Determinants of intracellular pH in gas gland cells of the swimbladder of the European eel Anguilla anguilla.
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.
Sötz
E
E
Institut für Zoologie und Limnologie, Universität Innsbruck, A-6020 Innsbruck, Austria.
Niederstätter
H
H
Pelster
B
B
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000400:Air Sacs / Q000166:cytology / Q000502:physiology*; D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000811:Anguilla; D000818:Animals; D000838:Anions; D001639:Bicarbonates / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012965:Sodium Chloride / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors
D000838:Anions; D001639:Bicarbonates; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; D012965:Sodium Chloride; D000584:Amiloride; C056369:5-(N-methyl-N-isobutyl)amiloride; D025262:Vacuolar Proton-Translocating ATPases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid