Receptor Regulation of Volume-Sensitive Osmolyte Efflux from Neural Cells.

Abstract

Cell volume regulation is a homeostatic imperative in the brain due to the restrictions of the skull. To counteract an osmolarity disturbance and restore normal cell volume, neural cells initiate volume regulatory mechanism by modifying the concentration of their intracellular inorganic and organic osmolytes. This dissertation illustrates the important ability of GPCRs to potentiate osmolyte efflux in response to hypo-osmotic stress. I discovered that the addition of sub-nanomolar concentrations of thrombin, mediated via the activation of PAR-1 receptor, can robustly enhance the release of the organic osmolyte taurine, through the volume sensitive organic osmolyte and anion channel (VSOAC) from neuro-tumor cells in response to hypo-osmotic stress. Biochemical and pharmacological studies demonstrated that both intracellular Ca2+ and PKC are required for receptor-mediated, but not basal (swelling-induced), taurine release. The results indicated that activation of PAR-1 receptors by thrombin lowers the threshold osmolarity or “set-point” for osmolyte efflux, thereby facilitating the ability of cells to respond to small reductions in osmolarity. Receptor activation (PAR-1 and mAChR) also enhanced the release of the inorganic osmolyte, Cl- (125I- used as a tracer). The kinetics and magnitude of 125I- release were greater than those of taurine. However, both osmolytes showed similar pharmacological profiles in response to VSOAC inhibitors. Although receptor stimulated-taurine efflux was inhibited by depletion of intracellular Ca2+ and/or inhibition of PKC, 125I- efflux was either unaffected or less dependent upon these two parameters. This differential regulation of the osmolyte release suggests either the presence of separate but pharmacologically similar, efflux channels or, receptor-specific activation of distinct signal transduction pathways that differentially contribute to the release of taurine and 125I-, both of which are released through a common membrane channel. I also demonstrated that cholesterol depletion with methyl-ß-cyclodextrin synergistically potentiated receptor-mediated taurine release. This stimulatory effect was not due to disruption of lipid rafts or changes in receptor signaling. Experiments with cholesterol analogs provided evidence that the potentiation by cholesterol depletion resulted from changes in the biophysical properties (fluidity) of the membrane. Together, this thesis provide new insight into mechanisms involved in the GPCR-regulation of osmolyte efflux following hypo-osmotic stress in neural cells.