This grant proposal concerns mechanisms by which red blood cells achieve and maintain their content of water and small ions. The experiments are divided into two parts. The first involves dog red blood cells, in which movements of sodium, calcium, protons, and potassium across the membrane are strongly dependent on cell volume. The proposed work seeks to examine the nature of the stimulus-response coupling, so as to clarify how certain membrane transporters are turned on and off by minor degrees of cell shrinkage or swelling. Methods will involve the use of cross-linking agents, membrane-expanding treatments, and selective proteolyic digestion to influence the relationship between cell volume and ion transport. Membrane protein separations will be performed by gel electrophoresis, using cells that have had specific transporters """"""""turned on"""""""" or """"""""turned off"""""""" by volume manipulation. Net and isotopic flux measurements will be used to monitor transport. The second project is a one-year, preliminary study designed to evaluate, by the techniques of electron probe microanalysis, a unifying hypothesis that would explain the generation of dense, viscous red blood cells in sickle cell disease, hereditary spherocytosis, and other hemolytic states. The hypothesis states that red blood cells in all these conditions are subject to fragmentation or budding. The vesicles that bud off the cells are believed to be richer in water and salts and poorer in hemoglobin content than the parent cells, thus leaving the latter dense, viscous, and poorly deformable. The work proposed here involves a one year feasibility study during which red blood cell fragments produced in vitro would be assayed for ion, water, and hemoglobin content by standard techniques of atomic absorption and/or flame photometry. The same fragments would be subjected to electron probe microanalysis, to determine whether the method has the sensitivity and specificity to approach the problem with blood samples from patients. The hypothesis cannot be evaluated in the blood of patients in any other way than by electron probe microanalysis: this is because cell fragments in the circulation may originate from platelets, leukocytes, and possibly other non-red cell sources. Only by combining the techniques of electron microscopy and microanalysis can one be sure of the origin of the fragments in which quantitative elemental determinations are to be made.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK011356-20
Application #
3224780
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1975-09-01
Project End
1990-08-31
Budget Start
1986-09-01
Budget End
1987-08-31
Support Year
20
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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
Colclasure, G C; Parker, J C (1993) ATP dependence of K-Cl cotransport in dog red blood cells. Am J Physiol 265:C1648-52
Parker, J C (1993) Urea alters set point volume for K-Cl cotransport, Na-H exchange, and Ca-Na exchange in dog red blood cells. Am J Physiol 265:C447-52
Parker, J C (1992) Volume-activated cation transport in dog red cells: detection and transduction of the volume stimulus. Comp Biochem Physiol Comp Physiol 102:615-8
Orringer, E P; Parker, J C (1992) Hydroxyurea and sickle cell disease. Hematol Pathol 6:171-8
Parker, J C; Colclasure, G C (1992) Actions of thiocyanate and N-phenylmaleimide on volume-responsive Na and K transport in dog red cells. Am J Physiol 262:C418-21
Colclasure, G C; Parker, J C (1992) Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells. J Gen Physiol 100:1-10
Parker, J C; Colclasure, G C (1992) Macromolecular crowding and volume perception in dog red cells. Mol Cell Biochem 114:9-11
Colclasure, G C; Parker, J C (1991) Cytosolic protein concentration is the primary volume signal in dog red cells. J Gen Physiol 98:881-92

Showing the most recent 10 out of 19 publications