The epithelial layer of the urinary bladder serves as a barrier to prevent the indiscriminate reabsorption of solutes excreted by the kidney. The bladder epithelium modifies urinary contents via specific transport proteins which reabsorb or secrete selected solutes. Certain disorders of the urinary bladder, such as interstitial cystitis, disrupt the epithelium and likely affect its barrier and transport functions. Na is a major solute that is reabsorbed across epithelial cells int he kidneys and urinary bladder in a two step process. Na exits the urinary space by crossing the apical cell membrane through an amiloride-sensitive epithelial Na channel; and then crosses the basolateral cell membrane via the Na,K- ATPase. Na channels in mammalian urinary bladder are activated by aldosterone and by bladder stretch by mechanisms which have not been delineated. The epithelial Na channel cloned from rat colon consists of at least three structurally related subunits, termed alpha-ENaC, beta-ENaC and gammaENaC (Epithelial Na Channel).
The aims of this proposal are to define the molecular structure and ontogeny of mouse ENaCs, and to characterize mechanisms by which bladder stretch and aldosterone regulate ENaCs in the mammalian urinary bladder. These studies will examine several related hypotheses; (1) mouse ENaCs are homologous to the ENaCs recently characterized in other species; (2) ENaCs are expressed in mammalian urinary bladder (3) ENaCs are expressed late in gestation or in the early neonatal period in mouse urinary bladder: (4) distention of the urinary bladder leads to the incorporation of intracellular vesicles containing ENaCs into the apical membrane of the epithelium; (5) ENaCs are activated by membrane stretch; (6) aldosterone does not alter expression of ENaC message and protein in the urinary bladder, but increases Na transport by activating ENaCs expressed at the apical plasma membrane. Full length cDNAs encoding mouse alpha-, beta-, and gammaENaC will be characterized and used to examine developmental expression of ENaCs in mice. Cellular distribution of ENaCs will be examined in collapsed and in distended rat urinary bladder epithelium by immunocytochemistry and immunoelectron microscopy, and correlated with changes in Na transport properties of the bladder. ENaCs will be expressed in Xenopus oocytes and changes in functional characteristics induced by membrane stretch will be characterized. Changes in Na transport(ENaC function) and ENaC expression in rat urinary bladder in response to aldosterone will be determined. Aldosterone regulated gene products which may control ENaC function will be identified by differential display PCR and by RNA arbitrarily primed PCR, and the functional role of these gene products in Na channel regulation will be examined. These proposed studies will provide critical new information regarding the physiology of solute transport in the urinary bladder, and provide a framework for characterization of altered solute transport in selected disorders of the urinary bladder.
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