The purpose of the present study was to elucidate the mechanisms of retina-to-blood transport of l-proline across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The vitreous humor/retina-to-blood transport of [3H]l-proline across the BRB was evaluated by microdialysis. Transport mechanisms of [3H]l-proline were investigated by cellular uptake using an in vitro model of the inner BRB (TR-iBRB2 cells). The mRNA level of system A was determined by quantitative real-time PCR analysis with specific primers. [3H]l-Proline and [14C]d-mannitol, which is a bulk flow marker, were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [3H]l-proline during the terminal phase was 1.6-fold greater than that of [14C]d-mannitol. The terminal elimination rate constant difference between [3H]l-proline and [14C]d-mannitol was reduced in the retinal presence of 3 mM l-proline and 5 mM α-methylaminoisobutyric acid, suggesting that l-proline is transported via a carrier-mediated retina-to-blood transport process across the BRB. [3H]l-Proline uptake by TR-iBRB2 cells appeared to be mediated through a saturable and Na+-dependent process. The corresponding Michaelis-Menten constant was 392 μM. This process was reduced by substrates for system A, suggesting that system A is involved in l-proline uptake. Of the isoforms of system A, ATA1, ATA2, and ATA3, ATA2 mRNA is predominantly expressed in TR-iBRB2 cells and isolated rat retinal endothelial cells. In conclusion, system A, most likely ATA2, is responsible for the retina-to-blood transport of l-proline across the inner BRB and may play a role in maintaining the concentration of small neutral amino acids in the retina.
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