The regulation of Na+ reabsorption in the cortical collecting duct (CCD) has been the focus of our current studies. Our interest stems from recent findings that single gene mutations expressed in this segment, e.g., subunits of the amiloride-sensitive epithelial Na+ channel (ENaC) or the 11beta-hydroxysteroid dehydrogenase, lead to severe hypertension. We will examine two interrelated aspects of ENaC regulation: first, the hypothesis of regulated exocytosis of ENaC subunits in response to arginine vasopressin (AVP) versus activation of preexisting but quiescent channels present in the membrane; and second, we will expand our examination of the involvement of protein kinase C (PKC) isoforms as inhibitors of sustained activation of the Na+ channel by AVP. Our recent work suggests that a specific PKC isoform (PKC-theta) may be involved in this inhibition. Exocytosis of the GABA transporter expressed in oocytes has been shown to involve specific PKC isoforms, and suggests a mechanism by which PKC inhibits the Na+ transport response, but not the water permeability response, to AVP in the rabbit CCD. Those portions of the project related to studies of channel regulation by ENaC subunit trafficking will be conducted in MDCK cells that have been stably transfected to express the three ENaC subunits, each uniquely epitope- tagged. We have developed episomal mammalian expression vectors as well as retroviral LTR constructs of all three rENaC subunits (as well as the water channel aquaporin 2) for expression in MDCK cells. Surface- labeling with 125/I-labeled Fab fragments against the epitopes will then be used to quantify the density of the subunits in the membrane and relate it to amiloride-sensitive short-circuit current. We will also use the isolated perfused CCD preparation to measure Na+ and water transport, and to examine the possibility that a specific PKC isoform is involved in inhibiting the AVP effect in the rabbit CCD. PKC isoforms that are activated by AVP in CCD cells will also be identified by Western blotting of cell fractions to determine which PKC isoforms redistribute in response to AVP. The overall hypotheses are: a) That both regulated exocytosis and activation of channels in the membrane are involved in the AVP response, and that PKC theta or another PKC isoform, directly or indirectly, modulates one or both mechanisms; and b) That a PKC isoform, and/or the activation of PLA2 or PLD by a PKC isoform, inhibit the AVP response in the rabbit but not the rat CCD. These observations will provide important information regarding the physiological or pathophysiological circumstances under which AVP would contribute to salt retention.
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