Relationship between a HCO3- -permeable conductance and a CLCA protein from rat pancreatic zymogen granules.
Ca(2+)-induced enzyme secretion in the exocrine pancreas is not completely understood. We have proposed that Ca(2+)-induced enzyme secretion in the exocrine pancreas involves activation of ion conductances in the membrane of zymogen granules (ZG). Here we have identified a Ca(2+)-activated anion conductance in rat pancreatic ZG membranes (ZGM). Ca(2+) (2.5-50 microM) increased the conductance for I(-), NO(3)(-), Br(-), or HCO(3)(-), but not for Cl(-), as determined by the rate of valinomycin-induced osmotic lysis of ZG suspended in isotonic K(+)-salts. 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonate (100 microM) or 25 microM dithiothreitol strongly inhibited Ca(2+)-dependent lysis. The permeability sequence, Ca(2+) dependence, and inhibitor sensitivity of ZG anion conductance are reminiscent of a family of epithelial Ca(2+)-activated anion channels (CLCA). CLCA expression was confirmed by RT-PCR with rat pancreatic mRNA and mouse CLCA1 primers. A PCR product (580bp) exhibited 81%, 77%, and 57% amino acid similarity to the three mouse isoforms mCLCA-1, -2, and -3 (mgob-5), respectively. Antibodies against bovine tracheal CLCA1 showed CLCA expression in ZGM by immunoblotting, immunoperoxidase light microscopy, and immunogold labeling. These findings suggest that a CLCA-related protein could account for the Ca(2+)-activated HCO(3)(-) conductance of rat pancreatic ZGM and contribute to hormone-stimulated enzyme secretion.
Thévenod
Frank
F
Department of Physiology and Pathophysiology, Medical Faculty, University of Witten/Herdecke, Stockumer Strasse 12, Thyssenhaus, D-58448 Witten, Germany. frank.thevenod@uni-wh.de
Roussa
Eleni
E
Benos
Dale J
DJ
Fuller
Catherine M
CM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D018118:Chloride Channels / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D004229:Dithiothreitol / Q000494:pharmacology; D004553:Electric Conductivity; D005089:Exocytosis; D007425:Intracellular Membranes / Q000737:chemistry; D017136:Ion Transport; D008297:Male; D008969:Molecular Sequence Data; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D016415:Sequence Alignment
D001639:Bicarbonates; D018118:Chloride Channels; C018564:dihydro-DIDS; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium; D004229:Dithiothreitol