1-O-hexadecyl-2-Q-methylglycerol, a novel inhibitor of protein kinase C, inhibits the respiratory burst in human neutrophils.
To assess the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the activation of the human neutrophil respiratory burst, we have utilized an ether lipid of the type 1-O-alkyl-2-O-methylglycerol (AMG), recently shown to be an inhibitor of this kinase. AMG-C16 (with an hexadecyl chain at the sn-1 position) was found to inhibit the respiratory burst induced by sub-optimal concentrations of phorbol 12,13-dibutyrate. Respiratory burst activity was recovered by subsequent addition of a supraoptimal dose of phorbol 12-myristate 13-acetate, indicating that in the presence of the inhibitor only the activation of the NADPH:O2 oxidoreductase via protein kinase C is inhibited, but not the oxidoreductase itself. The respiratory burst induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) was also inhibited in the presence of AMG-C16, the extent of inhibition being dependent on the concentration of fMLP. At the concentrations applied in these studies, AMG-C16 had no effect on cell viability, did not affect the formation of inositol phosphates induced by fMLP, and did not affect the characteristics of the Ca2+ fluxes induced by the same stimulus. In a cell-free assay system, AMG-C16 had no effect on the activity of cAMP-dependent or Ca2+/calmodulin-dependent protein kinase but inhibited protein kinase C in a dose-dependent fashion. To characterize the inhibitory action of AMG-C16 on the respiratory burst activity in more detail, we studied protein phosphorylation in relation to respiratory burst activity in neutrophil cytoplasts. We focused on the phosphorylation of the 47-kDa protein, because this protein is functionally associated with the NADPH:O2 oxidoreductase. At suboptimal concentrations of phorbol 12,13-dibutyrate, AMG-C16 inhibited phosphorylation of proteins, including that of the 47-kDa protein. Recovery of protein phosphorylation in parallel to recovery of respiratory burst activity was obtained by addition of increasing doses of phorbol 12,13-dibutyrate. Recovery of respiratory burst activity at intermediate concentrations of fMLP did not result in a proportional increase in 47-kDa protein phosphorylation; phosphorylation of the 47-kDa protein was recovered only at high concentrations of fMLP. From these data we conclude that protein kinase C is involved in the activation of the respiratory burst by phorbol esters and fMLP. However, with fMLP as a stimulus, a second signal seems to be triggered, which is insensitive to AMG-C16.
Kramer
I M
IM
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
van der Bend
R L
RL
Tool
A T
AT
van Blitterswijk
W J
WJ
Roos
D
D
Verhoeven
A J
AJ
D002118:Calcium / Q000097:blood; D002474:Cell-Free System; D005995:Glyceryl Ethers / Q000494:pharmacology*; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010710:Phosphates / Q000097:blood; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*
D005995:Glyceryl Ethers; D010710:Phosphates; C059039:1-O-hexadecyl-2-O-methylglycerol; D015240:Phorbol 12,13-Dibutyrate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D002118:Calcium