It was previously shown that ouabain decreases the potential difference across anin vitro preparation of bullfrog retinal pigment epithelium (RPE) when applied to the apical, but not the basal, membrane and that the net basal-to-apical Na+ transport is also inhibited by apical ouabain. This suggested the presence of a Na+−K+ pump on the apical membrane of the RPE. In the present experiments, ... [Show full abstract] intracellular recordings from RPE cells show that this pump is electrogenic and contributes approximately −10 mV to the apical membrane potential (V
AP). Apical ouabain depolarizedV
AP in two phases. The initial, fast phase was due to the removal of the direct, electrogenic component. In the first one minute of the response to ouabain,V
AP depolarized at an average rate of 4.4±0.42 mV/min (n=10, mean ±sem), andV
AP depolarized an average of 9.6±0.5 mV during the entire fast phase. A slow phase of membrane depolarization, due to ionic gradients running down across both membranes, continued for hours at a much slower rate, 0.4 mV/min. Using a simple diffusion model and K+-specific microelectrodes, it was possible to infer that the onset of the ouabain-induced depolarization coincided with the arrival of ouabain molecules at the apical membrane. This result must occur if ouabain affects an electrogenic pump. Other metabolic inhibitors, such as DNP and cold, also produced a fast depolarization of the apical membrane. For a decrease in temperature of ≃10°C, the average depolarization of the apical membrane was 7.1±3.4 mV (n=5) and the average decrease in transepithelial potential was 3.9±0.3 mV (n=10). These changes in potential were much larger than could be explained by the effect of temperature on anRT/F electrodiffusion factor. Cooling the tissue inhibited the same mechanism as ouabain, since prior exposure to ouabain greatly reduced the magnitude of the cold effect. Bathing the tissue in 0mm [K+] solution for 2 hr inhibited the electrogenic pump, and subsequent re-introduction of 2mm [K+] solution produced a rapid membrane hyperpolarization. We conclude that the electrogenic nature of this pump is important to retinal function, since its contribution to the apical membrane potential is likely to affect the transport of ions, metabolites, and fluid across the RPE.