Respiratory burst of rabbit peritoneal neutrophils

Abstract

Superoxide COY) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4P-phorbol1Zmyristate 13-acetate (PMA) was studied at 25 "C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of ' O;, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate '0, in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1 % Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphoraseoxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[pthio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase -oxidase transition was accompanied by a 10-fold increase in the K , for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with crosslinking reagents, like glutaraldehyde or l-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.Phagocytes exposed to a variety of soluble and particulate stimuli show a rapid production of the superoxide anion 'O;, by reduction of 02. The enzyme responsible is an NADPH oxidase complex. This reaction is known as the respiratory burst, since the oxidase is dormant in resting phagocytes and is powerfully activated upon the binding of stimulatory ligands on specific membrane receptors (for review, cf. Rossi, 1986; Bellavite, 1988; Segal, 1989). It is thought that in activated neutrophils the NADPH oxidase complex is composed of two redox components, an NADPHdependent dehydrogenase of molecular mass 65 kDa (Doussi2re and Vignais, 1985;Markert et al., 1985;Kakinuma et al., 1987), that probably contains FAD as prosthetic group, and a low-potential cytochrome b referred to as cytochrome 6 5 5 8 beca...

Activation of O2−‐generating oxidase in an heterologous cell‐free system derived from Epstein‐Barr‐virus‐transformed human B lymphocytes and bovine neutrophils

“…4B). It was recently recognized, with a cell-free system consisting of membranes and cytosol from rabbit neutrophils and nitroblue tetrazolium as electron acceptor, that the reduction of the dye was largely insensitive to superoxide dismutase, indicating that electrons are trapped by the dye instead of reacting with O2 (Laporte et al, 1990). This was interpreted in terms of better accessibility of the smallsized nitroblue tetrazolium to an intermediary component of the oxidase complex.…”

Human fat cells possess a plasma membrane-bound H2O2-generating system that is activated by insulin via a mechanism bypassing the receptor kinase.

“…The respiratory burst oxidase is a multicomponent electron transport system containing a characteristic b-type cytochrome (cytochrome b558) and possibly a flavoprotein (23,24,(45)(46)(47)(48)(49).…”

Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils

“…The question of whether the NADPH oxidase of neutrophils contains an intrinsic diaphorase activity has been recently discussed (Laporte et al, 1990;Fujii and Kakinuma, 1991). Whereas it is true that, in a fully competent oxidase complex, the electron flux is directed to O2 to generate 0; (Bellavite et al, 1984), conditions have been described which result in a by-pass of the electron flux from NADPH to artificial acceptors via the NADPH dehydrogenase component of the oxidase complex (see Laporte et al, 1990).…”

“…Whereas it is true that, in a fully competent oxidase complex, the electron flux is directed to O2 to generate 0; (Bellavite et al, 1984), conditions have been described which result in a by-pass of the electron flux from NADPH to artificial acceptors via the NADPH dehydrogenase component of the oxidase complex (see Laporte et al, 1990).…”