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The gamma-aminobutyric acid transporter and its interaction with taurine in the apical membrane of the bovine retinal pigment epithelium.

ABSTRACT: The characteristics of gamma-aminobutyric acid (GABA) uptake were investigated in apical membrane vesicles prepared from the bovine retinal pigment epithelium. An inwardly directed NaCl gradient stimulated GABA uptake markedly, and the time course of uptake exhibited an overshoot phenomenon indicating the presence of an active transport mechanism for GABA in these membranes. Other monovalent cations were not capable of substituting for Na+. In addition to this obligatory requirement for Na+, the GABA uptake also exhibited a Cl(-)-dependence, evident from the observations that the uptake was negligible in the presence of NaF or sodium gluconate in place of NaCl. NO3- and SCN- could substitute for Cl- to some extent. The uptake process was electrogenic, with a Na+/Cl-/GABA stoichiometry of 2:1:1 or 3:1:1. Substrate-specificity studies showed that the beta-amino acids such as taurine, hypotaurine and beta-alanine interacted with the GABA uptake process. Uptake of GABA could be completely inhibited by an excess of taurine and, similarly, uptake of taurine could be completely inhibited by an excess of GABA, suggesting that common transport processes operate in the uptake of these two compounds. However, a number of compounds which are specific inhibitors of GABA uptake inhibited taurine uptake only to a maximum of 50%. Kinetic analysis of GABA uptake in the concentration range 0.1-10 microM revealed that the uptake occurred via a single system and that taurine was a competitive inhibitor of this system. The Michaelis-Menten constant (Kt) for GABA was 0.94 microM and the apparent inhibition constant (Ki) for taurine was 230 microM. On the contrary, even though the kinetic analysis of taurine uptake in the concentration range 25-150 microM revealed participation of a single system in the uptake process, the inhibition of taurine uptake by GABA was not competitive. The presence of GABA decreased the maximal velocity of the taurine uptake process and also decreased the Kt for taurine. Based on these data, it is proposed that: (i) there are two distinct transport systems, namely the GABA transporter and the taurine transporter, in these membranes which accept both GABA and taurine as substrates, (ii) the affinities of these systems for taurine are very similar and cannot be kinetically distinguished under the experimental conditions employed, and (iii) the difference between the affinities of these system for GABA is much greater than for taurine.

SUBMITTER: Sivakami S

PROVIDER: S-EPMC1131046 | biostudies-other | 1992 Apr

REPOSITORIES: biostudies-other

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Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

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The gamma-aminobutyric acid transporter and its interaction with taurine in the apical membrane of the bovine retinal pigment epithelium.

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