Biosynthesis of hepoxilins: evidence for the presence of a hepoxilin synthase activity in rat insulinoma cells.
The 12(S)-lipoxygenase (12-LOX) pathway of arachidonic acid (AA) metabolism after dioxygenation to 12(S)-hydroperoxy-eicosatetraenoic acid is bifurcated in a reduction route to formation of 12(S)-hydroxy-eicosatetraenoic acid (12-HpETE) and an isomerization route to formation of hepoxilins. Interestingly, we found that the rat insulinoma RINm5F cells, which are devoid of cytoplasmic glutathione peroxidase (cGPx)/phospholipid hydroperoxide glutathione peroxidase (PHGPx), produce solely hepoxilin A(3) (HXA(3)). Since HXA(3) synthesis was abolished in heat-denatured or cGPx- or PHGPx-transfected cells, it was tempting to speculate that a HXA(3) synthase activity regulated by cGPx/PHGPx is present. To confirm this assumption we incubated AA with HeLa cells overexpressing the rat leukocyte-type 12-LOX. Neither HXA(3) nor 12(S)-HETE were detected due to abundance of cGPx/PHGPx. But, pretreatment of transfected cells with diethyl maleate, an inhibitor of glutathione and PHGPx, restored HXA(3) synthase and 12-LOX activities. Thus, we conclude, that cells containing rat leukocyte-type 12-LOX also possess an intrinsic HXA(3) synthase activity, which is activated by inhibition of cGPx/PHGPx. In normal cells HXA(3) is down-regulated by cGPx/PHGPx, but, it is persistently activated in oxidatively stressed cells deficient in cGPx/PHGPx, such as RINm5F.
Shankaranarayanan
Pattabhiraman
P
Eicosanoid Research Division and Centre for Experimental Gynecology and Breast Research, University Medical Centre Benjamin Franklin, Free University Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany.
Ciccoli
Roberto
R
Nigam
Santosh
S
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D007340:Insulinoma / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
C061163:11-glutathionylhepoxilin A3; D005979:Glutathione Peroxidase; D001092:Arachidonate 12-Lipoxygenase; D019746:Intramolecular Oxidoreductases; C473385:hepoxilin synthase; D015126:8,11,14-Eicosatrienoic Acid