GABA_(B1) AND GABA_(B2) RECEPTOR SUBUNITS CO-EXPRESSED IN CULTURED HUMAN RPE CELLS REGULATE INTRACELLULAR CA~(2+) VIA G_(I/O)-PROTEIN AND PHOSPHOLIPASE C PATHWAYS

摘要

GABA_B receptors associate with G_(i/o)-proteins that regulate voltage-gated Ca~(2+) channels and thus the intracellular Ca~(2+) concentration ([Ca~(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABA_B receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABA_B receptor sub-units, GABA_(B1) and GABA_(B2), are co-expressed in cultured human RPE cells, and then determine if the GABA_B receptor similarly regulates the [Ca~(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from five donor eye cups. Evidence for GABA_(B1) and GABA_(B2) mRNAs and proteins in the RPE cell cultures was investigated using real time polymerase chain reaction, western blots and immunofluorescence. The effects of the GABA_B receptor agonist baclofen, antagonist CGP46381, a G_(i/o)-pro-tein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca~(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABA_(B1) and GABA_(B2) mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred micromolars of baclofen caused a transient increase in the [Ca~(2+)]i of RPE cellsregardless of whether Ca~(2+) was added to the buffer. Baclo-fen-induced increases in the [Ca~(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABA_(B1) and GABA_(B2) are co-expressed in cell cultures of human RPE. GABA_B receptors in RPF regulate the [Ca~(2+)]i via a G_(i/o)-protein and phospholipase C pathway.