title: Inhibition of NADPH-oxidase activity in human polymorphonuclear neutrophils by lipophilic ascorbic acid derivatives.
abstract: As recently reported, B-003 (6-S-hexadecyl-2-methoxythioascorbic acid) shows strong inhibition of the N-formylmethionylleucyl phenylalanine (fMLP)-stimulated neutrophil superoxide production and degranulation ex vivo, which is not correlated with its antioxidant properties. Structure-activity studies with 12 derivatives. together with permeation studies, pointed to a process for uptake of B-003 but not its regioisomer B-015 into neutrophils and revealed the importance of the free acidic enolic hydroxyl group in the 3-position of ascorbic acid and of a long chain alkyl group having a chain length of C16-C18 for effective inhibition. We now report that B-003 also strongly suppressed C5a-, concanavalin A-, and calcium ionophore A23187-stimulated superoxide formation, whereas protein kinase C-mediated activation by phorbol ester remained unaffected. The fMLP- or C5a-induced calcium mobilization form intracellular stores of fura-2-loaded cells, as well as the fMLP- or A23187-triggered release of [14C] arachidonate from prelabeled neutrophils, was not affected by B-003. The observed release of GSH was not causally related to inhibition of the oxidative burst, because GSH depletion by 1-chloro-2,4-dinitrobenzene was without effect on the fMLP-stimulated superoxide formation or on the inhibitory effect of B-003. In a cell-free system, consisting of a light membrane fraction and a cytosol fraction from resting neutrophils, B-003 inhibited the arachidonate-induced assembly of the NADPH-oxidase under conditions where particulate NADPH-oxidase from phorbol ester-preactivated neutrophils and catalytically active cell-free assembled oxidase were not affected. The inhibitory effect was more pronounced when the system was incubated in the presence of the G protein activator guanosine-5'-O-(3-thio)triphosphate (GTP gamma S). [35S]GTP gamma S binding studies excluded displacement of the G protein activator from guanine nucleotide binding sites by B-003. In vitro assembly/co-sedimentation experiments in the presence of GTP gamma S revealed a 2-fold increase in a small cytosolic G protein with a molecular mass of 21 kDa (p21) in pelleted membranes, as detected by [35S]GTP gamma S protein blot probing, that was not affected by B-003. Structure-activity relationship studies of the effects of various 6-S-alkylascorbyl derivatives on the GTP gamma S/arachidonate-triggered assembly of the NADPH-oxidase showed strong dependence of the inhibition on the alkyl chain length, with long chain alkyl derivatives (C16 and C18) being most effective.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Faculty of Biology, University of Konstanz, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Schmid
authors.forename: V
authors.initials: V
authors.lastname: Figala
authors.forename: V
authors.initials: V
authors.lastname: Ullrich
mesh_terms: D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000378:metabolism; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: C087338:6-S-n-hexadecyl-3-O-methyl-6-thioascorbic acid; D006150:Guanine Nucleotides; D013481:Superoxides; D016718:Arachidonic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; C087337:6-S-hexadecyl-2-methoxythioascorbic acid; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate; D001205:Ascorbic Acid; D002118:Calcium