The coronary endothelium-derived hyperpolarizing factor (EDHF) stimulates multiple signalling pathways and proliferation in vascular cells

In the present study we determined whether the endothelium-derived hyperpol arizing factor (EDHF), in addition to its acute effects on vascular tone, a ctivates intracellular signalling pathways other than those associated with Ca2+-dependent K+ channels. EDHF was generated by rhythmic distension of p orcine coronary arteries under conditions of combined nitric oxide (NO) syn thase/cyclo-oxygenase blockade, and the EDHF-containing luminal incubate wa s applied to cultured human coronary endothelial or smooth muscle cells. In both cell types, the luminal incubate activated tyrosine kinases, the mito gen-activated protein (MAP) kinases, extracellular signal regulated kinases I and 2 (Erk1/2) and p38, as well as protein kinase B/Akt. The constituent responsible for Erk1/2 phosphorylation was identified as a cytochrome P450 (CYP) metabolite, as Erk1/2 activation was attenuated by pretreating the E DHF donor with the CYP 2C inhibitor sulfaphenazole as well as by CYP 2C ant isense oligonucleotides. Erk1/2 phosphorylation in detector cells was also observed following the transfer of supernatant from cultured endothelial ce lls treated with the CYP inducer P-naphthoflavone. The CYP 2C product 11, 1 2-epoxyeicosatrienoic acid (11, 12-EET) also activated tyrosine kinases, Er k1/2 and p38 MAP kinase. Overexpression of CYP 2C8 in native porcine corona ry artery endothelial cells resulted in an increase in endothelial 11,12-EE T production and Erk1/2 phosphorylation compared to that detected in untrea ted cells or cells transfected with an antisense CYP 2C8. Endothelial cell number was unaffected by transfection with LacZ or CYP 2C8 antisense but wa s significantly enhanced in cells overexpressing CYP 2C8. These observation s indicate that EDHF/11,12-EET is not simply a vasodilator and that its con tinuous release under pulsatile conditions in vivo may affect vascular cell signalling and proliferation.