Determinants of intracellular pH in gas gland cells of the swimbladder of the European eel Anguilla anguilla
2002
SUMMARY 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.
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