GABA(Aα1) and GABA(Aρ1) subunits are expressed in cultured human RPE cells and GABA(A) receptor agents modify the intracellular calcium concentration

PURPOSE: Gamma-aminobutyric acid(A) (GABA(A)) receptors (GABA(A)Rs), which are ionotropic receptors involving chloride channels, have been identified in various neural (e.g., mouse retinal ganglion cells) and nonneural cells (e.g., mouse lens epithelial cells) regulating the intracellular calcium concentration ([Ca(2+)]i). GABA(A)R β-subunit protein has been isolated in the cultured human and rat RPE, and GABA(Aα1) and GABA(Aρ1) mRNAs and proteins are present in the chick RPE. The purpose of this study was to investigate the expression of GABA(Aα1) and GABA(Aρ1), two important subunits in forming functional GABA(A)Rs, in the cultured human RPE, and further to explore whether altering receptor activation modifies [Ca(2+)]i. METHODS: Human RPE cells were separately cultured from five donor eye cups. Real-time PCR, western blots, and immunofluorescence were used to test for GABA(Aα1) and GABA(Aρ1) mRNAs and proteins. The effects of the GABA(A)R agonist muscimol, antagonist picrotoxin, or the specific GABA(Aρ) antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo3-AM. RESULTS: Both GABA(Aα1) and GABA(Aρ1) mRNAs and proteins were identified in cultured human RPE cells; antibody staining was mainly localized to the cell membrane and was also present in the cytoplasm but not in the nucleus. Muscimol (100 μM) caused a transient increase of the [Ca(2+)]i in RPE cells regardless of whether Ca(2+) was added to the buffer. Muscimol-induced increases in the [Ca(2+)]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM). CONCLUSIONS: GABA(Aα1) and GABA(Aρ1) are expressed in cultured human RPE cells, and GABA(A) agents can modify [Ca(2+)]i.