest ethanol effects in intact cells. Both ethanol-elicited calcium liberation and trypsinogen activation were significantly reduced in cells from type 2 inositol trisphosphate receptor knockout mice. More profound reductions were seen in cells from double inositol trisphosphate receptor (types 2 and 3) knockout mice. The inositol trisphosphate receptors, required for normal pancreatic stimulus-secretion coupling, are also responsible for the toxic ethanol action. Calmodulin protects by reducing calcium release sensitivity.

SUBMITTER: Gerasimenko JV

PROVIDER: S-EPMC3078340 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca2+ release through IP3 receptors.

Gerasimenko Julia V JV Lur György G Ferdek Pawel P Sherwood Mark W MW Ebisui Etsuko E Tepikin Alexei V AV Mikoshiba Katsuhiko K Petersen Ole H OH Gerasimenko Oleg V OV

Proceedings of the National Academy of Sciences of the United States of America 20110321 14

Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, whic ...[more]

PMID: 21436055

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Project description:InsP(3) binding to type-1, but not type-3, InsP(3) receptors is inhibited by calmodulin in a Ca(2+)-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca(2+) mobilization by type-1 InsP(3) receptors of cerebellum is inhibited by calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627-11632]. Using cell types expressing predominantly type-1, -2 or -3 InsP(3) receptors, we show that InsP(3)-evoked Ca(2+) mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC(50) approximately 15 microM) inhibited InsP(3)-evoked Ca(2+) release only in the presence of Ca(2+). The inhibition was unaffected by calcineurin inhibitors. The effect of calmodulin did not result from enhanced metabolism of InsP(3) because calmodulin also decreased the sensitivity of the Ca(2+) stores to adenophostin A, a non-metabolizable InsP(3)-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP(3) receptors was unaffected by Ca(2+)-calmodulin. Using a scintillation proximity assay to measure (125)I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP(3) receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound (125)I-calmodulin. The higher-affinity site (cyt11) was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that Ca(2+)-independent binding of calmodulin to a site within the first 159 residues of the type-1 InsP(3) receptor inhibits InsP(3) binding and may thereby regulate the kinetics of Ca(2+) release. Ca(2+)-dependent inhibition of Ca(2+) release by calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or Ca(2+)-calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit Ca(2+) release from any tetrameric receptor that includes a type-1 subunit.

Dataset Information

Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca2+ release through IP3 receptors.

Publications

Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca2+ release through IP3 receptors.

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