The long-term objective of our research is to understand the cellular and molecular mechanisms of electrogenic Na reabsorption by the renal inner medullary collecting duct (IMCD). Studies in our laboratory have shown that cGMP-gated cation channels and Na-selective channels mediate Na uptake across the apical membrane of IMCD cells and that Na reabsorption is stimulated by aldosterone and arginine vasopressin (AVP) and inhibited by atrial natriuretic peptide (ANP). However, little is known about the signaling pathways underlying hormonal regulation of Na reabsorption by the IMCD, the effects of osmolality on Na reabsorption and the relative contribution of Na-selective and cGMP-gated cation channels to net Na reabsorption. Accordingly, the goals of this proposal are to: 1) elucidate the cellular signaling pathways mediating hormone regulation of Na reabsorption; 2) identify the contribution of Na-selective and cGMP-gated cation channels to hormone regulated Na reabsorption and 3) obtain a cDNA clone encoding the cGMP-gated cation channel to study the regulation of channel gene expression by aldosterone and interstitial osmolality. Our preliminary molecular cloning studies indicate that the IMCD express a cGMP-gated cation channel gene that is related to cyclic nucleotide-gated cation channels in photoreceptor and olfactory cells. To achieve the first and second goal we will use our newly established IMCD cell line, which has the Na transport characteristics of native IMCD cells. The path clamp technique and measurements of amiloride-sensitive short circuit current will allow us to: 1) Characterize the effects of natriuretic peptides (i.e. ANP and urodilatin), AVP, aldosterone and osmolality on Na transport and on Na-selective and cGMP-gated cation channel activity and 2) Identify the signaling pathways of natriuretic peptide and AVP regulation of Na and cGMP-gated cation channel activity. To achieve the third goal we will use molecular techniques to: 1) Isolate a cDNA clone encoding the cGMP-gated cation channel, 2) Express and characterize a full length cDNA clone and 3) Study the regulation of the cGMP-gated cation channel expression by aldosterone and interstitial osmolality. Sequence information on the channel will provide important insights about functional domains, phosphorylation sites, pore forming domains and cGMP binding sites. This information will be useful in future studies examining structure-function relationships and signaling mechanisms involved in channel function and may lead to the isolation of cDNA clones coding for other Na-conducting channels. An important aspect of this application is that the proposed studies will provide a comprehensive and integrated understanding of the cellular and molecular mechanisms regulating Na reabsorption by the IMCD. Because Na reabsorption by the IMCD is regulated to meet the homeostatic needs of the organism, information on the mechanisms and regulation of Na reabsorption is important in understanding Na homeostasis and regulation of the extracellular fluid volume and blood pressure.
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