Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells.
Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO(3)(-) across the BLM, thus supporting HCO(3)(-) luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.
Perry
Clint
C
Univ. of Colorado and Denver Health Sciences Center, Dept. of Physiology and Biophysics, Mail Stop 8307, P.O. Box 6511, Aurora, CO 80045, USA.
Quissell
David O
DO
Reyland
Mary E
ME
Grichtchenko
Irina I
II
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D019921:Biotinylation; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D016764:Cell Polarity; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000378:metabolism; D018014:Gene Transfer Techniques; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D018613:Microscopy, Confocal; D008985:Monensin / Q000494:pharmacology; D010306:Parotid Gland / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014982:Xenopus laevis
D001639:Bicarbonates; D002147:Calmodulin; D018679:Cholinergic Agonists; D007211:Indoles; D008301:Maleimides; D047428:Protein Kinase Inhibitors; D011976:Receptors, Muscarinic; C422004:Slc4a4 protein, rat; C498902:Slc4a7 protein, rat; D027982:Sodium-Bicarbonate Symporters; D013449:Sulfonamides; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; D002217:Carbachol; D008985:Monensin; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate