Arachidonic acid mobilization and phosphoinositide turnover by the terminal complement complex, C5b-9, in rat oligodendrocyte x C6 glioma cell hybrids.
Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.
Shirazi
Y
Y
Department of Pathology, University of Maryland, School of Medicine, Baltimore 21201.
McMorris
F A
FA
Shin
M L
ML
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002999:Clone Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins / Q000502:physiology*; D005910:Glioma / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D006822:Hybrid Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D009457:Neuroglia / Q000378:metabolism*; D009836:Oligodendroglia / Q000201:enzymology / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011796:Quinacrine / Q000494:pharmacology; D051381:Rats
D000098:Acetophenones; D001095:Arachidonic Acids; D015938:Complement Membrane Attack Complex; D007546:Isoquinolines; D010716:Phosphatidylinositols; D010879:Piperazines; D016718:Arachidonic Acid; D000001:Calcimycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D003165:Complement System Proteins; D011493:Protein Kinase C; D011796:Quinacrine; C006821:4-bromophenacyl bromide; D002118:Calcium