Extracellular pH in the isolated retina of the toad in darkness and during illumination

1. Extracellular pH (pHo) was measured in the isolated retina preparation of the toad, Bufo marinus, using H(+)-selective microelectrodes. During superfusion with phosphate-buffered solution (pH 7.8), which had a low buffering capacity, pHo in the inner retina was 7.0-7.2 and there was a pHo gradient throughout the distal retina and into the bathing solution. 2. The retinal acidity appears to be due in part to the combined reactions of glycolysis and ATP hydrolysis, since anoxia greatly increased the pHo gradient, while superfusion with either glucose-free pyruvate solution or strophanthidin decreased this gradient. 3. Maintained illumination evoked both an acidification in the proximal retina and an alkalinization in the distal retina. Blocking synaptic transmission to second-order neurones (1.0 mM-aspartate) decreased the acidification but had little effect on the alkalinization, consistent with the notion that the alkalinization is of receptoral origin, while the acidification is of post-receptoral origin. 4. Retinal neurones extrude a significant amount of acid via Na(+)-H+ exchange, since 2.0 mM-amiloride, a blocker of Na(+)-H+ exchange, caused a sustained alkalinization in darkness and decreased the light-evoked changes in pHo, while 1.0 mM-4-acetamido-4'-isothiocyanatostilbene-2.2'-disulphonic acid (SITS), a blocker of Cl(-)-HCO3- exchange, produced a much smaller alkalinization. 5. Switching to a bicarbonate-buffered solution having a 75 times greater buffering capacity than the phosphate-buffered solution caused retinal pHo to become less acidic and significantly decreased the amplitude of the light-evoked pHo changes. 6. Addition of 2.0 mM-acetazolamide, a carbonic anhydrase inhibitor, to the bicarbonate-buffered solution increased both the pHo gradient and the light-evoked changes in pHo. These data are consistent with the idea that carbonic anhydrase, which is concentrated in Müller (glial) cells and to a lesser extent in horizontal cells, increases the effectiveness of the bicarbonate buffer system. 7. Switching from bicarbonate-buffered to phosphate-buffered solutions attenuated the b-wave of the electroretinogram, most likely by acidifying pHo. Overall, our results emphasize the importance of the bicarbonate buffer system in buffering pHo during periods of variable acid extrusion in light and in darkness.