Depolarization of isolated horizontal cells of fish acidifies their immediate surrounding by activating V‐ATPase

In order to interpret the formation of receptive field surrounds in retinal neurons, a proton‐mediated mechanism was proposed to mediate feedback from horizontal cells (HCs) to cone photoreceptors. To verify the idea that depolarized HCs release protons, we measured, by a fluorescence ratiometric technique, the pH of the immediate external surface (pHs) of HCs isolated from the carp or goldfish retina. When HCs stained by 5‐hexadecanoylaminofluorescein, a pH‐sensitive lipophilic dye, were depolarized by bath‐application of kainate or high‐K+ medium, pHs was lowered. The amount of pHs change was monotonically dependent on the degree of depolarization, as much as 0.21 ± 0.05 pH units by 100 mV depolarization (induced by 100 mm K+). Acidification was suppressed by 400 nm bafilomycin A1, a specific inhibitor of the vacuolar type H+ pump (V‐ATPase), suggesting that depolarization released protons from HCs via the voltage‐sensitive H+ pump. Immunocytochemical analysis, using an anti‐V‐ATPase antibody, revealed the existence of V‐ATPase in dissociated HCs. These results support the hypothesis that the feedback from HCs to cones could be proton mediated.