Oxygen metabolites induced by phorbol myristate acetate increase lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in human polymorphonuclear leukocytes

Abstract

To assess the general effects of protein kinase C (PKC) activation on cell membrane receptor mobility in human neutrophilic polymorphonuclear leukocytes (PMNLs), the lateral diffusion of fluoresceinated succinylated wheat germ agglutinin (S-WGA-FITC)-labeled membrane glycoconjugates was measured using fluorescence recovery after photobleaching (FRAP). Activation of PKC was achieved by incubating the PMNLs with different concentrations (5-100 nM) of phorbol myristate acetate (PMA). The membrane effects of dimethyl sulfoxide (DMSO), another possible membrane perturbant, were also studied. We found that PMA treatment (greater than or equal to 10 nM) increased the glycoconjugate diffusion coefficient (D) 2-2.5-fold. The mobile fraction (R) remained constant, around 30%. With DMSO, no effect on the diffusion was seen. The increase in lateral mobility due to cell stimulation with PMA was totally inhibited by catalase (200 units/ml) but only partly with superoxide dismutase (2000 units/ml). Exogenous hydrogen peroxide (0.01-5 mM) had no effect on glycoconjugate mobility in unstimulated cells. We therefore propose that activation of PKC mediates augmented mobility of glycoconjugate receptors in PMNL, a reaction that seems to be critically dependent on formation of reactive oxygen metabolites. The results indicate that endogenous formation of reactive metabolites upon receptor stimulation may have a general effect on receptor mobility.