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

Abstract

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. 86 Rb uptake by intact cells was measured in the presence or absence of ouabain. The ouabain-sensitive potassium (86 Rb) 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 (86 Rb) uptake rate measured in cells that had been preincubated with 200µM 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. 200µM 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 sodium-potassium transport. However, the response may also be partly dependent on activation of a protein kinase since the serine/threonine protein kinase inhibitors staurosporine (1µM) and H-89 (20µM) were both found to prevent the stimulatory effect of 200µM hydrogen peroxide on ouabain-sensitive potassium (86 Rb) uptake. Interestingly, neither H-89 nor staurosporine prevented the elevation of sodium content in cells that received 200µM 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 (86 Rb) uptake elicited by an increase in sodium permeability caused by amphotericin B.