ION TRANSPORT IN COLLECTING CELL CULTURES

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We aim to understand the ion transport properties and the mechanisms of its regulation in the different cell types of the cortical collecting duct (CCD). Specific aims are: 1. Continue characterizing the ion-transport mechanisms expressed in CCD cells in culture. Mixed CCD cultures will be used to identify mechanisms of ion transport, and the effects of ADH, aldosterone, and isoproterenol on the different transporters. 2. To study the effects of intra and extracellular pH on the cell biology and transport functions of CCD monolayers. 2.1. To assess the modifications in the proportion of the different cell types and the morphological changes in response to alterations in acid-base status of the cultures. 2.2. To establish the pH dependence of the transporters located in the apical and basolateral membranes of the different cells. We will examine the conductive and nonconductive ion-transport pathways of the three cell types under basal conditions and their responses to acute and chronic changes in pH of basolateral and/or apical solutions. 3. To characterize, structurally and functionally, homogeneous monolayers of principal cells and intercalated cells. 3.1. The ultrastructure and function of principal- cell monolayers will be studied: We will characterize at the cell-membrane level the baseline ion transport mechanisms and their regulation by hormones (ADH, aldosterone, isoproterenol). 3.2. Homogeneous cultures of intercalated cells, initially identified as either alpha or beta cells, will be studied with and without hormonal stimulation. Special consideration will be given to the possibility of transformation of a cell type to another. Monolayers of CCD cells will allow us to gather basic information on ion transport mechanisms at the cellular and single-membrane levels. They should also be useful tools for biochemical and molecular studies. Homogenous cultures will permit quantitative studies without the confounding influences of neighboring cells of different type. These studies could help our understanding of the pathogenesis of renal tubule ion-transport defects, such as renal tubular acidosis, concentration defects and K+ secretion abnormalities.