ISSN:
1432-1424
Keywords:
Na+/H+ exchange
;
salivary gland
;
muscarinic stimulation
;
anion channel
;
fluid secretion
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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+].
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00232912
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