Stimulation of active sodium-potassium transport by hydrogen peroxide in cultured rabbit nonpigmented ciliary epithelium.
OBJECTIVE
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 200microM 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. 200microM 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 (1microM) and H-89 (20microM) were both found to prevent the stimulatory effect of 200microM hydrogen peroxide on ouabain-sensitive potassium (86Rb) uptake. Interestingly, neither H-89 nor staurosporine prevented the elevation of sodium content in cells that received 200microM 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.
Chin
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Department of Ophthalmology & Visual Science, University of Louisville School of Medicine, KY 40202, USA.
Delamere
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D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002924:Ciliary Body / Q000166:cytology / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011817:Rabbits; D012413:Rubidium / Q000493:pharmacokinetics; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013449:Sulfonamides
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D028021:Sodium-Potassium-Chloride Symporters; D013449:Sulfonamides; D010042:Ouabain; D012964:Sodium; D006861:Hydrogen Peroxide; D000254:Sodium-Potassium-Exchanging ATPase; D019311:Staurosporine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D012413:Rubidium