Regulation of intracellular pH in salamander retinal rods

Abstract

1. We measured intracellular pH (pH(i)) in rods isolated from the retina of the axolotl salamander, Ambystoma mexicanum, using the fluorescent indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). 2. The light exposures associated with data acquisition had no marked effect on pH(i). There was no sharp change between the value obtained from the first exposure of dark-adapted rods and subsequent readings. Increasing the acquisition frequency from 1 to 10 min-1 either had no effect, or brought about a slow acidification, which was stopped or reversed when the low frequency was restored. 3. In nominally HCO3--free solution at pH 7.5, the rods had a steady-state pH(i) of 7.09 ± 0.02 (n = 46) and a buffering power (β1) of 24 ± 1 mM (pH unit)-1 (n = 48). The buffering power was virtually constant in the pH range 8.6-8.0. In the same range, pH(i) depended linearly on perfusion pH (pH(o)) with regression coefficients of 0.4-0.5. 4. There were no significant differences between the inner and enter segment of intact rods as regards steady-state pH(i) or responses to experimental treatments. 5. Recovery from an intracellular acid load imposed by sodium propionate or an NH4Cl prepulse in nominally bicarbonate-free perfusate was completely blocked by decreasing the extracellular Na+ concentration to 7 mM, and slowed by 86% by applying 1 mM amiloride. 6. Introduction of 2% CO2-13 mM HCO3- caused an alkalinization that was often preceded by a transient acidification. Steady-state pH(i) was on average 0.1 pH units higher than in nominally bicarbonate-free solution. The mean acid extrusion rate, calculated on the assumption that CO2-HCO3- behaves as an open system, was 19% higher (31 ± 2 mM h-1) than in a solution buffered only by Hepes (26 ± 2 mM h-1). 7. In the presence of CO2-HCO3-, 100 μM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) decreased the acid extrusion rate by 20% on average. Lowering the extracellular Cl- concentration to 7 mM raised pH(i), but did not significantly affect the acid extrusion rate. 8. We conclude that retinal rods regulate pH(i) by both Na+-H+ exchange and mechanism(s) involving HCO3--Cl- exchange. In the present conditions, the Na+-H+ exchanger appears as the dominant mechanism for acid extrusion.