Collecting duct alpha intercalated cells secrete protons and absorb bicarbonate. The ATP that is utilized for H+ secretion derives at least in part from glycolysis. Collecting duct glycolysis produces substantial amounts of lactic acid, but it is not understood how lactate exits the intercalated cell or what factors regulate this step. We have used functional assays to show that cells of the collecting duct cell line MDCK have a basolateral membrane lactate/H+ co-transporter. We have also shown that a monoclonal antibody to the rabbit erythrocyte lactate/H+ co-transporter recognizes similar proteins in the rabbit kidney on immunoblots, and immunocytochemically labels the basolateral membranes of rabbit kidney proximal tubule, medullary thick ascending limb, and collecting duct, especially intercalated cells. The amount of the transporter in the collecting ducts appears to be increased by carbohydrate loading the animal, suggesting that it is regulated as a function of metabolism. We have created a rabbit kidney cDNA library, screened it with this antibody, and have a positive clone. Based on these results, we ask the following questions: 1. Does lactate cross the basolateral membrane of intercalated cells via a lactate/H+ co-transporter? 2. What is the structure of this transporter? 3. What regulates the lactate/H+ co-transporter? We have three corresponding Specific Aims: 1. Determine the intracellular localization, size, and functional properties of the rabbit collecting duct lactate/H+ co-transporter. 2. Clone, sequence, and express the cDNA of the rabbit kidney lactate/H+ co-transporter. 3. Quantitate the changes in lactate/H+ co-transporter function, mRNA, and protein amount in response to changes in metabolism. The methods that will be used include perfused tubules, cultured collecting duct cells, immuno- and mRNA blotting, immunocytochemistry, digital imaging spectrofluorometry, and cDNA cloning and sequencing. The proposed experiments will advance our understanding of a fundamental aspect of intercalated cell function: the mechanism by which lactate is transported across the basolateral membrane.
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