Effects of sulfhydryl reagents on the regulation of cytosolic pH in rat sublingual acini.

Abstract

The effects of sulfhydryl (SH) reagents on the regulation of cytosolic pH (pHi) in rat sublingual mucous acini were examined using pH-sensitive dye 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The membrane permeable SH reagent N-ethylmaleimide (NEM) resulted in a cytosolic acidification (0.19 +/- 0.02 pH unit after 5-min treatment). In contrast, the impermeable SH reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (PCMB) and Cu2+ did not affect the resting pHi. Muscarinic stimulation with carbachol (CCh) induced a cytosolic acidification (0.15 +/- 0.01 pH unit after 5 min). Cu2+ enhanced the CCh-stimulated acidification 2-fold (0.30 +/- 0.02 pH unit after 5 min). Preincubation with SH reducing agent dithiothreitol (DTT) prevented the NEM-induced and Cu2+ -enhanced pHi decreases. The Cu2+ -enhanced but not the NEM-induced acidifications were reversed by addition of DTT after stimulation. Na+/H+ exchange was not influenced by Cu2+ or NEM, suggesting that the SH reagent-induced acidification is not caused by inhibition of exchanger activity. Anion channel inhibitor diphenylamine-2-carboxylate (DPC) abolished the NEM-induced and the Cu2+ -enhanced acidifications, suggesting that the acidification was mediated by HCO3- efflux via anion channels. Cu2+ did not affect the CCh-evoked increase in cytosolic free Ca2+ concentration ([Ca2+]i). In contrast, NEM triggered an elevation in [Ca2+]i, and DTT completely prevented this increase. The CCH-stimulated [Ca2+]i increases were prevented by blocking Ca2+ release from the intracellular pool with 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), and the NEM-stimulated [Ca2+]i increases were abolished by chelating cytosolic Ca2+ with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). TMB-8 prevented the CCh-stimulated, but not the Cu2+ -enhanced pHi decrease, and the NEM-induced pHi decrease was not blocked by BAPTA, suggesting that the effects of Cu2+ and NEM on anion channels are independent of Ca2+. Taken together, these results indicate that SH groups play a critical role in pHi regulation and the reactive SH groups are probably located on cytoplasmic site(s) of anion channels or an associated regulatory protein.