Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells

Shin Hatou, Masakazu Yamada, Yoko Akune, Hiroshi Mochizuki, Atsushi Shiraishi, Takeshi Joko, Teruo Nishida, Kazuo Tsubota

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

PURPOSE. The Na+-/K+-dependent ATPase (Na, K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. METHODS. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na, K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na, K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. RESULTS. Insulin increased the Na, K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na, KATPase α1-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na, K-ATPase α1-subunit. CONCLUSIONS. These results suggest that insulin increases the Na, K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by α1-subunit dephosphorylation.

Original languageEnglish
Pages (from-to)3935-3942
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume51
Issue number8
DOIs
Publication statusPublished - 2010 Aug

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Adenosine Triphosphatases
Endothelial Cells
Insulin
Ouabain
Protein Kinase C
Western Blotting
Immunohistochemistry
Protein Phosphatase 1
Corneal Endothelium
Protein Phosphatase 2
Protein C Inhibitor
Protein Kinase Inhibitors
Adenosine Triphosphate
Phosphates
Membranes
sodium-translocating ATPase

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells. / Hatou, Shin; Yamada, Masakazu; Akune, Yoko; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo; Tsubota, Kazuo.

In: Investigative Ophthalmology and Visual Science, Vol. 51, No. 8, 08.2010, p. 3935-3942.

Research output: Contribution to journalArticle

Hatou, Shin ; Yamada, Masakazu ; Akune, Yoko ; Mochizuki, Hiroshi ; Shiraishi, Atsushi ; Joko, Takeshi ; Nishida, Teruo ; Tsubota, Kazuo. / Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells. In: Investigative Ophthalmology and Visual Science. 2010 ; Vol. 51, No. 8. pp. 3935-3942.
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T1 - Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells

AU - Hatou, Shin

AU - Yamada, Masakazu

AU - Akune, Yoko

AU - Mochizuki, Hiroshi

AU - Shiraishi, Atsushi

AU - Joko, Takeshi

AU - Nishida, Teruo

AU - Tsubota, Kazuo

PY - 2010/8

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N2 - PURPOSE. The Na+-/K+-dependent ATPase (Na, K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. METHODS. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na, K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na, K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. RESULTS. Insulin increased the Na, K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na, KATPase α1-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na, K-ATPase α1-subunit. CONCLUSIONS. These results suggest that insulin increases the Na, K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by α1-subunit dephosphorylation.

AB - PURPOSE. The Na+-/K+-dependent ATPase (Na, K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. METHODS. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na, K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na, K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. RESULTS. Insulin increased the Na, K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na, KATPase α1-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na, K-ATPase α1-subunit. CONCLUSIONS. These results suggest that insulin increases the Na, K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by α1-subunit dephosphorylation.

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