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 (pH₁) in rat sublingual mucous acini were examined using the 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-chloromercurlbenzoic acid (PCMB) and Cu²⁺ did not affect the resting pH₁. 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 pH, 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⁺]₁). In contrast, NEM triggered an elevation in [Ca²⁺]₁, and DTT completely prevented this increase. The CCH-stimulated [Ca²⁺]₁ 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⁺]₁ 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 pH_i decrease, and the NEM-induced pH_i 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 pH_i regulation and the reactive SH groups are probably located on cytoplasmic site(s) of anion channels or an associated regulatory protein. [P.S.E.B.M. 1996, Vol 211]