Evidence for 5-lipoxygenase activity in human B cell lines. A possible role for arachidonic acid metabolites during B cell signal transduction.
Ligand binding to B lymphocytes via membrane Ig initiates a cascade of events beginning with the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate. Subsequent to the activation of protein kinase C and the induction of a rise in intracellular calcium by diacylglycerol and inositol 1,4,5-trisphosphate, there is gene transcription and eventually cellular activation. By mimicking the initial event of B cell activation with phorbol ester and calcium ionophore one can begin to identify the many mediators used in signaling between the membrane and the nucleus. We have examined the effect of calcium on arachidonic acid (AA) metabolism in several EBV-transformed human B cell lines. The cells were labelled with [3H]AA and stimulated with the calcium ionophore A23187. Analysis of the supernatant by reversed-phase HPLC demonstrated a dose-dependent release of an AA metabolite that coeluted with authentic 5-hydroxyeicosatetraenoic acid (5-HETE). In addition, the AA metabolite coeluted with standard 5-HETE under straight-phase chromatography. Further analysis by RIA confirmed the identification of 5-HETE and revealed an additional metabolite, 5-HETE lactone (5-HL). 5-HL is the intramolecular ester of 5-HETE generated in the presence of acid. We were unable to convert [3H] 5-HETE into 5-HL during sample preparation unless cells were present, suggesting that the 5-HL, is of cellular origin. These results suggest that the AA metabolites 5-HETE and its intramolecular ester 5-HL may play a role in B cell activation because they are produced subsequent to a rise in intracellular Ca2+, an event that occurs during cross-linking of membrane Ig.
Schulam
P G
PG
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
Shearer
W T
WT
D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000378:metabolism*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000502:physiology*; D001402:B-Lymphocytes / Q000201:enzymology*; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D002472:Cell Transformation, Viral; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D066298:In Vitro Techniques; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism
D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D016718:Arachidonic Acid; D000001:Calcimycin; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase