Project: Research project

Project Details


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+

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.
Effective start/end date12/31/894/30/95


  • Cell Biology


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