Release of superoxide and change in morphology by neutrophils in response to phorbol esters: antagonism by inhibitors of calcium-binding proteins.

Abstract

The ability of phorbol derivatives to function as stimulating agents for superoxide (02-) release by guinea pig neutrophils has been evaluated and compared to the known ability of each compound to activate protein kinase C. Those that activate the kinase also stimulate 02-release, while those that are inactive with respect to the kinase have no effect on 02-release. The same correlation was observed with respect to the ability of phorbol esters to induce morphological changes in neutrophils, i.e., vesiculation and reduction in granule content. Certain phenothiazines and naphthalene sulfonamides that are known antagonists of calcium-binding proteins blocked both phorbol ester-induced O~-release and morphological changes in these cells.

Mutation of the Cyba gene encoding p22phox causes vestibular and immune defects in mice

“…We used the ferricytochrome c assay to quantify the rates of superoxide generation in WT and nmf333/nmf333 neutrophils in the presence and absence of PMA, which activates superoxide generation through the PKC signaling pathway (26). Neutrophils from homozygous nmf333 mutant mice did not produce detectable levels of superoxide, either in the presence or absence of the stimulus ( Figure 2B), whereas PMA-activated WT cells generated superoxide at previously described levels (27).…”

Cofilin undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase complex. Evidence for a novel cycle of phosphorylation and dephosphorylation

“…1,2-Dioctanoyl-sn-glycerol was purchased from Calbiochem, La Jolla, Calif. A rabbit polyclonal antibody to purified cofilin from porcine brain was a gift from Paul Janmey (Harvard Medical School, Boston, Mass.). The protein kinase inhibitors H-7, ML-7, and KN-62 were obtained from Seikagaku America, St. Petersburg, Fla. Sources of all other materials are provided elsewhere (Robinson et al 1985Badwey et al 1989). The peptides CGQTVDDPYATFVK [(C)G61-K73] and CGVTITDPFKHFVG [(C)G61-G73] were produced by solid state synthesis on an HMP resin using Fmoc [N-(9-fluorenyl)methoxycarbonyl] chemistry by Analytical Biotech Services, Boston, Mass.…”

The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view

“…The latter group of agonists has been particularly valuable in sorting out the various components of the stimulatory pathways involved in O2-production. For example, certain tumor promoters that activate protein kinase C (PKC; e.g., PMA, mezerein, teleocidin; Castagna et al 1982) trigger rates of O2 release from neutrophils comparable to those ob- (1979,1984) and Badwey et al (1989b) served during phagocytosis (Robinson et al 1985;Badwey et al 1988; for review . Similarly, A1F 4 activates members of the heterotrimeric G protein family (e.g., Sternweis and Gilman 1982;Sondek et al 1994).…”

“…In addition to phagocytosable particles, a remarkably diverse group of soluble and quasi-soluble agents can stimulate O~JH202 release. These agonists include ligands that bind to specific receptors on the cell surface [e.g., fMet-Leu-Phe (fMLP)] (e.g., Williams et al 1977;Lehmeyer et al 1979), hydrophobic molecules that intercalate into and disorganize the plasmalemma (e.g., cis-unsaturated fatty acids, retinoids) (Badwey et al 1984;Robinson et al 1987), and chemicals that activate specific enzymes involved in the signaltransduction sequence(s) of these cells [e.g,, aluminum fluoride (A1F 4 )] (e.g., Curnutte et al 1979;Robinson et al 1985;Hartfield and Robinson 1990). The latter group of agonists has been particularly valuable in sorting out the various components of the stimulatory pathways involved in O2-production.…”