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.
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