Platelet-activating factor induces protein kinase activity in the particulate fraction of human neutrophils.
Platelet-activating factor (PAF) is a proinflammatory lipid that has both platelet- and phagocyte-stimulating properties. Because several known activators of calcium-, phospholipid-dependent protein kinase (protein kinase c, PKC) also stimulate neutrophil responses and because neutrophil stimuli such as phorbol diesters and the chemotactic peptide f-Met-Leu-Phe are reported to increase protein kinase activity in neutrophil (PMN) particulate fractions, we investigated the effect of PAF on neutrophil protein kinase activities. In neutrophils exposed to 10(-6) mol/L PAF, cytosolic PKC activity was 521 +/- 38 pmol 32P/10(7) PMN/min (mean +/- SEM), which was not significantly lower than cystolic activity in buffer-treated controls (558 +/- 32 pmol 32P/10(7) PMN/min, n = 14). PAF-exposed cells exhibited a concomitant rise in protein kinase activity associated with the particulate fraction with 53 +/- 4 pmol 32P/10(7) PMN/min compared with 32 +/- 2 pmol in control cells (n = 14). Particulate protein kinase activity was independent of the presence of calcium and phospholipid in the assay medium. The specific PKC inhibitor H-7 inhibited particulate protein kinase activity, however, which suggested that the enzyme activity assayed in this fraction may be PKC in a constitutively activated form. The increase in particulate protein kinase activity induced by PAF required the presence of cytochalasin B, was detectable within 5 seconds of exposure to PAF, and was not reversed by washing the cells free of extracellular PAF after initial exposure. Although PAF did not have a direct effect on PKC activity from cytosolic fractions from resting cells, the increase in particulate protein kinase activity induced by PAF was inhibited when the cells were first depleted of calcium by incubation with Quin 2. These results suggest that PAF induces an increase in particulate protein kinase activity in neutrophils by a calcium-dependent mechanism and that the induction of membrane-associated protein kinase activity may be involved in neutrophil-stimulating actions such as superoxide production, which occur at higher concentrations of PAF.
Gay
J C
JC
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232.
Stitt
E S
ES
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002118:Calcium / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010743:Phospholipids / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D013481:Superoxides / Q000096:biosynthesis
D007546:Isoquinolines; D010743:Phospholipids; D010879:Piperazines; D010972:Platelet Activating Factor; D013481:Superoxides; D003571:Cytochalasin B; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011494:Protein Kinases; D002118:Calcium