Biomedical subjects
Publications and source records attributed to P Bellavite.
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Studies were carried out on the mechanism responsible for the enhancement of the respiratory and secretory responses to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) exhibited by human neutrophils suspended in Na+-free, high-K+ buffered solution. The results demonstrate that: (a) the variation of Na+ concentration in the suspending solution induces in human neutrophils a marked modification of the recognition apparatus for the chemotactic peptide fMet-Leu-Phe, the lack of or low concentration of this ion increasing the number of the receptors and their specific affinity for the ligand; (b) the greater respiratory burst and secretion induced by fMet-Leu-Phe in human neutrophils suspended in Na+-free, high-K+ medium are due to the increased formation of receptor-ligand complexes at the cell membrane; (c) the greater respiratory response is partially due also to a higher efficiency of these receptor-ligand complexes. The molecular mechanism by which Na+ exerts a regulative role on the properties of the recognition apparatus for the chemotactic peptide and its possible significance are discussed.
NADPH oxidase from stimulated guinea pig granulocytes was extracted with deoxycholate. The solubilized enzyme was stable in 20% glycerol. Solubilized enzyme was free of myeloperoxidase activity. The properties of the deoxycholate solubilized enzyme indicated that it is a high molecular weight complex with a flavoprotein, calmodulin and cytochrome b possibly forming part of the complex. Maximum activity was between pH 7.0 and 7.5. The Km value was 15.8 microM for NADPH and 434 microM for NADH indicating that NADPH is the preferential substrate.
The paper reports a simple, sensitive and time-saving procedure for the assay of the function of the phagocytes on microsamples of whole blood. The method consists in the evaluation of the stimulation of superoxide anion (O-2) production (as superoxide dismutase-sensitive cytochrome c reduction) by leukocytes in whole blood challenged with (a) phagocytosable particles (opsonized zymosan); (b) particles that become phagocytosable by virtue of the opsonizing capacity of the plasma of blood samples (zymosan); and (c) a soluble agent such as phorbol myristate acetate. Preliminary studies indicate that this procedure can be used as a routine test because it enables information to be obtained about the respiratory responsiveness of phagocytes and about cellular and humoral defects of phagocytosis.
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The subcellular distribution of the NADPH oxidase of guinea-pig peritoneal-elicited macrophages was investigated. Post-nuclear supernatants obtained from PMA-stimulated macrophages were fractionated in discontinuous sucrose gradients. The NADPH oxidase was found to be enriched at the interface between 20 and 34 per cent sucrose. This interface was also enriched in 5'-nucleotidase, a plasma membrane marker and in glucose-6-phosphatase and NADPH-cytochrome c reductase, two endoplasmic reticulum markers. The distribution in the gradient of beta-glucuronidase, a marker of lysosomes and of succinate dehydrogenase, a marker of mitochondria was clearly different from that of NADPH oxidase and of the markers of plasma membrane and of endoplasmic reticulum. These results indicated that in stimulated-elicited macrophages the NADPH oxidase is associated with a membrane fraction. With the fractionation technique employed it was not possible to clarify whether the oxidase is located in the plasma membrane or in the endoplasmic reticulum. In order to clarify this matter the isolation of phagosomes was performed. NADPH oxidase was found to be enriched in the phagosomal fraction. Phagosomes were also found to be enriched in the plasma membrane marker 5'-nucleotidase. Glucose-6-phosphatase,, a marker of endoplasmic reticulum, and beta-glucuronidase, a marker of lysosomes were not enriched in the phagosomal fraction. The results obtained clearly suggest that the activated NADPH oxidase of peritoneal elicited macrophages of guinea pig is located in the plasma membrane.
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1. The so called "soluble" oxidase(s) are not involved in the respiratory burst of guinea pig and human granulocytes and of guinea pig peritoneal resident and elicited macrophages. 2. The activation of the oxidation of NADPH by a membrane bound NAD(P)H oxidase is the main mechanism responsible for the activation of the respiration of phagocytes. 3. The oxidase is inactive in resting cells and the activated form works on the plasma membrane. 4. More than one mechanism is operative in the oxidation of NAD(P)H by cell free particles in vitro. These mechanisms vary in relation to the conditions of assay (pH and concentration of substrate). 5. Under optimal conditions in vitro the enzymatic oxidation of NADPH practically involves the univalent pathway of oxygen reduction with stoichiometry of two nanomoles of O2 formed for one nanomole of NADPH oxidized. 6. Also in intact cells all O2 is first univalently reduced to O2 and then discharged outside the cell or in the phagocytic vacuoles. 7. The main reactions involved in the O2 balance in intact cells are the univalent reduction of O2, the dismutation of O2 to H2O2 and the degradation of the peroxide through catalatic and peroxidatic mechanisms. 8. The total oxygen univalently reduced by the activated oxidase is 2-4 folds the net oxygen consumed by the cells, depending on the mechanism of H2O2 degradation. 9. All the rate of extrarespiration is accounted for by the rate of oxidation of physiological concentration of NADPH by the membrane-bound enzyme. This adequacy can be observed only under appropriate experimental conditions, because the high activity of the oxidase is not a permanent state.
Recent studies have demonstrated that the activated NADPH oxidase, the enzyme responsible for the stimulation of O2 consumption with O2 formation during phagocytosis, is located in the plasma membrane of leukocytes. The present work deals with whether the activation induced by phagocytosis involves the enzyme of the entire membrane or only that of the portion of the membrane that interacts with the phagocytosable particle and forms the phagosome. The results presented show that the activity of the NADPH oxidase of phagosomal membrane, isolated by centrifugation of homogenates on discontinuous sucrose gradients, is increased 12.6-fold with respect that of homogenate. In contrast, the activities of 5'-nucleotidase and of acid p-nitrophenyl phosphatase, enzyme markers of the plasma membrane not activated during phagocytosis and uniformly distributed on the entire membrane, are increased only about three-fold with respect to that of homogenate. These results indicate that during phagocytosis and activation of NADPH oxidase is a segmentary response that involves only the enzyme that forms the phagocytic vacuole. This fact is relevant for the function of toxic intermediates of oxygen reduction that are discharged in direct contact with the engulfed agent.
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1. The NADPH-oxidizing activity of a 100 000 X g particulate fraction of the postnuclear supernatant obtained frm guinea-pig phagocytosing poymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O2- generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH. 2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1:1. 3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O2- generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation. 4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O2- formed during the oxidation of NADPH. 5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reactin at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH. 6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH. 7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible. 8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase. 9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O2; an enzymatic, apparently non-univalent, O2 reduction and a non-enzymatic chain reaction. 10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O2 is optimal at pH 7.0 with 1 mM NADPH.
One of the most impressive property of the leucocytes is that of changing the oxidative metabolism during various functions. When challenged with phagocytosable particles or with membrane perturbing agents such as chemotactic factors, detergents, lectins and other ligands, granulocytes and mononuclear phagocytes undergo a dramatic increase of oxygen consumption which is associated with the production of superoxide anion (O-2), of hydrogen peroxide (H2O2) and of hydroxyl radical (OH.). These events are referred to as "respiratory burst' . Most of the functions of the inflammatory cells (killing of micro-organisms, tissue damage, amplification of the inflammatory process) are linked to the production, to the fate and to the chemical reactivity of these highly reactive compounds. The authors examine the following aspects: (i) the mechanism responsible for the respiratory burst; (ii) the conditions present in the inflammatory site that induces the metabolic activation of leucocytes; (iii) the variability of the respiratory burst in different types of leucocytes; (iv) the fate, the interrelationships and the reactivity of the intermediate products of oxygen reduction; (v) the relationships between the inflammatory process and the production of free radicals by the inflammatory cells.
The paper presents an experimental procedure for a simultaneous assay of oxygen consumption, O2- release and H2O2 accumulation at a very early stage of the respiratory burst that is induced by phagocytosis in guinea pig polymorphonuclear leucocytes. The main findings are as follows: (a) The oxygen consumption that is measurable does not correspond to all oxygen that is reduced. The relationship between the actual oxygen consumed and the amount that is reduced depends on the fate of the intermediate products O2- and H2O2. (b) O2- is measurable extracellularly by the reduction of cytochrome c. When cytochrome c oxidizes the extracellular O2-, molecular oxygen is formed. This fact is shown by a decrease of oxygen consumption. The molar ratio between the O2- detected and the oxygen given back is 1. (c) The amount of O2- released from the cells accounts for only a small part of oxygen actually reduced. (d) H2O2 is detectable only in the presence of NaN3. In this condition almost all oxygen consumed is recovered in the form of H2O2. The molar ratio O2/H2O2 is near unity. The amount of H2O2 derived from dismutation of O2- released is only an aliquot of the total H2O2 accumulated. Thus, most of H2O2 is derived from intracellular sources. (e) In the absence of inhibitors of H2O2 degrading reactions, no detectable accumulation of peroxide occurs. Under these conditions, the main part of H2O2 formed is degraded in almost equal amount by catalase and myeloperoxidase, while only a small aliquot is degraded by NaN3 insensitive reactions.
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