Activation of electropermeabilized neutrophils by adenosine 5'-[gamma-thio]triphosphate (ATP[S]). Role of phosphatases in stimulus-response coupling

Abstract

Electrically permeabilized human neutrophils were used to study the mechanism of activation of the NADPH oxidase by chemotactic factors. The respiratory burst elicited by formyl-methionyl-leucyl-phenylalanine (fMLP) was strictly dependent on the addition of ATP. The response was also supported by adenosine 5'-[gamma-thio]triphosphate (ATP[S]), but not by the non-hydrolysable analogue (p[NH]ppA). In the presence of ATP, displacement of fMLP from its receptor by antagonist peptides resulted in the abrupt termination of the O2-consumption burst. In contrast, the response persisted after displacement of fMLP when ATP[S] was present. This finding is consistent with the formation of biologically active thiophosphoproteins which are resistant to cleavage by cellular phosphatases. Accordingly, lower concentrations of ATP[S], as compared with ATP, were required to support the fMLP response. The data indicate that protein phosphatases control the extent and duration of the response in cells stimulated with chemoattractants. Unlike ATP, sub-millimolar concentrations of ATP[S] elicited a spontaneous respiratory burst in the absence of fMLP or other stimuli. This effect was inhibited by p[NH]ppA and was not observed in intact (non-permeabilized) cells, indicating interaction of ATP[S] with an intracellular adenine-nucleotide-binding site, possibly a protein kinase. These results suggest that protein kinases are active in neutrophils in the absence of exogenous stimuli, but that accumulation of the essential phosphoprotein(s) is normally prevented by the ongoing vigorous phosphatase activity. It is conceivable that control of the respiratory burst is exerted by inhibition of phosphatase activity, instead of or in addition to the more commonly postulated activation of protein kinases.