pH changes in frog rods upon manipulation of putative pH-regulating transport mechanisms

Abstract

Rod intracellular pH (pHi) in the intact frog retina was measured fluorometrically with the dye 2′,7′-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein under treatments chosen to affect putative pH-regulating transport mechanisms in the plasma membrane. The purpose was to relate possible pHi changes to previously reported effects on photoresponses. In nominally bicarbonate-free Ringer, application of amiloride (1 mM) or substitution of 95 mM external Na+ by K+ or choline triggered monotonic but reversible acidifications, consistent with inhibition of Na+/H+ exchange. Bicarbonate-dependent mechanisms were characterized as follows: (1) Replacing half of a 12 mM phosphate buffer by bicarbonate caused a sustained rise of pHi. (2) Subsequent application of the anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS, 0.2 mM) set off a slow acidification. (3) Substitution of external Cl− by gluconate (95 mM) caused a rapid pHi rise both in normal Na+ and low-Na+ perfusion. (4) This effect was inhibited by DIDS. The results support a consistent explanation of parallel electrophysiological experiments on the assumption that intracellular acidifications reduce and alkalinizations (in a certain range) augment photoresponses. It is concluded that both Na+/H+ exchange and bicarbonate transport control rod pHi, modulating the light-sensitive current. Part of the bicarbonate transport is by Na+-independent HCO3−/Cl− exchange, but a further Na+-coupled bicarbonate import mechanism is implicated.