RESUMO
The effect of modification of maleimide derivatives on superoxide production by guinea-pig neutrophils induced by a variety of different soluble stimuli was studied. Pretreatment of neutrophils by showdomycin, a very slowly penetrating-SH reagent, did not affect superoxide production by all of the stimuli used, suggesting no exposure of sulfhydryl groups involved in superoxide-generating system on the cell surface. Pretreatment with N-ethylmaleimide (MalNEt), a considerably penetrating-SH reagent, markedly inhibited superoxide production stimulated by formyl-methionyl-leucyl-phenylalanine (HCO-Met-Leu-Phe), cytochalasin E or digitonin, but not superoxide production stimulated by the ionophore A23187 or sodium fluoride. The oxygen consumption stimulated by HCO-Met-Leu-Phe or cytochalasin E was inhibited by MalNEt pretreatment, whereas the oxygen consumption stimulated by A23187 was not inhibited by MalNEt. The inhibition by MalNEt of superoxide production did not appear to be due to the interference with binding of the affected stimuli, since MalNEt pretreatment did not inhibit the release of lysozyme, granule enzyme, induced by HCO-Met-Leu-Phe, cytochalasin E or digitonin. Particulate fractions from MalNEt-pretreated neutrophils before exposure to the stimulus exhibited the inhibition of the enhancement of NADPH-dependent superoxide production induced by HCO-Met-Leu-Phe, cytochalasin E or digitonin, but not A23187, whereas treatment of neutrophils with MalNEt after activation by these stimuli had no effect on the NADPH oxidase activity in particulate fractions. Direct exposure of particulate fractions from A23187-stimulated neutrophils to MalNEt showed no actual susceptibility of NADPH oxidase to MalNEt inhibition. These findings suggest that the inhibitory effect of MalNEt is caused by the modification of the process of the activation by the affected stimuli of the superoxide system, probably NADPH oxidase and that at least two mechanisms exist for activation of superoxide-generating system in guinea-pig neutrophils on the basis of the susceptibility to MalNEt inhibition.
