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Activation of the phagocytic cell superoxide-generating NADPH oxidase requires interaction of cytosolic and membrane-associated components. With most stimuli activation of the oxidase is accompanied by multisite phosphorylation of the 47-kDa cytosolic oxidase factor (p47) which translocates from cytosol to membranes. Native p47 is a highly basic protein that undergoes stepwise charge shifts with successive phosphorylation events. Phosphorylation of p47 was studied by immunoprecipitation from neutrophil cytosol and membrane fractions followed by two-dimensional gel electrophoresis and autoradiography. In the resting cell p47 was not phosphorylated. In the cytosol of phorbol myristate acetate-activated neutrophils eight distinct p47 phosphoproteins were present. The membrane fraction from these activated cells contained a family of p47 phosphoproteins of electrophoretic mobilities identical to those seen in cytosol plus an additional, more acidic p47 phosphoprotein not present in cytosol. Very early after activation (30 s) only the four most acidic p47 phosphoproteins were present in the membrane fraction. Only at later times (5-15 min) was the full spectrum of p47 phosphoproteins present in the membrane fraction. In contrast, the full spectrum of p47 phosphoproteins was present in the cytosol over the entire time course we studied. In neutrophils from patients with cytochrome b558-deficient chronic granulomatous disease p47 phosphorylation was incomplete and p47 translocation to membrane did not occur. These studies demonstrated that the cytochrome was essential for formation of the three most acidic p47 phosphoproteins and greatly augmented formation of the fourth most acidic p47 phosphoprotein found in normal neutrophils. The temporal correlation between specific p47 phosphorylation events and p47 translocation to membrane is consistent with a model of oxidase activation in which a series of p47 phosphorylation events which occurs in cytosol precedes and may be required for p47 interaction with membrane.

Professional phagocytes contain a unique NADPH oxidase responsible for the production of microbicidal oxidants. Activation of this oxidase requires participation of cytosolic and membrane proteins, but the interactions of these components are incompletely understood. Patients with autosomal recessive Chronic Granulomatous Diseases (CGD) are characterized by functional defects in phagocyte oxidase activity resulting from a deficiency of either a 47 kDa (p47) or a 67 kDa (p67) cytosolic oxidase component. Cytosols from such patients are valuable for biochemical studies of the oxidase, but are not generally available because CGD is a rare disorder. The present study illustrates means of producing cytosols functionally and immunochemically deficient in either p47 or p67. Cytosol from monocytes cultured for 6 days is immunochemically deficient in p47 but not p67, while cytosol from HL-60 cells induced with retinoic acid for 3 days is deficient in p67 but not p47. Each of these cytosols fail to generate superoxide when added to neutrophil membranes in a cell-free assay but complement each other when combined. Complementation studies in which these cytosols were mixed in the cell-free assay with p47- or p67- deficient CGD cytosol established the functional characteristics of the experimentally produced cytosols.

A vascular permeability factor (VPF) derived from serum-free conditioned medium of cultured human malignant gliomas (HG-VPF) has been described previously. The rapid kinetics of HG-VPF activity in an in vivo assay of vascular permeability suggests a direct action upon the vascular endothelial cell. To determine whether HG-VPF was capable of inducing a physiologically significant alteration in isolated endothelial cells, cytosolic calcium [Ca++]i was measured in vitro in these cells before and after their exposure to media containing this substance. This was accomplished by preloading cultured endothelial cells with a fluorescent intracellular Ca++ probe fura-2/AM. It was found that HG-VPF induced a rapid and transient elevation of [Ca++]i in normal endothelial cells derived from human umbilical vein, bovine adrenal medulla, bovine pulmonary artery, and rat brain. This effect was inhibited by chelating extracellular calcium [Ca++]e with ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetra-acetic acid (EGTA), indicating that the HG-VPF-induced response resulted from the influx of extracellular calcium. The addition of cations that act as nonspecific calcium channel blockers (Li+, Co++, Mn++, La ) completely inhibited VPF activity, further supporting the role of [Ca++]e influx. The HG-VPF activity was not, however, blocked by verapamil, a calcium antagonist that appears to be specific for voltage-gated calcium channels. Furthermore, exposure of endothelial cells to 120 mM [K+]e did not result in a calcium transient. Coincubation of endothelial cells with dexamethasone inhibited HG-VPF-induced rises in [Ca++]i, while having no effect upon cyclic nucleotide-induced changes in calcium. The present studies indicate that vascular extravasation induced by human glioma-derived VPF may be mediated by a direct action upon vascular endothelial cells. Furthermore, the observed dexamethasone-induced inhibition of this process suggests a specific cellular action for corticosteroids. This, together with previous observations that dexamethasone suppresses both the production of VPF by tumor cells in vitro and its permeability-inducing activity in vivo, may explain the efficacy of glucocorticoids in the treatment of neoplastic vasogenic brain edema. Finally, studies with a polycationic peptide (protamine) known to induce blood-brain barrier disruption in vivo revealed similar effects upon endothelial cytosolic calcium levels. As HG-VPF is a positively charged macromolecule, a common interaction between these substances and the negatively charged endothelial cell surface in the induction of permeability is suggested. Nonspecific cross-linking of charged groups of the endothelial glycocalyx and specific HG-VPF receptor binding are both valid mechanisms of HG-VPF-mediated calcium changes. Their potential relevance in the setting of microvascular permeability is discussed.