CELLULAR MECHANISMS OF MINERALOCORTICOID ACTION

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Maintenance of extracellular fluid volume homeostasis is essential for hemodynamic stability, and abnormalities of renal sodium handling have been linked to cardiovascular disease and hypertension. Ultimate regulation of sodium excretion in the kidney occurs in the distal nephron and is modulated by the mineralocorticoid aldosterone. The long term goals of this research are to understand the cellular mechanisms by which aldosterone regulates sodium reabsorption. The initial physiologic event in aldosterone stimulation of sodium reabsorption is an increase in apical membrane permeability due to an increase in number and open probability of conductive sodium channels. These channels are known to be gated by heterotrimeric G proteins localized near the channel. This study proposes that previously inactive or quiescent channels are activated by aldosterone stimulation of G-protein localization. G-proteins are thought to be targeted to membranes by sequential carboxylmethylation and lapidation of terminal amino acids. Both acylation and carboxylmethylation have been implicated in the action of aldosterone and are essential for stimulation of sodium transport. This study will characterize by electrophoresis the apical membrane proteins which are carboxylmethylated and/or acylated and prenylated in response to aldosterone. Immunoprecipitation by antibodies to G-protein subunits and to sodium channel complex will be employed to determine the identify of these post-translationally modified proteins and their relationship to the channel. Studies with inhibitors of lapidation and methylation reactions will be carried out to determine whether these reactions are required for membrane localization. Since these proteins may be transcriptionally as well as post-translationally regulated, Western blotting of metabolically labelled proteins will be performed to assess whether regulatory G- proteins are increased in abundance in either cytosol or membranes following aldosterone stimulation.