Muscarinic receptor‐induced acidification in sublingual mucous acinar cells: loss of pH recovery in Na + −H + exchanger‐1 deficient mice

Intracellular pH (pH i ) plays an important role in regulating fluid and electrolyte secretion by salivary gland acinar cells. The pH‐sensitive, fluorescent dye 2′,7′‐bis(carboxyethyl)−5(6)‐carboxylfluorescein (BCECF) was used to characterize the mechanisms involved in regulating pH i during muscarinic stimulation in mouse sublingual mucous acinar cells. In the presence of HCO 3 − , muscarinic stimulation caused a rapid decrease in pH i (0.24 ± 0.02 pH units) followed by a slow recovery rate (0.042 ± 0.002 pH units min −1 ) to the initial resting pH i in sublingual acinar cells. The muscarinic receptor‐induced acidification in parotid acinar cells was of a similar magnitude (0.25 ± 0.02 pH units), but in contrast, the recovery rate was ≈4‐fold faster (0.181 ± 0.005 pH units min −1 ). The agonist‐induced intracellular acidification was inhibited by the anion channel blocker niflumate, and was prevented in the absence of HCO 3 − by treatment with the carbonic anhydrase inhibitor methazolamide. These results indicate that the muscarinic‐induced acidification is due to HCO 3 − loss, probably mediated by an anion conductive pathway. The Na + −H + exchange inhibitor 5‐( N‐ ethyl‐ N‐ isopropyl)amiloride (EIPA) amplified the magnitude of the agonist‐induced acidification and completely blocked the Na + ‐dependent pH i recovery. To examine the molecular nature of the Na + −H + exchange mechanism in sublingual acinar cells, pH regulation was investigated in mice lacking Na + −H + exchanger isoforms 1 and 2 (NHE1 and NHE2, respectively). The magnitude and the rate of pH i recovery in response to an acid load in acinar cells isolated from mice lacking NHE2 were comparable to that observed in cells from wild‐type animals. In contrast, targeted disruption of the Nhe1 gene completely abolished pH i recovery from an acid load. These results demonstrate that NHE1 is critical for regulating pH i during a muscarinic agonist‐stimulated acid challenge and probably plays an important role in regulating fluid secretion in the sublingual exocrine gland. In NHE1‐deficient mice, sublingual acinar cells failed to recover from an acid load in the presence of bicarbonate. These results confirm that the major regulatory mechanism involved in pH i recovery from an acid load is not Na + −HCO 3 − cotransport, but amiloride‐sensitive Na + −H + exchange via isoform 1.