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

Abstract

In the present study we determined whether the endothelium-derived hyperpolarizing factor (EDHF), in addition to its acute effects on vascular tone, activates intracellular signalling pathways other than those associated with Ca2+-dependent K+ channels. EDHF was generated by rhythmic distension of porcine coronary arteries under conditions of combined nitric oxide (NO) synthase/cyclo-oxygenase blockade, and the EDHF-containing luminal incubate was applied to cultured human coronary endothelial or smooth muscle cells. In both cell types, the luminal incubate activated tyrosine kinases, the mitogen-activated protein (MAP) kinases, extracellular signal regulated kinases 1 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 EDHF donor with the CYP 2C inhibitor sulfaphenazole as well as by CYP 2C antisense oligonucleotides. Erk1/2 phosphorylation in detector cells was also observed following the transfer of supernatant from cultured endothelial cells treated with the CYP inducer beta-naphthoflavone. The CYP 2C product 11,12-epoxyeicosatrienoic acid (11,12-EET) also activated tyrosine kinases, Erk1/2 and p38 MAP kinase. Overexpression of CYP 2C8 in native porcine coronary artery endothelial cells resulted in an increase in endothelial 11,12-EET production and Erk1/2 phosphorylation compared to that detected in untreated cells or cells transfected with an antisense CYP 2C8. Endothelial cell number was unaffected by transfection with LacZ or CYP 2C8 antisense but was significantly enhanced in cells overexpressing CYP 2C8. These observations indicate that EDHF/11,12-EET is not simply a vasodilator and that its continuous release under pulsatile conditions in vivo may affect vascular cell signalling and proliferation.

Dynamic Modulation of Interendothelial Gap Junctional Communication by 11,12-Epoxyeicosatrienoic Acid

“…5 In the present study, we observed that the endothelial cell uncoupling observed 5 to 10 minutes after cell stimulation with either bradykinin or 11,12-EET was not detectable in cells pretreated with inhibitors of MEK, which prevent the activation of ERK1/2. Both bradykinin and 11,12-EET activated ERK1/2 in endothelial cells 30,31 and induced a MEK inhibitor-sensitive shift in the mobility of Cx43 in SDS-PAGE, changes that are indicative of Cx43 phosphorylation. Such observations suggest that the activation of ERK1/2 leads to the phosphorylation of endothelial connexins and promotes cellular uncoupling.…”

Cytochrome P450 2C9-induced Endothelial Cell Proliferation Involves Induction of Mitogen-activated Protein (MAP) Kinase Phosphatase-1, Inhibition of the c-Jun N-terminal Kinase, and Up-regulation of Cyclin D1

“…CYP 2C9-derived EETs rather than ROS are therefore the most likely mediators of the enhanced cyclin D1 expression and endothelial cell proliferation. DISCUSSION We have demonstrated previously that the CYP 2C9 product, 11,12-EET, activates multiple signal transduction pathways in native and cultured endothelial cells and that overexpression of a CYP 2C epoxygenase enhances endothelial cell numbers (7). Therefore, we set out to elucidate the intracellular signal transduction cascade linking enhanced CYP expression and EET production with proliferation.…”

“…Effects of 11,12-EET and H 2 O 2 on Basal JNK Activity and MKP-1 Expression-Since CYP 2C9 generates EETs as well as ROS, both of which can affect the activation of MAP kinases as well as cell proliferation (7,19,20), we compared the effects of 11,12-EET and H 2 O 2 on JNK activity and MKP-1 expression. Stimulation of endothelial cells with 11,12-EET (1 M) elicited a marked and time-dependent decrease in basal JNK activity, which was pronounced 1 h after stimulation (Fig.…”

“…We have reported previously that EETs activate MAP kinases and enhance endothelial cell growth (7). Therefore, the aim of the present investigation was to determine the cellular events linking CYP 2C with endothelial cell proliferation.…”

Norepinephrine-Induced Stimulation of p38 Mitogen-Activated Protein Kinase Is Mediated by Arachidonic Acid Metabolites Generated by Activation of Cytosolic Phospholipase A2 in Vascular Smooth Muscle Cells

“…well as its metabolites 12(S)-HETE and 11,12-epoxyeicosatrienoic acid activate p38 MAPK in some cell types (Paine et al, 2000;Alexander et al, 2001;Fleming et al, 2001;Reddy et al, 2002). Whether AA and its metabolites mediate the activation of p38 MAPK by a physiological agonist has not been investigated.…”