This proposal is part of a long range plan of investigation to define the ionic basis of human neutrophil function. The studies are designed to examine the membrane properties of resting cells as a prerequisite for a better understanding of stimulus-induced events. The principal part of this research plan involves a systematic study of the pathways and transport systems for chloride movements across the resting cell membrane: anion exchange, active transport, and electrodiffusion. These sections include a detailed analysis and biochemical characterization of the kinetic parameters of the anion exchange mechanism with respect to its pH-dependence, anion selectivity, and substrate and inhibitor interactions. The physiologic function of this counter-transport system, as a Cl-/HCO3- exchanger, will be explored in relation to intracellular pH (pHi) recovery from alkaline loads, control of steady-state pHi, and cell volume regulation in anisotonic media. Similar types of analyses will be applied in characterizing the active transport system for the intracellular accumulation of Cl- and in probing the mechanism of uptake as a possible ATP-driven Cl- pump. The properties of passive leak Cl- fluxes will be assessed by examining volume-induced changes in Cl- permeability elicited by cell swelling in hypotonic media. The knowledge gained from the behavior of Cl- channels using this model and from other studies noted above will be valuable in elucidating the alterations in Cl- fluxes that take place in neutrophils activated by chemotactic factors and other stimuli. A major goal of this project is to document a stimulus-induced increase in Cl- conductance as the underlying basis for the membrane depolarization elicited by a variety of agents. All of this work, which involves measurements of isotopic Cl- fluxes, indirect probes of intracellular pH and membrane potential, and patch-clamp techniques, is intended to complement and extend original observations on the resting ion distributions and fluxes of Na+, K+, and Cl-, intracellular pH regulation, and membrane potential of isolated human neutrophils. Thus, this research should provide further insights into the fundamental nature of a cell that subserves critical roles both in host defense against microorganisms and in the pathogenesis of tissue injury in a number of diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038094-02
Application #
3294128
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1987-05-01
Project End
1992-04-30
Budget Start
1988-05-01
Budget End
1989-04-30
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Simchowitz, L; Vogt, S K (1993) Substrate and inhibitor specificity of the lactate carrier of human neutrophils. J Membr Biol 131:23-34
Stoddard, J S; Steinbach, J H; Simchowitz, L (1993) Whole cell Cl- currents in human neutrophils induced by cell swelling. Am J Physiol 265:C156-65
Simchowitz, L; Textor, J A; Cragoe Jr, E J (1993) Cell volume regulation in human neutrophils: 2-(aminomethyl)phenols as Cl- channel inhibitors. Am J Physiol 265:C143-55
Simchowitz, L; Textor, J A (1992) Lactic acid secretion by human neutrophils. Evidence for an H+ + lactate- cotransport system. J Gen Physiol 100:341-67
Dale, W E; Simchowitz, L (1991) The role of Na(+)-Ca2+ exchange in human neutrophil function. Ann N Y Acad Sci 639:616-30
Simchowitz, L; Davis, A O (1991) Internal alkalinization by reversal of anion exchange in human neutrophils: regulation of transport by pH. Am J Physiol 260:C132-42
Simchowitz, L; Textor, J A; Vogt, S K (1991) Use of tributyltin to probe contribution of Cl(-)-HCO3- exchange to regulation of steady-state pHi in human neutrophils. Am J Physiol 261:C906-15
Simchowitz, L; Bibb, J A (1990) Functional analysis of the modes of anion transport in neutrophils and HL-60 cells. Annu Rev Physiol 52:381-97
Simchowitz, L; Davis, A O (1990) Intracellular pH recovery from alkalinization. Characterization of chloride and bicarbonate transport by the anion exchange system of human neutrophils. J Gen Physiol 96:1037-59
Simchowitz, L; Foy, M A; Cragoe Jr, E J (1990) A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils. J Biol Chem 265:13449-56

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