Electrogenic Na+-ascorbate cotransport in cultured bovine pigmented ciliary epithelial cells

Area of Science:

• Ophthalmology
• Cell Biology
• Biochemistry

Background:

• Ascorbic acid (AA) is highly concentrated in mammalian aqueous humor.
• This suggests an active transport mechanism across the ciliary epithelium.
• The specific transporters and their mechanisms remain largely uncharacterized.

Purpose of the Study:

• To investigate the transport mechanism of ascorbic acid (AA) in bovine pigmented ciliary epithelial cells.
• To characterize the role of sodium (Na+) in AA uptake.
• To determine the stoichiometry and electrogenicity of the AA transporter.

Main Methods:

• Utilized [14C]AA and [14C]dehydroascorbate (DHA) for uptake studies in cultured bovine ciliary epithelial cells.
• Investigated the effect of extracellular Na+ concentration, inhibitors (phloretin, isoascorbate), and intracellular Na+ levels on AA uptake.
• Measured membrane potential changes in response to AA addition using microelectrode techniques.

Main Results:

• Observed a 40-fold intracellular accumulation of AA, dependent on extracellular Na+.
• DHA uptake was Na+-independent and showed a different time course, indicating distinct transport systems.
• AA uptake kinetics exhibited saturation and sigmoidal Na+ dependence, suggesting a Na+-coupled transporter with a stoichiometry of 2:1 or higher (Na+:AA).
• AA addition caused membrane depolarization, confirming the electrogenic nature of the transporter.

Conclusions:

• Bovine pigmented ciliary epithelial cells actively transport ascorbic acid via a Na+-dependent mechanism.
• The transporter likely translocates at least two Na+ ions per AA molecule, indicating an electrogenic transport system.
• This study elucidates a key mechanism for vitamin C accumulation in the eye, with implications for ocular health and disease.