Requirement of phosphatidylinositol 3-kinase activation and calcium influx for leukotriene B4-induced enzyme release.
Leukotriene B(4) (LTB(4)) is a potent lipid mediator involved in host defense and inflammatory responses. It causes chemotaxis, generation of reactive oxygen species, and degranulation. However, only little is known of the molecular mechanisms by which LTB(4) induces these biological activities. To analyze the intracellular signaling pathways to mediate lysosomal enzyme release through the cloned LTB(4) receptor (BLT1), we transfected BLT1 to rat basophilic leukemia cells (RBL-2H3). LTB(4) dose-dependently released beta-hexosaminidase, and the release was mostly inhibited when the cells were pretreated with pertussis toxin, indicating that the degranulation is mediated by G(i) proteins. LTB(4) activated phosphatidylinositol 3-kinase (PI3-K) through G(i), and inhibition of PI3-K by wortmannin or LY290042 inhibited degranulation. Granulocytes from PI3-Kgamma-deficient mice showed reduced LTB(4)-induced degranulation, suggesting that this isozyme of PI3-K is involved in the degranulation. LTB(4) also caused calcium release from intracellular stores and calcium influx from the outside milieu through G(i), but only the calcium influx is critical for the lysosomal enzyme release. Calcium influx and PI3-K activation are both downstream events of G(i), since they were inhibited by pertussis toxin. These two events are in essence independent each other, because calcium depletion did not affect PI3-K, and inhibition of PI3-K did not attenuate calcium influx significantly. Thus, our results have clearly shown that LTB(4) binds BLT1 and activates G(i)-like protein, and both PI3-Kgamma activation and a sustained calcium elevation by calcium influx are necessary for enzyme release in these cells.
Ito
Nobuko
N
Department of Biochemistry and Molecular Biology, University of Tokyo, Japan.
Yokomizo
Takehiko
T
Sasaki
Takehiko
T
Kurosu
Hiroshi
H
Penninger
Josef
J
Kanaho
Yasunori
Y
Katada
Toshiaki
T
Hanaoka
Kazuo
K
Shimizu
Takao
T
D000818:Animals; D001491:Basophils / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015550:Cell Degranulation / Q000502:physiology; D002614:Chelating Agents / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D006098:Granulocytes / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007975:Leukotriene B4 / Q000378:metabolism*; D051379:Mice; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D037342:Pertussis Toxin / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D051381:Rats; D018102:Receptors, Leukotriene B4 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D018129:Phosphatidylinositol Phosphates; D018102:Receptors, Leukotriene B4; C060974:phosphatidylinositol 3,4,5-triphosphate; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D007975:Leukotriene B4; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009195:Peroxidase; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D001619:beta-N-Acetylhexosaminidases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium