Role Of The G-protein And Tyrosine Kinase-Rho/ROK Pathways In15-hydroxyeicosatetraenoic Acid (15-HETE) Induced Pulmonary Vasoconstriction In Hypoxic Rats

Objective: We studied the contribution of ROK as well as G-protein andtyrosine kinase to the15-HETE induced pulmonary vasoconstriction.Methods: In order to observe the effects of G-protein and tyrosine kinase onhypoxia-induced vasoconstriction, organization bath rings research was used to testthe tension of pulmonary arterial rings which were pre-treated with blockers inhypoxic rats. RNA interference technology was used to make the expression of15-LOspecifically partial deficiency and comfirm the effect of15-HETE on the ROKexpression. Western blot and RT-PCR were employed to detect the expression of ROKin both protein and mRNA levels which were effected by G-protein and tyrosinekinase/ROK pathways. The immunocytochemistry was used to detect whether thetranslocation of ROK2protein from the nucleus to the cytoplasm during hypoxiaexposure relies on the activity of15-LO. RP-HPLC was employed to measure thelevels of endogenous12-and15-HETE.Results: The increased tension of pulmonary artery induced by hypoxia could bedecreased by G-protein inhibitors, tyrosine kinase blockers and15-LO inhibitorNDGA, indicating that the G-protein and tyrosine kinase–Rho/ROK pathways wereinvolved in hypoxia-induced vasoconstriction. Hypoxia significantly increased thelevel of P-MYPT1in hypoxic pulmonary artery which represent the activity of Rho,while NDGA could reverse this process. The changes of ROK expression regulated byhypoxia were time-dependent, ROK expression at both protein and mRNA levelsreached the plateau level in24h-exposure to hypoxia. The expression of both ROK1and ROK2decreased after the15-LO was silenced under both normoxic and hypoxic conditions. Hypoxia and exogenous15-HETE increased the expression of ROK atboth protein and mRNA levels, which could be reversed by NDGA. Meanwhile, theup-regulation of ROK expression induced by15-HETE appears to be mediated by theG-protein and tyrosine kinase pathways. ROK2protein located in nucleus undernormoxic condition and then translocated to the cytoplasm during hypoxia exposureor treated with15-HETE, indicating that this process relies on the activity of15-LO.While ROK1protein had no noticeable translocation at the same condition withROK2. Hypoxia increased the level of endogenous15-HETE in rat lung tissue, whichwas much higher than the12-HETE at the same condition.Conclusions: These results suggest that15-HETE may mediate theup-regulation of ROK expression through G-protein and tyrosine kinase pathwaysunder hypoxic condition, leading to PA vasoconstriction. The translocation of ROK2from the nucleus to the cytoplasm during hypoxia exposure relies on the mechanismfor15-HETE production.