During hyperosmolar stress, brain volume is regulated acutely by the uptake of electrolytes and chronically by the accumulation of organic solutes referred to as """"""""organic osmolytes"""""""". The mechanisms by which this occurs are largely unknown. Studies outlined in this proposal will utilize glial cell culture models to elucidate the cellular and molecular basis underlying brain volume regulation during hypernatremia and clinical correction of the hypernatremic state. Studies carried out in our laboratories over the last 12 months have demonstrated that cultured rat glial cells exhibit a pattern of volume regulation remarkably similar to that of the intact brain. For example, hypernatremic stress results in activation of acute volume regulatory electrolyte uptake pathways that return cell volume rapidly (5-10 minutes) to its original value. With prolonged exposure to hyperosmolality, the electrolytes are extruded and the organic osmolyte, inositol, is accumulated via upregulation of Na+-dependent inositol transport. Return to normotonic conditions result in cell swelling and a slow loss of inositol from the cells. The cellular and molecular mechanisms underlying these processes will be elucidated using a variety of approaches. Specifically, we will characterize acute volume regulatory electrolyte uptake pathways and their control using membrane transport assays, laser light scattering measurements of cell volume changes and fluorescence ratio imaging of intracellular Ca2+ signals. The mechanisms by which Na+- dependent inositol transport is upregulated will be assessed by transport assays and northern blot analysis of cDNA probes for the transporter. We will also determine how acute (electrolyte uptake) and chronic (organic osmolyte accumulation) volume regulatory pathways are temporally coordinated using transport assays and whole-cell patch clamp methods. The inositol efflux pathway activated during reacclimation of hyperosmolar cells to normotonic conditions will be characterized using transport assays. Studies outlined in this grant will provide the first detailed cellular and molecular understanding of how cells in the central nervous system (CNS) adapt to acute and chronic hyperosmolality and their correction. Such investigations are essential for understanding the CNS complications of osmolality disturbances and in the development of more effective therapies to treat plasma hyperosmolality.
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