Endothelium-derived relaxing factor (EDRF) is a recently discovered paracrine hormone released from the endothelium lining the vasculature. EDRF is released in vivo by a number of factors including bradykinin, acetylcholine and shear stress. EDRF has been shown to cause diuresis and natriuresis without a change in glomerular filtration rate. These data could be explained by either a pressure diuresis or an direct inhibition of tubular transport caused by EDRF. A direct inhibition of sodium and water absorption by EDRF in individual nephron segments has not been reported to date. Our hypothesis is that EDRF produced by renal endothelial cells inhibits fluid and sodium transport in the collecting duct system. This is based on 1) our preliminary data showing that EDRF inhibits transport in cultured collecting duct cells; 2) the proximity of the renal vessels and nephrons; and 3) the fact that EDRF stimulates cGMP production in vascular tissue, while cGMP inhibits transport in the collecting duct.
Our specific aims are to investigate: 1) the effects of EDRF on basal sodium and water transport; 2) the effects of EDRF on vasopressin-stimulated transport of sodium and water; 3) whether the second messenger cascade of EDRF involves activation of soluble guanylate cyclase and consequent increase of cGMP; 4) the roles of cGMP-dependent protein kinase and phosphodiesterase in mediating the effects of EDRF; and 5) whether the sodium channel is phosphorylated as part of the response to EDRF. We will use a variety of techniques to address these specific aims.
In aims #1 and #2 we will measure transepithelial sodium and water flux.
In aim #3 we will assay cyclic nucleotides and measure soluble and particulate guanylate cyclase activity.
In aim #4 we will measure kinases and phosphodiesterases.
In aim #5 we will immunoprecipitate the amiloride-sensitive sodium channel and use Western blot analysis to determine whether a subunit of the channel is phosphorylated. The collecting duct system plays a pivotal role in the fine regulation of solute and water excretion, and consequently extracellular fluid volume. Extracellular fluid volume is one of the primary determinants of blood pressure. There are few data concerning the effects of EDRF on transport along the nephron, and yet it may prove to be one of the most important regulators of collecting duct sodium and water absorption.
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