Atrial natriuretic peptides (ANP) induce large increases in renal sodium excretion. Evidence is accumulating that ANP induces natriuresis in part by directly altering sodium transport in the inner medullary collecting duct (IMCD). Recently, we have demonstrated that ANP inhibits transport-dependent oxygen consumption and sodium uptake in suspensions of IMCD cells, as well as apical sodium uptake in confluent monolayers of IMCD cells in culture. Further studies have implicated cGMP as an intracellular second messenger mediating effects of ANP on IMCD cells. This proposal seeks to define the effect of ANP on IMCD sodium transport and to characterize the steps between binding of peptide to the cell surface and alterations in sodium handling. We will examine the mechanisms of IMCD sodium transport in the presence and absence of ANP by measuring unidirectional and net transepithelial sodium fluxes as well as transepithelial resistances in confluent monolayers of IMCD cells grown on filter bottom cups. Because ANP inhibits sodium entry in IMCD cells, transepithelial flux measurements will be supplemented with measurements of sodium entry in cell suspensions and confluent monolayers. Transport measurements in both suspensions and in confluent monolayers will provide new information about IMCD sodium transport and its regulation by ANP. We will evaluate the mechanisms by which ANP binding leads to altered sodium handling. We will characterize IMCD ANP receptors using equilibrium binding and affinity crosslinking techniques. We will determine whether cGMP mimics the effects of ANP on sodium handling by IMCD cells in suspension and in confluent monolayers. We will determine whether ANP raises cGMP levels by stimulating guanylate cyclase or by inhibiting phophodiesterase. Finally, we will determine whether cGMP, by activating protein kinases, regulates transport via phosphorylations of specific proteins. We will determine how unique features of the medullary environment such as hypertonicity and low oxygen tension, as well as volume regulatory hormones alter sodium transport in IMCD cells and its regulation by ANP. The studies will define the role of ANP in the regulation of sodium transport in the inner medullary collecting duct, the final arbiter of urinary sodium excretion.
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