The priming effect of 12(S)-hydroxyeicosatetraenoic acid on lymphocyte phospholipase D involves specific binding sites

We have previously shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HET E)-enrichment primed human peripheral blood mononuclear cells for phospholi pase D activation by mitogens. Given that 12(S)-HETE-enriched cells stimula ted with concanavalin A released free 12(S)-HETE in the extracellular mediu m, and that the priming effect of 12(S)-HETE on phospholipase D was suppres sed by the nonpermeant drug, suramin, we hypothesized an extracellular mech anism for 12(S)-HETE-induced PLD activation. Using [H-3]12(S)-HETE as a lig and and a rapid filtration technique, we have pointed out the presence of s pecific low-affinity 12(S)-HETE binding sites on intact human mononuclear c ells and lymphocytes. [H-3]12(S)-HETE binding was efficiently displaced by other monohydroxylated and n-3 fatty acids but not by oleate and arachidona te, and was also significantly inhibited by suramin and pertussis toxin. Fu rthermore, 12(S)-HETE-induced PLD activation was strongly inhibited by pert ussis toxin and genistein, but was not PKC-dependent. In addition, 12(S)-HE TE also potentiated the ConA-induced tyrosine phosphorylation. of a 46-50 k Da protein, which was inhibited by genistein. Collectively, these results s uggest that 12(S)-HETE binding sites on human lymphocytes may be coupled to phospholipase D through pertussis toxin sensitive G-proteins and tyrosine kinases.