As an outgrowth of studying cellular regulation of inorganic solutes (Na+, K+, and Ca2), we began investigating organic solute regulation and this has become the major focus of this renewal. We and others showed that hyperosmolar stress causes the renal inner medulla, the brain, and cultured renal epithelial cells to accumulate organic solutes known as osmolytes. In renal epithelial cells four major osmolytes exist, glycerophosphorylocholine (GPC), betaine, myo-inositol, and sorbitol. Our studies of whole animals and MDCK cells indicated that each osmolyte can be regulated independently but the cellular mechanisms remain largely unknown. The principal objective of this proposal is to elucidate the basic cellular processes involved in the adaptation to hyperosmolar stress. Two model cell systems known to accumulate the four major osmolytes will be studied, inner medullary collecting duct (IMCD) cells. Studies will focus on two of the four major osmolytes, GPC and inositol.
The specific aims are: 1) To characterize the physiological response to hyperosmolality. We will examine the time-course, solute-dependence (i.e., NaC1, glucose, mannitol, urea), inhibitor- sensitivity (e.g., sorbinil), and hormone-dependence (e.g., vasopressin) of osmolyte accumulation. Osmolytes will be identified and quantified using NMR and HPLC. Parallel experiments will identify the correlations in cell volume and inorganic ion transport. 2) GPC is the most abundant renal osmolyte and is the most responsive to changes in osmolality. However, unlike the other osmolytes, the biochemical pathway for synthesis of GPC is completely unknown. Using our expertise in NMR, HPLC, and TLC we will uncover the cellular mechanism of GPC formation. This will involve an analysis of choline and glucose uptake, identification of intermediate metabolites, and the identification and characterization of the major enzyme(s) (e.g., GPC synthetase, lysophospholipase, PLA2) which regulate GPC synthesis. 3) A Na-dependent inositol transporter appears responsible for inositol accumulation under hyperosmolar conditions. This transporter will be characterized and, subsequently, the cDNA will be cloned and sequenced. The ability to produce probes and antibodies to this transporter. These studies will provide insight into the cellular mechanisms responsible for the adaptation of cells to osmotic stress and will have important implications for a variety of pathophysiological states known to involve osmotic imbalance or changes in polyols and methylamines such as hypernatremia and diabetes mellitus.
