Determinants of intracellular pH in gas gland cells of the swimbladder of the European eel Anguilla anguilla

Abstract

Gas gland cells of the European eel (Anguilla anguilla) were cultured on collagen-coated coverslips, and intracellular pH was measured using the pH-sensitive fluorescent probe 2',7'-bis-(2-carboxypropyl)-5-(6)-carboxyfluorescein (BCPCF). The contributions of various proton-translocating mechanisms to homeostasis of intracellular pH (pHi) were assessed by adding specific inhibitors of the various proton-translocating mechanisms at a constant extracellular pH (pHe) of 7.4 and after artificial acidification of the cells using the ammonium pulse technique. The greatest decrease in pHi was observed after addition of 5-(N-ethyl-N-isobutyl)-amiloride (MIA), an inhibitor of Na(+)/H(+) exchange. Na(+)/H(+) exchange was active under steady-state conditions at an extracellular pH of 7.4, and activity increased after intracellular acidification. Incubation of gas gland cells with 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of anion exchange, also caused a decrease in pHi, but this decrease was not as pronounced as in the presence of MIA. Furthermore, at low pHi, the effect of DIDS was further reduced, suggesting that bicarbonate-exchanging mechanisms are involved in maintaining a steady-state pHi but that their importance is reduced at low pH. Bafilomycin A(1), a specific inhibitor of the V-ATPase, had no effect on steady-state pHi. However, recovery of intracellular pH after an artificial acid load was significantly impaired in the presence of bafilomycin. Our results suggest that Na(+)/H(+) exchange and anion exchange are important for the regulation of pHi at alkaline values of pHe. When pHi is low, a situation probably often encountered by gas gland cells during gas secretion, Na(+)/H(+) exchange continues to play an important role in acid secretion and a V-ATPase appears to contribute to proton secretion.