The influence of acetylshikonin, a natural naphthoquinone, on the production of leukotriene B4 and thromboxane A2 in rat neutrophils.
Both A23187 and formyl-Met-Leu-Phe (fMLP) induced the release of arachidonic acid and the production of thromboxane B(2) and leukotriene B(4) from rat neutrophils that were inhibited by acetylshikonin in a concentration-dependent manner. Acetylshikonin blocked exogenous arachidonic acid-induced leukotriene B(4) and thromboxane B(2) production in neutrophils and inhibited the enzymatic activity of ram seminal vesicles cyclooxygenase and human recombinant 5-lipoxygenase, whereas it had no effect on cytosolic phospholipase A(2) activity, in cell-free systems. 3-Morpholinosydnonimine- and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE)-mediated dihydrorhodamine 123 oxidation (to assess the lipid peroxide and peroxynitrite scavenging activity) was reduced by acetylshikonin. The membrane recruitment of cytosolic phospholipase A(2) was inhibited, but the phosphorylation of cytosolic phospholipase A(2) was enhanced, by acetylshikonin in the A23187-induced response. Acetylshikonin alone stimulated extracellular signal regulated kinase (ERK) phosphorylation and enhanced this response in cells stimulated with A23187 and fMLP. The phosphorylation of ERKs and cytosolic phospholipase A(2) was attenuated by U0126, a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Acetylshikonin facilitated both A23187- and fMLP-mediated translocation of 5-lipoxygenase to the membrane. Acetylshikonin attenuated both fMLP- and ionomycin-mediated [Ca(2+)](i) elevation. These results indicate that the inhibition of eicosanoid production by acetylshikonin is due to the attenuation of cytosolic phospholipase A(2) membrane recruitment via the decrease in [Ca(2+)](i) and to the blockade of cyclooxygenase and 5-lipoxygenase activity.
Hsu
Mei-Feng
MF
Department of Biochemistry, China Medical University, Taichung 404, Taiwan, Republic of China.
Chang
Ling-Chu
LC
Huang
Li-Jiau
LJ
Kuo
Sheng-Chu
SC
Lee
Hsiao-Yun
HY
Lu
Min-Chi
MC
Wang
Jih-Pyang
JP
D000818:Animals; D000880:Anthraquinones / Q000008:administration & dosage / Q000494:pharmacology*; D000893:Anti-Inflammatory Agents / Q000008:administration & dosage / Q000494:pharmacology*; D001094:Arachidonate 5-Lipoxygenase / Q000187:drug effects / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007975:Leukotriene B4 / Q000096:biosynthesis*; D008297:Male; D051379:Mice; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D054467:Phospholipases A2 / Q000187:drug effects / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012669:Seminal Vesicles / Q000187:drug effects / Q000201:enzymology; D012756:Sheep; D013928:Thromboxane A2 / Q000096:biosynthesis*
D000880:Anthraquinones; D000893:Anti-Inflammatory Agents; D007975:Leukotriene B4; D016718:Arachidonic Acid; D000001:Calcimycin; D013928:Thromboxane A2; D009240:N-Formylmethionine Leucyl-Phenylalanine; D001094:Arachidonate 5-Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases; D054467:Phospholipases A2; C073944:acetylshikonin; D002118:Calcium