Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

Summary

The regulation of intracellular pH (pH _i ) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO ⁻₃ -containing medium (25 mm NaHCO₃/5% CO₂; pH 7.4) have a steady-state pH _i of 7.25±0.02. Acid loading of acinar cells using the NH ⁺₄ /NH₃ prepulse technique resulted in a Na⁺-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, K _i ∼ 0.42 μm) pH _i recovery, the kinetics of which were not influenced by the absence of extracellular Cl⁻. The rate and magnitude of the pH _i recovery were dependent on the extracellular Na⁺ concentration, indicating that Na⁺/H⁺ exchange plays a critical role in maintaining pH _i above the pH predicted for electrochemical equilibrium. When the NH ⁺₄ /NH₃ concentration was varied, the rate of pH _i recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na⁺/H⁺ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl⁻-free medium did not significantly alter resting pH _i , suggesting the absence of Cl⁻/HCO ⁻₃ exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t1/2 < 30 sec; 0.16 ± 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37±0.05 pH unit) or in the absence of extracellular Na⁺ (0.34±0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO ⁻₃ -free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl⁻. The Ca²⁺ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid) to chelate intracellular Ca²⁺ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na⁺/H⁺ exchange activity; (ii) is most likely mediated by HCO ⁻₃ efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca²⁺].