Activation of guinea-pig and bovine neutrophil NADPH oxidase by N,N'-dicyclohexylcarbodiimide.
Dicyclohexylcarbodiimide (DCCD) is a potent stimulant of superoxide generation in guinea-pig peritoneal and bovine blood neutrophils. The dependence of DCCD-elicited respiratory burst on the composition of the medium was investigated. At 37 degrees C, the superoxide generation was short-lived and a rapid losses of enzymatic activity was observed; at 0 degree C, the activity could be preserved for hours. Superoxide generation by whole cells was accompanied by exocytic degranulation. Prolonged incubation with DCCD at 37 degrees C resulted also in a progressive loss of cellular integrity evidenced by the release of a fraction of lactate dehydrogenase. Km values of the particulate NADPH oxidase isolated from DCCD-triggered guinea-pig and bovine cells were 31.7 and 50.0 microM, respectively. Cells pre-equilibrated with the potential sensitive dye Di-S-C3-(5) exhibited changes in the transmembrane potential upon stimulation. Stimulation with DCCD resulted also in the release of membrane-associated calcium, indicated by quenching of the fluorescence of chlortetracycline-loaded neutrophils. Both effects were observed also in human neutrophils which did not generate superoxide upon exposure to DCCD. The mechanism of DCCD-induced responses is discussed.
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D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002234:Carbodiimides / Q000494:pharmacology*; D002417:Cattle; D003470:Culture Media; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D006168:Guinea Pigs; D008274:Magnesium / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010101:Oxygen Consumption / Q000187:drug effects; D013481:Superoxides / Q000097:blood; D013696:Temperature
D002234:Carbodiimides; D003470:Culture Media; D013481:Superoxides; D004024:Dicyclohexylcarbodiimide; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D008274:Magnesium; D005033:Ethylmaleimide; D002118:Calcium