Referenced in: none

Automatically associated channels: Kv10.1

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

Authors: G H Zhang, J E Melvin

Journal, date & volume: Proc. Soc. Exp. Biol. Med., 1996 Feb , 211, 190-8

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/8599027

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.