Stimulation of superoxide anion production in guinea pig polymorphonuclear leukocytes by hypotonic conditions in combination with protein kinase C activators

Abstract

Conditions for Superoxide anion (O − 2) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, O − 2 production was significantly enhanced by concurrent treatment with low concentrations of 1-oleoyl-2-acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced O − 2 production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic O − 2 production with hypotonicity. Phosphorylation of 46-kDa protein(s), which was commonly enhanced in parallel with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 μ m OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in O − 2 production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase.