The primary overall objectives are the characterization and identification of passive cotransport pathways for Na, K, and Cl in erythrocytes of humans and sheep. These pathways are widespread, and have prominent functional significance in epithelia and other tissues as well. Human red cells appear to have two separate pathways, one for Na/K/Cl and one for K/Cl, while sheep cells (LK genotype) have only the one for K/Cl. We proposed to study various aspects of the mechanisms of these transport pathways, including their degree of asymmetry, the role of Cl, and their dependence on membrane voltage. We hope to label specifically the K/Cl pathway in sheep cells by taking advantage of an antigen associated with the pathway. Antibodies against this antigen prevent stimulation of transport by N-ethylmaleimide (NEM). This feature should permit us to label the sites with 3H-NEM, identify the protein, and characterize it partially. We plan to study the regulatory mechanisms associated with cotransport by investigating the roles of metabolism, phosphorylation of membrane proteins, interaction of peripheral proteins with the transport pathway, and the relationship of all these and the sensitivity of transport to changes in cell volume. We will also study developmental changes in cotransport, principally in experiments on reticulocytes in culture. We will look at changes in transport and numbers of antigens during maturation. We will determine the role, if any, in maturation of three newly discovered features of sheep reticulocytes: a reticulocyte-specific protein, microtubules, and coated vesicles. We will attempt to modify the process of maturation with various agents, including antibodies and calmodulin inhibitors. In all of the studies in this proposal, we plan to use both alloimmune antibodies and monoclonal antibodies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK033640-07
Application #
3232052
Study Section
General Medicine B Study Section (GMB)
Project Start
1984-04-01
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
7
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Syracuse University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Bize, I; Munoz, P; Canessa, M et al. (1998) Stimulation of membrane serine-threonine phosphatase in erythrocytes by hydrogen peroxide and staurosporine. Am J Physiol 274:C440-6
Holtzman, E J; Kumar, S; Faaland, C A et al. (1998) Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C. elegans. Am J Physiol 275:F550-64
Dunham, P B; Blostein, R (1997) L antigens of sheep red blood cell membranes and modulation of ion transport. Am J Physiol 272:C357-68
Kelley, S J; Dunham, P B (1996) Mechanism of swelling activation of K-Cl cotransport in inside-out vesicles of LK sheep erythrocyte membranes. Am J Physiol 270:C1122-30
Krarup, T; Dunham, P B (1996) Reconstitution of calyculin-inhibited K-Cl cotransport in dog erythrocyte ghosts by exogenous PP-1. Am J Physiol 270:C898-902
Colclasure, G C; Parker, J C; Dunham, P B (1995) Creatine kinase is required for swelling-activated K-Cl cotransport in dog red blood cells. Am J Physiol 268:C660-8
Parker, J C; Dunham, P B; Minton, A P (1995) Effects of ionic strength on the regulation of Na/H exchange and K-Cl cotransport in dog red blood cells. J Gen Physiol 105:677-99
Dunham, P B (1995) Effects of urea on K-Cl cotransport in sheep red blood cells: evidence for two signals of swelling. Am J Physiol 268:C1026-32
Bize, I; Dunham, P B (1995) H2O2 activates red blood cell K-Cl cotransport via stimulation of a phosphatase. Am J Physiol 269:C849-55
Suvitayavat, W; Dunham, P B; Haas, M et al. (1994) Characterization of the proteins of the intestinal Na(+)-K(+)-2Cl- cotransporter. Am J Physiol 267:C375-84

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