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

Shinshi Chin, Nicholas A Delamere

Research output: Contribution to journalArticle

8 Citations (Scopus)

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. 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.

Original languageEnglish (US)
Pages (from-to)254-260
Number of pages7
JournalCurrent Eye Research
Volume18
Issue number4
DOIs
StatePublished - 1999
Externally publishedYes

Fingerprint

Hydrogen Peroxide
Potassium
Epithelium
Sodium
Ouabain
Rabbits
Staurosporine
Protein Kinases
Cell Line
Sodium-Potassium-Exchanging ATPase
Atomic Spectrophotometry
Active Biological Transport
Protein-Serine-Threonine Kinases
Amphotericin B
Protein Kinase Inhibitors
Centrifugation
Permeability
Hydrolysis
Adenosine Triphosphate
Membranes

Keywords

  • Ciliary epithelium
  • Hydrogen peroxide
  • Oxidation
  • Protein kinases
  • Rabbit
  • Sodium pump

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Stimulation of active sodium-potassium transport by hydrogen peroxide in cultured rabbit nonpigmented ciliary epithelium. / Chin, Shinshi; Delamere, Nicholas A.

In: Current Eye Research, Vol. 18, No. 4, 1999, p. 254-260.

Research output: Contribution to journalArticle

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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. 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.",
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N2 - 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.

AB - 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.

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KW - Sodium pump

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