The effect of AVP on the collecting duct is a critical determinant of renal water excretion and, in concert with other hormones, a determinant of sodium excretion as well. Whereas AVP shares with other agonists (beta-adrenergic and prostanoids) the ability to stimulate adenylyl cyclase, it is unique in its water reabsorbing ability. We hypothesize that activation of other signaling pathways and/or protein kinases account for this differential response. Such pathways are also likely to be activated in pathophysiologic conditions characterized by resistance to AVP action, such as high Ca2+ and increased lithium. We also suggest that the effect of AVP on Na+ reabsorption is mediated through a receptor and subsequent cellular signals that are distinct from those that subserve the hydroosmotic response to the hormone. In order to test these hypotheses will employ dissected nephron segments and two established cell lines of collecting duct cells, one from the inner medulla and one from the cortical collecting duct. In these tissues, the distribution and relative abundance of neurohypophyseal hormone receptor (V2, Vm and oxytocin) will be assessed by reverse transcription-PCR- sequencing approach employing the conserved oligonucleotide primer from recently cloned receptor sequences. The central role of cAMP and cAMP dependent PKA will be analyzed in the two cell lines by their transfection with inhibition of the cAMP dependent PK (PKI) gene or by a gene that encodes for a mutated regulatory subunit of the kinase. The effects of such transfections will be monitored in a recently designed chamber for measurements of the hydroosmotic response to the hormone and by transmembrane current changes confirmed by 22Na uptake for the Na+ reabsorptive effect by comparing transfected cells with the wild-type. To understand why agonists that increase cyclase do not increase water reabsorption their effects on other second messenger systems and on other second messenger systems and on the protein kinase cascade will be assessed with attention to cell polarity. Finally, the more preuse mechanism of AVP resistance in two clinically relevant settings, high cell Ca+ and lithium will be explored by genetic manipulation of the cells by transfecting with mutants of G alpha Gq as well as, whenever available and feasible, dominant inhibitors of specific protein kinase. These experiments should contribute to the definition of the site(s) responsible for AVP resistance, and more globally to the overall action of AVP on the collecting duct.
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