Maintenance of cell volume is critically important for normal functioning of most mammalian cells. Transport processes exist to bring about a precise balance between high rates of solute influx and efflux, and obligatorily coupled water transport, across the plasma membrane. Cell swelling is a particular problem for cells in both health and disease and must be continually guarded against. Reduction of extracellular fluid osmolarity, such as observed in hyponatremia, or net cellular accumulation of solute, such as may occur with altered plasma solute levels or hormonal imbalances, results in water movement into cells and hence cell swelling. Swelling activates transport processes at the plasma membrane to return volume towards normal, a process referred to as regulatory volume decrease. These processes are particularly evident in the proximal straight tubule of the kidney where swelling activates potassium and chloride conductive pathways in the basolateral membrane to allow efflux of potassium and chloride from the cell, accompanied by obliged water efflux, to return volume to near normal. The overall goal of the present proposal is to elucidate the mechanisms and signalling pathways underlying regulatory volume decrease in the proximal straight tubule of the rabbit kidney. This will be accomplished by first fully elucidating the effect of cell swelling on the basolateral membrane potassium and chloride conductances using electrophysiological techniques, and the mechanism of activation of these conductive pathways, i.e. exocytic insertion of pathways vs activation of latent pathways already in the plasma membrane, using fluorescent probes to study membrane turnover. Whereupon, the regulatory pathways controlling these conductances will be elucidated. Focus will be on the role of calcium, based on measurements of intracellular calcium activity using fluorescent calcium probes. The interaction with calcium and calmodulin acceptor proteins in regulating these pathways, and the role of phosphatidylinositol turnover pathway--shown in preliminary studies to be activated during cell swelling--will be directly assessed. The studies will provide new insights into the mechanisms of volume regulation in cells and the underlying biochemical pathways that regulate these processes.
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