Maintenance of the solute composition and volume of intra- and extracellular fluid compartments in the CNS is crucial for normal brain functioning. Small changes in solute composition can dramatically alter neuronal signaling and information processing. Because of the rigid confines of the skull and complex brain architecture changes in total brain volume can cause devastating neurological damage. It is not surprising to find, therefor, that the composition and volume of brain fluid compartments are controlled tightly under both normal and pathological condiitons. CNS osmotic and ionic balance are regulated by solute and water transport across the blood-brain barrier, choroid plexus an plasma membrane of glial cells and neurons. Despite its clinical and physiological significance, the underlying mechanisms of brain osmotic homeostasis are incompletely understood. Studies carried out in the first 2 years of NS30591 funding, however, have provided the first detailed cellular and molecular description of how cultured CNS glial cells adapt to acute and chronic hypertonicity and their correction. Several of our findings have important clinical implications and suggest new strategies for correcting plasma osmolality disorders and treating brain volume disturbances. Glial cells exposed chronically to hypertonicity accumulate the organic osmolyte myo-inositol via enhanced Na+/myo-inositol cotransporter gene expression. When returned to normotonic conditions, the cells swell and remain swollen for prolonged periods of time due in part to slow downregulation of the cotransporter. Myo-inositol is lost from the swollen cells via activation of a volume-sensitive anion channel we have described and termed VSOAC (Volume-Sensitive Organic osmolyte /Anion Channel). Studies outlined in the current proposal will complete the cloning and characterization of the brain cotransporter and will quantify osmotically regulated cotransporter gene transcription. Cotransporter protein downregulation will be characterized by western analysis. Pulse-chase labeling of cellular mRNA and in vitro mRNA degradation assays will characterize postulated post-transcriptional regulation of cotransporter message levels. Key components of our cell culture model will be tested in the whole animal to integrate cellular and molecular data into a description of brain osmoregulatory behavior. Using a hypernatremic rat model, we will quantify cotransporter mRNA levels in the brain by Northern analysis and in situ hybridization and we will measure myo-inositol transport in brain plasma membrane vesicles. The properties and regulation of VSOAC will be characterized by patch clamp, video microscopy and Xenopus oocyte expression techniques. Studies outlined in this grant provide a central foundation for the comprehensive understanding of brain volume homeostasis. Such an understanding is essential for treating a variety of disease status.
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