Stimulation of active sodium-potassium transport by hydrogen peroxide in cultured rabbit nonpigmented ciliary epithelium

Purpose. Studies were conducted to examine the effect of hydrogen peroxide on active sodium-potassium transport in a cell line derived from nonpigmented ciliary epithelium of the rabbit eye. Methods. Studies were carried out using a rabbit nonpigmented ciliary epithelium cell line. ⁸⁶Rb uptake by intact cells was measured in the presence or absence of ouabain. The ouabain-sensitive potassium (⁸⁶Rb) uptake rate was used as an index of the rate of active sodium-potassium transport. Cell sodium content was measured by atomic absorption spectrophotometry. Na,K-ATPase activity was determined by measuring ATP hydrolysis in the presence or absence of ouabain, using membrane material isolated by centrifugation of cell homogenates. Results. Ouabain-sensitive potassium (⁸⁶Rb) uptake rate measured in cells that had been preincubated with 200 μM hydrogen peroxide for either 30 min or 60 min was increased to 196% and 181% of the control uptake rate, respectively. Lesser concentrations of hydrogen peroxide caused lesser degrees of stimulation. 200 μM hydrogen peroxide caused an increase of cell sodium content. Such a change of cell sodium content is likely to be responsible, at least in part, for the observed stimulation of active sodiumpotassium transport. However, the response may also be partly dependent on activation of a protein kinase since the serine/threonine protein kinase inhibitors staurosporine (1 μM) and H-89 (20 μM) were both found to prevent the stimulatory effect of 200 μM hydrogen peroxide on ouabain-sensitive potassium (⁸⁶Rb) uptake. Interestingly, neither H-89 nor staurosporine prevented the elevation of sodium content in cells that received 200 μM hydrogen peroxide. Conclusions. Taken together, these findings suggest a low concentration of hydrogen peroxide causes increased sodium entry into the cell and also activates a protein kinase-dependent mechanism for sodium pump stimulation. The protein kinase-dependent mechanism does not appear to be triggered by an increased rate of sodium entry since staurosporine did not prevent the stimulation of ouabain-sensitive potassium (⁸⁶Rb) uptake elicited by an increase in sodium permeability caused by amphotericin B.