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Research Article

PKCβ- and PKC-dependent endocytosis of NBCe1-A and NBCe1-B in salivary parotid acinar cells

¹University of Colorado and Denver Health Sciences Center ²University of Missouri - Columbia

Submitted 16 January 2009 ; revised 18 September 2009 ; accepted in final form 20 September 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 the rat parotid acinar cells, ParC5 and ParC10. We showed that YFP-tagged NBCe1-A and GFP-tagged NBCe1-B are co-localized with ECad in the basolateral membrane (BLM) but not with apical membrane marker ZO1. 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 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 general PKC inhibitor GF109203X, the PKCβ specific inhibitor GÖ6976, and the PKC specific inhibitor rottlerin. We verified the involvement of PKC by blocking CCh-induced internalization of NBCe1-A-CFP in cells transfected with dominant-negative kinase-dead (Lys376Arg) PKC-GFP. Our data suggest that NBCe1-A and NBCe1-B undergo constitutive and CCh-stimulated endocytosis regulated by conventional PKCs (PKCβ) and by novel, PKC, 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 tenidap significantly decreases an initial phase of luminal anion secretion measured as a transient short circuit current (Isc) 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.

internalization; model of saliva secretion; muscarinic; Ussing chamber