PURPOSE. Cultured rabbit nonpigmented ciliary epithelium (NPE) transports ascorbic acid (ASC) inward through a sodium-dependent mechanism. This study was conducted to test whether Na-K transport is activated to export the additional sodium, which enters the cell in cotransport with ASC. METHODS. Studies were conducted using a cell line derived from rabbit NPE. ASC uptake was measured using [14C]ascorbic acid. The ouabain-sensitive potassium (86Rb) uptake rate was measured as an index of active Na-K transport. Cellular sodium was measured by atomic absorption spectrophotometry or SBFI fluorescence. RESULTS. In the presence of 200 μM ASC, ouabain-sensitive potassium (86Rb) uptake rate increased ~70%; lesser concentrations of ASC produced lesser increases. Phloridzin (100 μM) inhibited ASC uptake and inhibited the stimulatory effect of external ASC on 86Rb uptake. Dehydroascorbic acid (DHA) did not increase 86Rb uptake. Neither DHA nor ASC altered the Na,K-ATPase activity measured in isolated membrane material. External ASC appeared to stimulate active sodium transport through a mechanism involving an increase of cytoplasmic sodium. In the presence of 200 μM ASC, cellular sodium increased ~26%; studies with cells, sodium loaded by nigericin treatment, suggested that this sodium increase could account for the degree of 86Rb uptake stimulation observed in ASC- treated cells. However, the cellular sodium increase could not be explained simply on the basis of sodium entry through the ASC transporter. An additional sodium-entry pathway seemed to be activated in cells that accumulated ASC. Dimethylamiloride (DMA) abolished both the cellular sodium increase and the 86Rb uptake stimulation caused by ASC. DMA did not prevent ASC uptake. CONCLUSIONS. ASC significantly stimulated active Na-K transport in cultured NPE. The mechanism appeared to involve activation of a DMA- sensitive sodium entry pathway, which caused cytoplasmic sodium concentration to increase.
|Original language||English (US)|
|Number of pages||8|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Jan 1 1998|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience