Adenosine triphosphate-dependent calcium pump in the plasma membrane of guinea pig and human neutrophils

Abstract

Changes in cytosolic free Ca may function as a second messenger in neutrophils. Since the plasma membrane seems to be a major regulator of intracellular Ca in many cells, we characterized an energy-dependent Ca transport system in plasma membrane-enriched fractions ("podosomes") from phorbol myristate acetate-stimulated guinea pig and human neutrophils. The active Ca transport system in guinea pig podosomes exhibited a high affinity for Ca (Michaelis constant [Km]Ca 280 +/- 120 nM) and a maximum velocity of 0.83 nmol Ca/mg protein per min. Uptake showed an absolute requirement for Mg ATP (Km ATP 67 microM), whereas other trinucleotides were inactive. Ca uptake was optimal at pH 7, was azide insensitive and temperature dependent. Vanadium, an inhibitor of the Ca/Mg ATPase of heart sarcolemma, inhibited Ca pump activity by 50% at 1 microM. Ca transport was not affected in a NaCl-containing medium, an observation arguing against the presence of a Na/Ca exchange system. Calmodulin at 0.5-10 micrograms/ml stimulated the Ca pumping activity of EGTA-washed podosomes. Calmodulin depletion decreased the affinity of the Ca pump for Ca (Km Ca 2.07 microM) and its readdition restored it (Km Ca 0.55 microM). ATP-dependent Ca transport by podosomes and phagocytic vesicles was inactivated by exposure to trypsin or to the nonpenetrating sulfhydryl reagent rho-chloromercuribenzene sulfonate. Human podosomes had a Ca uptake system with properties similar to those of the guinea pig. These findings demonstrate the presence of a Ca pump in the neutrophil plasma membrane, which is active at physiological concentrations of free cytosolic Ca. By changing Ca concentrations at the cell periphery, this pump could control various motile functions of the neutrophil, such as locomotion or degranulation.