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

1. Intracellular pH (pH(i)) plays an important role in regulating fluid and electrolyte secretion by salivary gland acinar cells. The pH-sensitive, fl uorescent dye 2',7'-bis(carboxyethyl)-5(6)carboxylfluorescein (BCECF) was u sed to characterize the mechanisms involved in regulating pH(i) during musc arinic stimulation in mouse sublingual mucous acinar cells. 2. In the presence of HCO3-, 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 acin ar cells. The muscarinic receptor-induced acidification in parotid acinar c ells was of a similar magnitude (0.25 +/- 0.02 pH units), but in contrast, the recovery rate was similar to 4-fold faster (0.181 +/- 0.005 pH units mi n(-1)). 3. The agonist-induced intracellular acidification was inhibited by the ani on channel blocker niflmate, and was prevented in the absence of HCO3- by t reatment with the carbonic anhydrase inhibitor methazolamide. These results indicate that the muscarinic-induced acidification is due to HCO3- loss, p robably mediated by an anion conductive pathway. 4. The Na+-H+ exchange inhibitor 5-(Nethyl-N-isopropyl)amiloride (EIPA) amp lified the magnitude of the agonist-induced acidification and completely bl ocked the Na+-dependent pH(i) recovery. 5. To examine the molecular nature of the Na+-H+ exchange mechanism in subl ingual acinar cells, pH regulation was investigated in mice lacking Na+-Hexchanger 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 iso lated from mice lacking NHE2 were comparable to that observed in cells from wild-type animals. In contrast, targeted disruption of the Nhe1 gene compl etely abolished pH(i) recovery from an acid load. These results demonstrate that NHE1 is critical for regulating pH(i) during a muscarinic agonist-sti mulated acid challenge and probably plays an important role in regulating f luid secretion in the sublingual exocrine gland. 6. In NHE1-deficient mice, sublingual acinar cells failed to recover from a n 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+-HCO3- cotransport, but amiloride-sensitive Na+-H+ exchange via isof orm 1.