PKCαβγ- and PKCδ-dependent endocytosis of NBCe1-A and NBCe1-B in salivary parotid acinar cells

Perry C, Baker OJ, Reyland ME, Grichtchenko II. PKC alpha beta gamma- and PKC delta-dependent endocytosis of NBCe1-A and NBCe1-B in salivary parotid acinar cells. Am J Physiol Cell Physiol 297: C1409-C1423, 2009. First published September 30, 2009; doi:10.1152/ajpcell.00028.2009.-We examined membrane trafficking of NBCe1-A and NBCe1-B variants of the electrogenic Na+-HCO3- 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 PKC alpha beta gamma-specific inhibitor Go-6976, and the PKC delta-specific inhibitor rottlerin. We verified the involvement of PKC delta by blocking CCh-induced internalization of NBCe1-A-cyan fluorescent protein (CFP) in cells transfected with dominant-negative kinase-dead (Lys376Arg) PKC delta-GFP. Our data suggest that NBCe1-A and NBCe1-B undergo constitutive and CCh-stimulated endocytosis regulated by conventional PKCs (PKC alpha beta gamma) and by novel PKC delta 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.