PKC{alpha}{beta}{gamma}- and PKC{delta}-dependent endocytosis of NBCe1-A and NBCe1-B in salivary parotid acinar cells.
We examined membrane trafficking of NBCe1-A and NBCe1-B variants of the electrogenic Na(+)-HCO(3)(-) cotransporter (NBCe1) encoded by the SLC4A4 gene, using confocal fluorescent microscopy in rat parotid acinar cells (ParC5 and ParC10). We showed that yellow fluorescent protein (YFP)-tagged NBCe1-A and green fluorescent protein (GFP)-tagged NBCe1-B are colocalized with E-cadherin in the basolateral membrane (BLM) but not with the apical membrane marker zona occludens 1 (ZO-1). We inhibited constitutive recycling with monensin and W13 and detected that NBCe1-A and NBCe1-B accumulated in vesicles marked with the early endosomal marker early endosome antigen-1 (EEA1), with a parallel loss from the BLM. We observed that NBCe1-A and NBCe1-B undergo massive carbachol (CCh)-stimulated redistribution from the BLM into early endosomes. We showed that internalization of NBCe1-A and NBCe1-B was prevented by the general PKC inhibitor GF-109203X, the PKCalphabetagamma-specific inhibitor Gö-6976, and the PKCdelta-specific inhibitor rottlerin. We verified the involvement of PKCdelta by blocking CCh-induced internalization of NBCe1-A-cyan fluorescent protein (CFP) in cells transfected with dominant-negative kinase-dead (Lys376Arg) PKCdelta-GFP. Our data suggest that NBCe1-A and NBCe1-B undergo constitutive and CCh-stimulated endocytosis regulated by conventional PKCs (PKCalphabetagamma) and by novel PKCdelta in rat epithelial cells. To help develop a more complete model of the role of NBCe1 in parotid acinar cells we also investigated the initial phase of the secretory response to cholinergic agonist. In an Ussing chamber study we showed that inhibition of basolateral NBCe1 with 5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-carboxamide (tenidap) significantly decreases an initial phase of luminal anion secretion measured as a transient short-circuit current (I(sc)) across ParC10 cell monolayers. Using trafficking and functional data we propose a model that describes a physiological role of NBC in salivary acinar cell secretion.
Department of Physiology and Biophysics, University of Colorado Denver, Aurora, 80045, USA.
Clint
C
Perry
Olga J
OJ
Baker
Mary E
ME
Reyland
Irina I
II
Grichtchenko
D000818:Animals; D001120:Arginine; D002217:Carbachol / Q000494:pharmacology; D002461:Cell Line, Transformed; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005799:Genes, Dominant; D007211:Indoles / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008239:Lysine; D008985:Monensin / Q000494:pharmacology; D009154:Mutation; D000078183:Oxindoles; D010306:Parotid Gland / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D027982:Sodium-Bicarbonate Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014018:Tissue Distribution
D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D000078183:Oxindoles; D020033:Protein Isoforms; C422004:Slc4a4 protein, rat; D027982:Sodium-Bicarbonate Symporters; D002217:Carbachol; D008985:Monensin; D001120:Arginine; C062866:tenidap; D011493:Protein Kinase C; D008239:Lysine; D013755:Tetradecanoylphorbol Acetate