Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro.
Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.
Pollack
R J
RJ
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104.
Lok
J B
JB
Donnelly
J J
JJ
D000818:Animals; D003470:Culture Media / Q000494:pharmacology; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism / Q000502:physiology; D005978:Glutathione / Q000502:physiology*; D066298:In Vitro Techniques; D008842:Microfilariae / Q000254:growth & development; D009854:Onchocerca / Q000254:growth & development*; D009930:Organic Chemicals; D009994:Osmolar Concentration; D010710:Phosphates / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013997:Time Factors
D003470:Culture Media; D009930:Organic Chemicals; D010710:Phosphates; D013438:Sulfhydryl Compounds; C046666:tryptose; D005978:Glutathione; D003545:Cysteine