HCO3- secretion by murine nasal submucosal gland serous acinar cells during Ca2+-stimulated fluid secretion

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca2+-activated Cl- secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca2+-activated Cl- secretion was accompanied by secretion of HCO3-, possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH(i)) and cell volume. Resting pH(i) was 7.17 +/- 0.01 in physiological medium (5% CO2-25 mM HCO3-). During carbachol (CCh) stimulation, pH(i) fell transiently by 0.08 +/- 0.01 U concomitantly with a fall in Cl- content revealed by cell shrinkage, reflecting Cl- secretion. A subsequent alkalinization elevated pH(i) to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO2-HCO3- -free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO3- efflux by ion substitution or exposure to the Cl- channel inhibitor niflumic acid (100 mu M) strongly inhibited agonist-induced acidifi cation by > 80% and > 70%, respectively. The Na+/H+ exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidifi cation. Gene expression profiling suggested that serous cells express NHE isoforms 1-4 and 6-9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pH(i) recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO3- during Ca2+-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl- channel, with HCO3- secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na+-dependent pH(i) regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na+-free media.