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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK025519-22
Application #
6517007
Study Section
General Medicine B Study Section (GMB)
Program Officer
Ketchum, Christian J
Project Start
1979-06-01
Project End
2003-12-31
Budget Start
2002-04-01
Budget End
2003-12-31
Support Year
22
Fiscal Year
2002
Total Cost
$245,341
Indirect Cost
Name
University of Alabama Birmingham
Department
Physiology
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Xie, Yi; Schafer, James A (2004) Inhibition of ENaC by intracellular Cl- in an MDCK clone with high ENaC expression. Am J Physiol Renal Physiol 287:F722-31
Schafer, James A (2002) Abnormal regulation of ENaC: syndromes of salt retention and salt wasting by the collecting duct. Am J Physiol Renal Physiol 283:F221-35
Morris, Ryan G; Schafer, James A (2002) cAMP increases density of ENaC subunits in the apical membrane of MDCK cells in direct proportion to amiloride-sensitive Na(+) transport. J Gen Physiol 120:71-85
Schafer, J A; Li, L; Sun, D (2000) The collecting duct, dopamine and vasopressin-dependent hypertension. Acta Physiol Scand 168:239-44
Schafer, J A (2000) Interaction of modeling and experimental approaches to understanding renal salt and water balance. Ann Biomed Eng 28:1002-9
Wilborn, T W; Sun, D; Schafer, J A (1998) Expression of multiple alpha-adrenoceptor isoforms in rat CCD. Am J Physiol 275:F111-8
Morris, R G; Tousson, A; Benos, D J et al. (1998) Microtubule disruption inhibits AVT-stimulated Cl- secretion but not Na+ reabsorption in A6 cells. Am J Physiol 274:F300-14
Hirsch, J R; Cermak, R; Forssmann, W G et al. (1997) Effects of sodium nitroprusside in the rat cortical collecting duct are independent of the NO pathway. Kidney Int 51:473-6
Schafer, J A; Watkins, M L; Li, L et al. (1997) A simplified method for isolation of large numbers of defined nephron segments. Am J Physiol 273:F650-7
Sun, D; Schafer, J A (1996) Dopamine inhibits AVP-dependent Na+ transport and water permeability in rat CCD via a D4-like receptor. Am J Physiol 271:F391-400

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