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

Ha-Van Nguyen, Gary E. Shull* and James E. Melvin

1. 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.

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 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).

3. The agonist-induced intracellular acidification was inhibited by the anion channel blocker niflumate, and was prevented in the absence of HCO₃^- by treatment with the carbonic anhydrase inhibitor methazolamide. These results indicate that the muscarinic-induced acidification is due to HCO₃^- loss, probably mediated by an anion conductive pathway.

4. 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.

5. 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.

6. 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⁺-HCO₃^- cotransport, but amiloride-sensitive Na⁺-H⁺ exchange via isoform 1.

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