Characterization of the Na, K-ATPase pump in cultured bovine corneal endothelial cells

Naphtali Savion, Nahid Farzame

Research output: Contribution to journalArticlepeer-review

Abstract

Bovine corneal endothelial cells in culture possess Na, K-ATPase pump sites, as measured by [3H]ouabain binding, and demonstrated an active ouabain-sensitive 86Rb+ uptake. The binding of [3H]ouabain, a specific inhibitor of Na, K-ATPase, was used to quantitate the density of Na, K-ATPase pump sites in bovine corneal endothelial cell cultures. [3]ouabain binding was time-dependent and reached saturation after 1-2 hr. The specific binding represented more than 99% of the total cell-associated [3H]ouabain, and about 85% of this binding was abolished in the presence of K+ ions. The binding was concentration-dependent and saturated at a ouabain concentration of 2 × 10-8m with a dissociation constant (Ka) of 1·0 × 10-8m. The number of [3H]ouabain binding sites was maximal in sparse, actively growing cultures and decreased accompanying the development of a confluent monolayer. A pump density of 2·2 × 106 pump sites cell-1 was estimated for sparse cultures, declining to 0·8 × 106 pump site cell-1 at confluence. The activity of the Na, K-ATPase pump in bovine corneal endothelial cell cultures was evaluated by measuring 86Rb+ influx. Sparse and confluent cultures demonstrated 86Rb+ ouabain-sensitive uptake at a rate of 4·2 nmol (106 cells)-1 min-1 and 1·5 nmol (106 cells)-1 min-1, respectively. The ouabain-sensitive 86Rb+ uptake was linear for at least 30 min, while the ouabain-insensitive 86Rb+ uptake was slower and declined during the 30-min time period. The density of Na, K-ATPase pump sites in cultured bovine corneal endothelial cells is comparable to the density reported for intact rabbit corneal endothelium in situ, and the pump activity is comparable to that reported for several transporting epithelia. Therefore, this system of cultured bovine corneal endothelial cells provides an excellent model to study the Na, K-ATPase pump, its role in the corneal endothelium fluid transport and its reaction to various drugs.

Original languageEnglish
Pages (from-to)355-363
Number of pages9
JournalExperimental Eye Research
Volume43
Issue number3
DOIs
StatePublished - Sep 1986

Keywords

  • Na, K-ATPase
  • Rb uptake
  • [H]ouabain binding
  • cell culture
  • cornea
  • endothelium

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