Inhibition of NADPH-oxidase activity in human polymorphonuclear neutrophils by lipophilic ascorbic acid derivatives

E Schmid, V Figala and V Ullrich

Faculty of Biology, University of Konstanz, Germany.

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)

Volume 45, Issue 5, pp. 815-825, 05/01/1994
Copyright © 1994 by American Society for Pharmacology and Experimental Therapeutics