The objective of this proposal is to determine the molecular regulation of the Na-K-2Cl cotransporter (NKCCl) in vascular endothelial cells. This transporter is activated by cell shrinkage and rapidly restores cell volume, thus playing a potentially important role in minimizing gap formation in the endothelial monolayer and thereby maintaining the integrity of the blood-tissue barrier. The transporter is also activated by growth factors to produce rapid increases in cell volume necessary to compensate for loss of adjacent cells and for cell growth. The mechanism by which cell volume and growth factors regulate NKCCl in endothelial cells or other cells is unknown. Current evidence suggests that NKCCl is regulated by phosphorylation but the identity of the responsible kinase and its mode of regulation is unknown.
The aims of this proposal are to demonstrate the existence of a volume-sensitive kinase that phosphorylates NKCCl in vitro, identify the kinase, prove that it regulates NKCCl in vivo, and to demonstrate that the kinase is regulated by an upstream, volume-sensitive kinase that is also regulated by growth factors. This will be accomplished with in vitro kinase assays using genetically engineered fusion proteins encompassing portions of NKCCl, affinity purification, and in vivo assays of NKCCl phosphorylation and activity after specific inhibition of kinases with antisense oligonucleotides and dominant kinase-deficient mutants. The results will provide important information on the function and regulation of this important transporter in endothelial cells and define a specific regulatory kinase pathway that is under dual control by cell volume and growth factors.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL047449-06
Application #
6183494
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Project Start
1993-05-01
Project End
2003-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
6
Fiscal Year
2000
Total Cost
$302,538
Indirect Cost
Name
Emory University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Garg, Puneet; Martin, Christopher F; Elms, Shawn C et al. (2007) Effect of the Na-K-2Cl cotransporter NKCC1 on systemic blood pressure and smooth muscle tone. Am J Physiol Heart Circ Physiol 292:H2100-5
Lomashvili, K; Garg, P; O'Neill, W C (2006) Chemical and hormonal determinants of vascular calcification in vitro. Kidney Int 69:1464-70
Jiang, Gengru; Akar, Fatma; Cobbs, Scott L et al. (2004) Blood pressure regulates the activity and function of the Na-K-2Cl cotransporter in vascular smooth muscle. Am J Physiol Heart Circ Physiol 286:H1552-7
Lomashvili, Koba A; Cobbs, Scott; Hennigar, Randolph A et al. (2004) Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin. J Am Soc Nephrol 15:1392-401
Akar, F; Jiang, G; Paul, R J et al. (2001) Contractile regulation of the Na(+)-K(+)-2Cl(-) cotransporter in vascular smooth muscle. Am J Physiol Cell Physiol 281:C579-84
Akar, F; Skinner, E; Klein, J D et al. (1999) Vasoconstrictors and nitrovasodilators reciprocally regulate the Na+-K+-2Cl- cotransporter in rat aorta. Am J Physiol 276:C1383-90
Klein, J D; Lamitina, S T; O'Neill, W C (1999) JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro. Am J Physiol 277:C425-31
O'Neill, W C (1999) Physiological significance of volume-regulatory transporters. Am J Physiol 276:C995-C1011
Shrode, L D; Klein, J D; Douglas, P B et al. (1997) Shrinkage-induced activation of Na+/H+ exchange: role of cell density and myosin light chain phosphorylation. Am J Physiol 272:C1968-79
Jena, M; Minore, J F; O'Neill, W C (1997) A volume-sensitive, IP3-insensitive Ca2+ store in vascular endothelial cells. Am J Physiol 273:C316-22

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