Biochemical characterization of isolated rat retinal cells: The γ-aminobutyric acid system

Abstract

Rat retinae were chemically dissociated with collagenase and eight populations of cells (layers I–VIII, from bottom to top, respectively) were separated by centrifugation through a linear metrizamide density gradient. Each cell type was examined to determine its content of γ-aminobutyric acid (GABA), glutamate decarboxylase, GABA uptake capacity and GABA binding sites. The cells in layers I and II had the highest concentration of GABA (a six-fold increase in GABA concentration as compared to the whole retina). Glutamate decarboxylase, the enzyme which catalyzes the decarboxylation of glutamate to form GABA, was also found in highest concentration in the cells of layers I and II. Only the cells in layer VII had the ability to take up exogenous GABA. Initial rate studies demonstrated a high and low affinity site for GABA uptake with dissociation constants of 4·7 and 50 μ m , respectively. GABA uptake was Na + -dependent and Ca 2+ -independent. Diaminobutyric acid and GABA were the two most potent inhibitors of [ 3 H]GABA uptake with K I values of 3 and 5 μ m , respectively. Muscimol and 3-aminopropanesulfonic acid, inhibitors of GABA binding, did not significantly effect GABA uptake at concentrations up to 10 m m . Specific [ 3 H]muscimol binding, used to quantitate GABA binding to GABA receptor sites, was present only in layer VI. Specific [ 3 H]muscimol binding to the membranes isolated from layer VI was saturable with a dissociation constant of 6·5 n m and was competitively inhibited by muscimol and GABA with K I values of 3 n m and 10 n m , respectively. [ 3 H]Muscimol binding was Ca 2+ - and Na + -independent and was not inhibited by diaminobutyric acid, nipecotic acid or β -alanine at concentrations up to 100 μ m .