11,12-Epoxyeicosatrienoic acid stimulates heme-oxygenase-1 in endothelial cells☆
Introduction
11,12-Epoxyeicosatrienoic acid (11,12-EET), derived through metabolism of arachidonic acid (AA) by cytochrome P-450 (CYP450) epoxygenase enzymes, has various biological activities: it dilates arterial vessels, causes inhibition of platelet aggregation, stimulates angiogenesis [1], inhibits both apical and basolateral Na(+)–K(+)–2Cl(−) cotransport in renal epithelial cells [2], is an important modulator of cardiac electrical excitability [3], induces polymorphonuclear leukocyte (PMN) aggregation [4], has antimigratory effects on smooth muscle cells [5], inhibits apoptosis [6], possesses fibrinolytic properties [7], is part of the endogenous antipyretic system [8]. EETs act as an endothelium-derived hyperpolarizing factor (EDHF) in some vascular beds [9], thus contributing to endothelium-dependent vasodilatation independently from nitric oxide.
Carbon monoxide (CO), a naturally produced gas by the action of heme-oxygenase (HO) on heme, causes endothelium-independent vasodilatation mediated both through cGMP and a direct effect on big-conductance calcium-activated K channels. The HO/CO system also modulates the proliferative response of vascular smooth muscle cells [10], has anti-atherosclerotic [11], and anti-hypertensive effects [12].
The major objective of this study was to determine the interactions between EETs and the HO/CO system in endothelial cells. Our results demonstrate that 11,12-EET is a potent inducer of HO-1 and, thus, CO production may mediate its effects in arterial vessels.
Section snippets
Chemicals
Metalloporphyrins were obtained from Porphyrin Products (Logan, UT, USA). 11,12-EET and 8,9-EET were provided by Dr. J.R. Falck. All other chemicals were obtained from Sigma Chemical (St. Louis, MO, USA). Stannous mesoporphyrin (SnMP) was dissolved in a solution of 50 mM NaCO3.
Cell culture conditions
Human dermal microvessel endothelial cells were grown in MCDB131 medium (GIBCO-BRL, City, ST, CO) supplemented with 10% FBS, 10 ng/mL epithelial growth factor (Sigma Chemicals, Saint Louis, MO, USA), and 1 μg/mL
Confocal microscopy analysis of HO-1 and HO-2 in endothelial cells
Fig. 1 shows the effect of 11,12-EET on HO-1 and HO-2 proteins. In EET-treated cells, membrane localization and cytoplasmic immunostaining of HO-1 (green signal) was increased (n = 4; p < 0.05) (Panel C compared to Panel A). HO-2 protein did not change with 11,12-EET incubation (Panels B and D). Staining intensity was computed as integrated optical density, measured in four different experiments, and digitally fixed images were analyzed as described before [16]. The integrated optical density of
Discussion
The results of this study showed for the first time that 11,12-EET causes induction of HO-1 in endothelial cells in culture and activation of HO in the aorta. This novel observation was substantiated by immunoflorescent microscopy, Western blot analysis, and enzymatic activity.
EETs are produced from AA in the endothelium. They are potent vasodilators of the arterial circulation, particularly of the coronary vessels. EETs also increase intracellular Ca2+ concentration in endothelial cells [17],
References (38)
- et al.
Effects of epoxyeicosatrienoic acids on polymorphonuclear leukocyte function
Life Sci
(2002) - et al.
Activation of Galpha's mediates induction of tissue-type plasminogen activator gene transcription by epoxyeicosatrienoic acids
J Biol Chem
(2001) - et al.
Epoxyeicosatrienoic acids and their sulfonimide derivatives stimulate tyrosine phosphorylation and induce mitogenesis in renal epithelial cells
J Biol Chem
(1998) - et al.
Transfection of an active cytochrome P-450 arachidonic acid epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor
J Biol Chem
(1999) - et al.
Endogenous non-cyclooxygenase metabolites of arachidonic acid modulate growth and mRNA levels of immediate-early response genes in rat mesangial cells
J Biol Chem
(1991) - et al.
Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes
J Biol Chem
(1997) - et al.
Heme oxygenase-1 attenuates ischemia/reperfusion-induced apoptosis and improves survival in rat renal allografts
Kidney Int
(2003) - et al.
Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-Delta 12,14-prostaglandin J2
J Biol Chem
(2003) - et al.
Cytochrome P450 2C9-derived epoxyeicosatrienoic acids induce angiogenesis via cross-talk with the epidermal growth factor receptor (EGFR)
FASEB J
(2003) - et al.
NO inhibits Na+–K+–2Cl− cotransport via a cytochrome P-450-dependent pathway in renal epithelial cells (MMDD1)
Am J Physiol Renal Physiol
(2003)
Stereospecific activation of cardiac ATP-sensitive K(+) channels by epoxyeicosatrienoic acids: a structural determinant study
Mol Pharmacol
Inhibition of vascular smooth muscle cell migration by cytochrome p450 epoxygenase-derived eicosanoids
Circ Res
Cytochrome p450 epoxygenase metabolism of arachidonic acid inhibits apoptosis
Mol Cell Biol
Role of cytochrome P-450 in endogenous antipyresis
Am J Physiol Regul Integr Comp Physiol
Endothelium-derived hyperpolarizing factor in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by activating smooth muscle BK(Ca) channels
Circulation
Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide
J Clin Invest
Heme oxygenas eand atherosclerosis
Treatment with tin prevents the development of hypertension in spontaneously hypertensive rats
Science
Retrovirus-mediated HO gene transfer into endothelial cells protects against oxidant-induced injury
Am J Physiol
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This work was supported by NIH grants HL55601 and HL34300 (NGA) and GM31278 (JRF) a grant from the Italian Ministry for University and Scientific and Technological Research (MURST).