Intracellular pH regulation and buffer capacity in CO₂/HCO₃/^--buffered media in cultured epithelial cells from rainbow trout gills

The influence of a CO₂/HCO₃/^--buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH(i)) was monitored by continuous spectrofluorometric recording from cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH = 7.70 were transferred to normal CO₂/HCO₃/^--buffered medium {P(CO₂) = 3.71 mmHg, [HCO₃/^-] = 6.1 mmol l^-1, extracellular pH (pH(e)) = 7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (~7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5-8.0 slykes) based on intracellular [HCO₃/^-], and was unaffected by most drugs and treatments. However, after transfer to high P(CO₂) = 11.15 mmHg, [HCO₃/^-] = 18.2 mmol l^-1 at the same pH(e) = 7.70, the final regulated pHi was elevated (~7.53). The rate of correction of alkalosis caused by washout of this high P(CO₂), high-HCO₃/^- medium was unaffected by removal of extracellular Cl^-3. Removal of extracellular Na⁺ lowered resting pH(i) and greatly inhibited the rate of pH(i) recovery from acidosis. Bafilomycin A₁ (3 μmol l^-1) had no effect on these responses. However amiloride (0.2 mmol l^-1) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH(i), the latter differing from the response in HEPES where amiloride also lowered resting pH(i). Similarly 4-acetamido-4-isothiocyanatostilbene-2,2'-disulfonic acid, sodium salt (0.1 mmol l^-1) did not affect resting pH(i) but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl^- also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H⁺ extrusion rate was unaffected. These results are consistent with the presence of Na⁺-(HCO₃/^-)(N) cotransport and/or Na⁺-dependent HCO₃/^-/Cl^- exchange, in addition to Na⁺/H⁺ exchange, as mechanisms contributing to 'housekeeping' pH(i) regulation in gill cells in CO₂/HCO₃/^- media, whereas only Na⁺/H⁺ exchange is seen in HEPES. Both Na⁺-independent Cl^-/HCO₃/^- exchange and V-type H⁺-ATPase mechanisms appear to be absent from these cells cultured in isotonic media.