Synergistic stimulation of neutrophils. Possible involvement of 5-hydroxy-6,8,11,14-eicosatetraenoate in superoxide release.
Neutrophils stimulated with optimal amounts of tumor-promoters that activate protein kinase C (e.g. mezerein, phorbol 12,13-dibutyrate) are known to release large quantities of superoxide: approximately 40-50 nmol O2-/min/10(7) cells. Previous studies have shown that treatment of neutrophils with the calcium ionophore A23187, or with 5-hydroxy-6,8,11,14-eicosatetraenonate (5-HETE), dramatically increased the ability of these cells to release O2- in response to suboptimal concentrations of the stimulants mentioned. In this manuscript, we provide data relevant to the basis of this augmentation of O2- release. The synergy with ionophore A23187 exhibited a partial requirement for extracellular Ca2+, whereas that with 5-HETE exhibited a near absolute requirement for that cation. Neutrophils stimulated with optimal amounts of tumor-promoters are known to exhibit a redistribution of protein kinase C activity from the soluble to a particulate fraction. A redistribution of kinase activity was not observed in cells stimulated synergistically. On the other hand, ionophore A23187 and 5-HETE increased the binding of a suboptimal amount of [3H] phorbol 12,13-dibutyrate to intact neutrophils by approximately 25 and 50%, respectively. Inhibitors of protein kinase C (i.e. sphingosine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) substantially blocked O-2 release from neutrophils stimulated either synergistically or with optimal levels of tumor-promoters. These data suggest a role for 5-HETE in modulating O-2 release by neutrophils and are discussed in relation to models of the interactions of protein kinase C with membranes.
Badwey
J A
JA
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115.
Robinson
J M
JM
Horn
W
W
Soberman
R J
RJ
Karnovsky
M J
MJ
Karnovsky
M L
ML
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D004357:Drug Synergism; D006168:Guinea Pigs; D006893:Hydroxyeicosatetraenoic Acids / Q000097:blood*; D007546:Isoquinolines / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D013110:Sphingosine / Q000494:pharmacology; D013449:Sulfonamides; D013481:Superoxides / Q000097:blood*
D002413:Cations, Divalent; D006893:Hydroxyeicosatetraenoic Acids; D007546:Isoquinolines; D010703:Phorbol Esters; D010879:Piperazines; D013449:Sulfonamides; D013481:Superoxides; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C043204:N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; D011493:Protein Kinase C; D013110:Sphingosine