OXYGEN METABOLITES INDUCED BY PHORBOL-MYRISTATE ACETATE INCREASE LATERAL DIFFUSION OF WHEAT-GERM AGGLUTININ-LABELED GLYCOCONJUGATES IN HUMAN POLYMORPHONUCLEAR LEUKOCYTES

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 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.