Epoxyeicosatrienoic and dihydroxyeicosatrienoic acids dilate human coronary arterioles via BK(Ca) channels: implications for soluble epoxide hydrolase inhibition

Epoxyeicosatrienoic acids (EETs) are metabolized by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs) and are putative endothelium-derived hyperpolarizing factors (EDHFs). EDHFs modulate microvascular tone; however, the chemical identity of EDHF in the human coronary microcirculation is not known. We examined the capacity of EETs, DHETs, and sEH inhibition to affect vasomotor tone in isolated human coronary arterioles (HCAs). HCAs from right atrial appendages were prepared for videomicroscopy and immunohistochemistry. In vessels preconstricted with endothelin-1, three EET regioisomers (8,9-, 11,12-, and 14,15-EET) each induced a concentration-dependent dilation that was sensitive to blockade of large-conductance Ca2+-activated K+ (BK(Ca)) channels by iberiotoxin. EET-induced dilation was not altered by endothelial denudation. 8,9-, 11,12-, and 14,15-DHET also dilated HCA via activation of BK(Ca) channels. Dilation was less with 8,9- and 14,15-DHET but was similar with 11,12-DHET, compared with the corresponding EETs. Immunohistochemistry revealed prominent expression of cytochrome P-450 (CYP450) 2C8, 2C9, and 2J2, enzymes that may produce EETs, as well as sEH, in HCA. Inhibition of sEH by 1-cyclohexyl-3-dodecylurea (CDU) enhanced dilation caused by 14,15-EET but reduced dilation observed with 11,12-EET. DHET production from exogenous EETs was reduced in vessels pretreated with CDU compared with control, as measured by liquid chromatography electrospray-ionization mass spectrometry. In conclusion, EETs and DHETs dilate HCA by activating BK(Ca) channels, supporting a role for EETs/DHETs as EDHFs in the human heart. CYP450s and sEH may be endogenous sources of these compounds, and sEH inhibition has the potential to alter myocardial perfusion, depending on which EETs are produced endogenously.     

EETs relax airway smooth muscle via an EpDHF effect: BK(Ca) channel activation and hyperpolarization

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.     

Epoxyeicosatrienoic acid-induced relaxation is impaired in insulin resistance

We assessed the effect of epoxyeicosatrienoic acids (EETs) in intact mesenteric arteries and Ca(2+)-activated K(+) (BK(Ca)) channels of isolated vascular smooth muscle cells from control and insulin-resistant (IR) rats. The response to 11,12-EET and 14,15-EET was assessed in small mesenteric arteries from control and IR rats in vitro. Mechanistic studies were performed in endothelium intact or denuded arteries and in the presence of pharmacological inhibitors. Moreover, EET-induced activation of the BK(Ca) channel was assessed in myocytes in both the cell-attached and the inside-out (I/O) patch-clamp configurations. In control arteries, both EET isomers induced relaxation. Relaxation was impaired by endothelium denudation, N(omega)-nitro-L-arginine, or iberiotoxin (IBTX), whereas it was abolished by IBTX + apamin or charybdotoxin + apamin. In contrast, the EETs did not relax IR arteries. In control myocytes, the EETs increased BK(Ca) activity in both configurations. Conversely, in the cell-attached mode, EETs had no effect on BK(Ca) channel activity in IR myocytes, whereas in the I/O configuration, BK(Ca) channel activity was enhanced. EETs induce relaxation in small mesenteric arteries from control rats through K(Ca) channels. In contrast, arteries from IR rats do not relax to the EETs. Patch-clamp studies suggest impaired relaxation is due to altered regulatory mechanisms of the BK(Ca) channel.     

Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochrome P-450 epoxygenase

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium (BK(Ca)) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BK(Ca) channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14(S),15(R)-EET. Adenovirus containing the cytomegalovirus promoter-Escherichia coli beta-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [14C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine (10(-5) M) increased 14,15-EET release twofold and BK(Ca) channel activity fourfold in cell-attached patches. Methacholine-induced increases in BK(Ca) channel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10(-5) M). 14(S),15(R)-EET was more potent than 14(R),15(S)-EET in relaxing bovine coronary arteries and activating BK(Ca) channels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14(S),15(R)-EET from endogenous arachidonic acid to activate BK(Ca) channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BK(Ca) channels in coronary SMCs.     

Cyclic AMP-dependent modulation of cardiac L-type Ca2+ and transient outward K+ channel activities by epoxyeicosatrienoic acids

The three major enzyme systems, cyclo-oxygenase, lipoxygenase, and cytochrome P450 (P450/CYP), metabolize arachidonic acid (AA) to biologically active compounds. P450 and its associated monooxygenase activities have been identified in mammalian cardiac tissue, including humans. The four regioisomeric eicosanoids, 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) of AA metabolites derived by P450 epoxygenases have shown to possess potent biological effects in numerous tissues. In the coronary circulation the EETs are leading candidates for endothelial-derived hyperpolarizing factors that hyperpolarize vascular smooth muscle cells by opening Ca2+-activated K+ channels. Recently, the effects of the CYP pathways and their metabolites on cardiac ischemia-reperfusion injury have been evaluated in animal models. Some of these AA metabolites are cardioprotective and some are detrimental. However, EETs appear to be cardioprotective in CYP2J2 transgenic mice and in a canine ischemic model. Multiple effects of EETs on cardiac ion channels have been observed, such as activation of ATP-sensitive K+ channels and L-type Ca2+ channels in cardiomyocytes and inhibition of cardiac Na+ channels and L-type Ca2+ channels reconstructed in planar lipid bilayers. This brief review summarizes EET-induced modulation of cardiac ion channels.     

Role of endothelial Ni(2+)-sensitive Ca(2+) entry pathway in regulation of EDHF in porcine coronary artery

Elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells is proposed to be required for generation of vascular actions of endothelium-derived hyperpolarizing factor (EDHF). This study was designed to determine the endothelial Ca(2+) source that is important in development of EDHF-mediated vascular actions. In porcine coronary artery precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid (CPA) caused endothelium-dependent relaxations in the presence of N(G)-nitro-L-arginine (L-NNA). The L-NNA-resistant relaxant responses were inhibited by high K(+), indicating an involvement of EDHF. In the presence of Ni(2+), which inhibits Ca(2+) influx through nonselective cation channels, the BK-induced EDHF relaxant response was greatly diminished and the CPA-induced response was abolished. BK and CPA elicited membrane hyperpolarization of smooth muscle cells of porcine coronary artery. Ni(2+) suppressed the hyperpolarizing responses in a manner analogous to removal of extracellular Ca(2+). EDHF-mediated relaxations and hyperpolarizations evoked by BK and CPA in porcine coronary artery showed a temporal correlation with the increases in [Ca(2+)](i) in porcine aortic endothelial cells. The extracellular Ca(2+)-dependent rises in [Ca(2+)](i) in endothelial cells stimulated with BK and CPA were completely blocked by Ni(2+). These results suggest that Ca(2+) influx into endothelial cells through nonselective cation channels plays a crucial role in the regulation of EDHF.     

川芎嗪对猪冠状动脉平滑肌细胞大电导钙激活钾通道的作用

本工作旨在研究川芎嗪对猪冠状动脉平滑肌细胞钾通道的作用,为阐明其扩张冠状动脉血管的机制提供实验依据。采用膜片钳细胞贴附式和内面向外式记录方式观察川芎嗪对猪冠状动脉平滑肌细胞大电导钙激活钾通道(large-conductance Ca2+- activated potassium channels,BKCa channels)的作用,分别用蛋白激酶A(protein kinase A,PKA)抑制剂H-89和蛋白激酶G (protein kinase G,PKG)抑制剂KT-5823处理细胞,再观察川芎嗪对BKCa通道作用的变化。结果表明在研究的0．73-8．07 mmol/L浓度范围,川芎嗪可以剂量依赖性地激活BKCa通道,使通道的开放概率从(0．01±0．003)增加到(0．03±0．01)-(．21± 0．18)(P<0．01,n=10),使通道平均关闭时间从(732．33±90．67)ms降低到(359．67±41．30)-(2．96±0．52)ms(P<0．01, n=10)。川芎嗪的这种激活作用在浴液游离钙离子浓度接近0 mmol/L时也存在。PKA的特异性抑制剂H-89(3 μmol/L)和 PKG的特异性抑制剂KT-5823(1 μmol/L)对川芎嗪激活BKCa通道的作用无影响。以上结果提示:川芎嗪能直接激活冠状动脉平滑肌BKCa通道,这种作用可能是川芎嗪扩张冠状动脉血管的一种重要机制。     

Protein phosphatase 2A and Ca2+-activated K+ channels contribute to 11,12-epoxyeicosatrienoic acid analog mediated mesenteric arterial relaxation

Epoxyeicosatrienoic acids (EETs) are considered to be endothelium-derived hyperpolarizing factors, and are potent activators of the large-conductance, Ca(2+)-activated K(+) (BK(Ca)) channel in vascular smooth muscle. Here, we investigate the signal transduction pathway involved in the activation of BK(Ca) channels by 11,12-EET and 11,12-EET stable analogs in rat mesenteric vascular smooth muscle cells. 11,12-EET and the 11,12-EET analogs, 11-nonyloxy-undec-8(Z)-enoic acid (11,12-ether-EET-8-ZE), 11-(9-hydroxy-nonyloxy)-undec-8(Z)-enoic acid (11,12-ether-EET-8-ZE-OH) and 11,12-trans-oxidoeicosa-8(Z)-enoic acid (11,12-tetra-EET-8-ZE), caused vasorelaxation of mesenteric resistance arteries. Mesenteric myocyte whole-cell (perforated-patch) currents were substantially (approximately 150%) increased by 11,12-EET and 11,12-EET analogs. Single-channel recordings were conducted to identify the target for 11,12-EET. 11,12-EET and 11,12-EET analogs also increased mesenteric myocyte BK(Ca) channel activity in cell-attached patches. Similar results were obtained in cell-free patches. Baseline mesenteric myocyte BK(Ca) channel activity (NPo) in cell-free patches averaged less than 0.001 at +50 mV and 11,12-EET (1 micromol/L) increased NPo to 0.03+/-0.02 and 11,12-EET analogs (1 micromol/L) increased NPo to 0.09+/-0.006. Inhibition of protein phosphatase 2A (PP2A) activity with okadaic acid (10 nmol/L) completely reversed 11,12-EET stimulated BK(Ca) channel activity and greatly attenuated 11,12-ether-EET-8-ZE mesenteric resistance artery vasorelaxation. 11,12-EET and 11,12-EET analogs increased mesenteric myocyte PP2A activity by 3.5-fold. Okadaic acid and the EET inhibitor, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) inhibited the 11,12-EET mediated increase in PP2A activity. These findings provide initial evidence that PP2A activity contributes to 11,12-EET and 11,12-EET analog activation of mesenteric resistant artery BK(Ca) channels and vasorelaxation.     

14,15-Dihydroxyeicosatrienoic acid relaxes bovine coronary arteries by activation of K(Ca) channels

Epoxyeicosatrienoic acids (EETs) cause vascular relaxation by activating smooth muscle large conductance Ca(2+)-activated K(+) (K(Ca)) channels. EETs are metabolized to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase. We examined the contribution of 14,15-DHET to 14,15-EET-induced relaxations and characterized its mechanism of action. 14,15-DHET relaxed U-46619-precontracted bovine coronary artery rings but was approximately fivefold less potent than 14,15-EET. The relaxations were inhibited by charybdotoxin, iberiotoxin, and increasing extracellular K(+) to 20 mM. In isolated smooth muscle cells, 14,15-DHET increased an iberiotoxin-sensitive, outward K(+) current and increased K(Ca) channel activity in cell-attached patches and inside-out patches only when GTP was present. 14,15-[(14)C]EET methyl ester (Me) was converted to 14,15-[(14)C]DHET-Me, 14,15-[(14)C]DHET, and 14,15-[(14)C]EET by coronary arterial rings and endothelial cells but not by smooth muscle cells. The metabolism to 14,15-DHET was inhibited by the epoxide hydrolase inhibitors 4-phenylchalcone oxide (4-PCO) and BIRD-0826. Neither inhibitor altered relaxations to acetylcholine, whereas relaxations to 14,15-EET-Me were increased slightly by BIRD-0826 but not by 4-PCO. 14,15-DHET relaxes coronary arteries through activation of K(Ca) channels. Endothelial cells, but not smooth muscle cells, convert EETs to DHETs, and this conversion results in a loss of vasodilator activity.     

20-Iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide: photoaffinity labeling of a 14,15-epoxyeicosatrienoic acid receptor

Endothelium-derived epoxyeicosatrienoic acids (EETs) relax vascular smooth muscle by activating potassium channels and causing membrane hyperpolarization. Recent evidence suggests that EETs act via a membrane binding site or receptor. To further characterize this binding site or receptor, we synthesized 20-iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide (20-I-14,15-EE8ZE-APSA), an EET analogue with a photoactive azido group. 20-I-14,15-EE8ZE-APSA and 14,15-EET displaced 20-(125)I-14,15-epoxyeicosa-5(Z)-enoic acid binding to U937 cell membranes with K(i) values of 3.60 and 2.73 nM, respectively. The EET analogue relaxed preconstricted bovine coronary arteries with an ED(50) comparable to that of 14,15-EET. Using electrophoresis, 20-(125)I-14,15-EE8ZE-APSA labeled a single 47 kDa band in U937 cell membranes, smooth muscle and endothelial cells, and bovine coronary arteries. In U937 cell membranes, the 47 kDa radiolabeling was inhibited in a concentration-dependent manner by 8,9-EET, 11,12-EET, and 14,15-EET (IC(50) values of 444, 11.7, and 8.28 nM, respectively). The structurally unrelated EET ligands miconazole, MS-PPOH, and ketoconazole also inhibited the 47 kDa labeling. In contrast, radiolabeling was not inhibited by 8,9-dihydroxyeicosatrienoic acid, 5-oxoeicosatetraenoic acid, a biologically inactive thiirane analogue of 14,15-EET, the opioid antagonist naloxone, the thromboxane mimetic U46619, or the cannabinoid antagonist AM251. Radiolabeling was not detected in membranes from HEK293T cells expressing 79 orphan receptors. These studies indicate that vascular smooth muscle, endothelial cells, and U937 cell membranes contain a high-affinity EET binding protein that may represent an EET receptor. This EET photoaffinity labeling method with a high signal-to-noise ratio may lead to new insights into the expression and regulation of the EET receptor.     

三磷酸肌醇对猪冠状动脉平滑肌细胞大电导钙激活钾通道的作用

应用膜片钳单通道电流记录技术,研究三磷酸肌醇(trisphosphateinositol,IP3)对猪冠状动脉平滑肌细胞大电导钙激活钾通道(large-conductanceCa2+-activatedpotassiumchannels,BKchannels)的作用。结果显示:在内面向外式(inside-out)膜片下,IP3(10~50μmol/L)可以浓度依赖性地增加通道的开放概率,而对电流幅值无明显影响,开放概率的增加是通过明显缩短平均关闭时间实现的(n=11,P<0.01);洗去药物后通道活性可以恢复到对照水平;IP3对通道的激活作用不随时间而衰减;IP3的降解产物对通道没有明显的激活作用。结果表明:在inside-out膜片下,IP3能够激活猪冠状动脉平滑肌细胞BK通道。     

Simultaneous resolution of underivatized regioisomers and stereoisomers of arachidonate epoxides by capillary electrophoresis

cis-Epoxyeicosatrienoic acids (EETs) and their hydrolysis products (threo-DHETs) have been proposed to be endothelial-dependent hyperpolarizing factors (EDHFs) which upregulate blood flow when tissue perfusion is impaired. Various EET regioisomers and enantiomers are formed from arachidonate by inducible cytochrome P450 epoxygenase isoforms, and tissue EET profiles may vary with diet, time, and disease. Because EET actions and metabolism may be regio- and stereospecific, convenient methods to measure profiles of EET isomers in tissues are needed. In the current studies, we describe two simple capillary electrophoretic methods for resolving EETs. The first method involves capillary electrophoresis with a mixture of neutral and anionic beta-cyclodextrins, which in one step baseline-resolves underivatized EET regioisomers and their enantiomers. Low picogram amounts of EET enantiomers were identified based on migration times and UV spectra. The method was also used to assess the antipode purity of EET standards, and to determine murine hepatic levels of EET enantiomers. The second method involves capillary electrochromatography, which also baseline-resolves underivatized EET and DHET regioisomers in one step. We conclude that in EET assays the major advantages of capillary electrophoresis over reversed-phase HPLC are improved peak efficiency, sensitivity, and resolution, plus precise coelution of deuterated and nondeuterated EETs.     

12-lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation

Noncyclooxygenase metabolites of arachidonic acid (AA) have been proposed to mediate endothelium-dependent vasodilation in the coronary microcirculation. Therefore, we examined the formation and bioactivity of AA metabolites in porcine coronary (PC) microvascular endothelial cells and microvessels, respectively. The major noncyclooxygenase metabolite produced by microvascular endothelial cells was 12(S)-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product. 12(S)-HETE release was markedly increased by pretreatment with 13(S)-hydroperoxyoctadecadienoic acid but not by the reduced congener 13(S)-hydroxyoctadecadienoic acid, suggesting oxidative upregulation of 12(S)-HETE output. 12(S)-HETE produced potent relaxation and hyperpolarization of PC microvessels (EC(50), expressed as -log[M] = 13.5 +/- 0.5). Moreover, 12(S)-HETE potently activated large-conductance Ca(2+)-activated K(+) currents in PC microvascular smooth muscle cells. In contrast, 12(S)-HETE was not a major product of conduit PC endothelial AA metabolism and did not exhibit potent bioactivity in conduit PC arteries. We suggest that, in the coronary microcirculation, 12(S)-HETE can function as a potent hyperpolarizing vasodilator that may contribute to endothelium-dependent relaxation, particularly in the setting of oxidative stress.     

Erythrocyte-derived epoxyeicosatrienoic acids

Red blood cells (RBCs) are reservoirs for cis- and trans-epoxyeicosatrienoic acids (EETs) that can be released. The sources of EET release from RBCs include direct synthesis from arachidonic acid, peroxidation of phospholipids and EETs esterified into cellular phospholipids. The release of EETs from RBCs can be through cytosolic phospholipase A2 (PLA2), secretory PLA2 and other responses associated with ATP release from RBCs. The erythrocyte ATP, purinergic receptors, ATP-binding cassette transporters, PLA2 and cytoskeleton rearrangement may all participate in EET release in the microcirculatory deformation of RBCs. EETs are vasodilatory and are candidate endothelium-derived hyperpolarizing factors. Due to the anti-hypertensive, fibrinolytic, and anti-thrombotic properties of EETs, their release from RBCs is replete with implications for the control of circulation and rheological characteristics of the circulating blood.     

Testosterone-induced relaxation of coronary arteries: activation of BKCa channels via the cGMP-dependent protein kinase

Androgens are reported to have both beneficial and detrimental effects on human cardiovascular health. The aim of this study was to characterize nongenomic signaling mechanisms in coronary artery smooth muscle (CASM) and define the ionic basis of testosterone (TES) action. TES-induced relaxation of endothelium-denuded porcine coronary arteries was nearly abolished by 20 nM iberiotoxin, a highly specific inhibitor of large-conductance, calcium-activated potassium (BK(Ca)) channels. Molecular patch-clamp studies confirmed that nanomolar concentrations of TES stimulated BK(Ca) channel activity by ～100-fold and that inhibition of nitric oxide synthase (NOS) activity by N(G)-monomethyl-L-arginine nearly abolished this effect. Inhibition of nitric oxide (NO) synthesis or guanylyl cyclase activity also attenuated TES-induced coronary artery relaxation but did not alter relaxation due to 8-bromo-cGMP. Furthermore, we detected TES-stimulated NO production in porcine coronary arteries and in human CASM cells via stimulation of the type 1 neuronal NOS isoform. Inhibition of the cGMP-dependent protein kinase (PKG) attenuated TES-stimulated BK(Ca) channel activity, and direct assay determined that TES increased activity of PKG in a concentration-dependent fashion. Last, the stimulatory effect of TES on BK(Ca) channel activity was mimicked by addition of purified PKG to the cytoplasmic surface of a cell-free membrane patch from CASM myocytes (～100-fold increase). These findings indicate that TES-induced relaxation of endothelium-denuded coronary arteries is mediated, at least in part, by enhanced NO production, leading to cGMP synthesis and PKG activation, which, in turn, opens BK(Ca) channels. These findings provide a molecular mechanism that could help explain why androgens have been reported to relax coronary arteries and relieve angina pectoris.     

Human coronary endothelial cells convert 14,15-EET to a biologically active chain-shortened epoxide

Cytochrome P-450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) play an important role in the regulation of vascular reactivity and function. Conversion to the corresponding dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolases is thought to be the major pathway of EET metabolism in mammalian vascular cells. However, when human coronary artery endothelial cells (HCEC) were incubated with (3)H-labeled 14,15-EET, chain-shortened epoxy fatty acids, rather than DHET, were the most abundant metabolites. After 4 h of incubation, 23% of the total radioactivity remaining in the medium was converted to 10,11-epoxy-hexadecadienoic acid (16:2), a product formed from 14,15-EET by two cycles of beta-oxidation, whereas only 15% was present as 14,15-DHET. Although abundantly present in the medium, 10,11-epoxy-16:2 was not detected in the cell lipids. Exogenously applied (3)H-labeled 10,11-epoxy-16:2 was neither metabolized nor retained in the cells, suggesting that 10,11-epoxy-16:2 is a major product of 14,15-EET metabolism in HCEC. 10,11-Epoxy-16:2 produced potent dilation in coronary microvessels. 10,11-Epoxy-16:2 also potently inhibited tumor necrosis factor-alpha-induced production of IL-8, a proinflammatory cytokine, by HCEC. These findings implicate beta-oxidation as a major pathway of 14,15-EET metabolism in HCEC and provide the first evidence that EET-derived chain-shortened epoxy fatty acids are biologically active.     

The coronary vasculature and the myocardium

The effect of bradykinin (BK) on myocardial inotropic state was tested on 5 isolated rat heart preparations, in which a proper ballon was placed to record left ventricular pressure, whose developed systolic value was taken as an index of contractility. A reduction of developed left ventricular pressure was observed when BK was added to the perfusion oxygenated Tyrode solution. However, when BK was given after 1-amino-benzotriazole, an inhibitor of Cytochrome P-450 (Cyt P-450), developed pressure did not change. Since Cyt P-450 is known to act on arachidonic acid inducing the production of epoxiecocistrienoic acids (EETs) which hyperpolarizes myocardial fibres, it was argued that the reduction in contractility by bradykinin was the result of the hyperpolarizing effect of EETs. The fact that the concentration of Cyt P-450 is higher in the vascular endothelial cells than in the sarcolemma of the myocytes and the observation that the coronary resistance decreases together with the contractility suggest that the endothelium plays a pivotal role in mediating the negative inotropic effect of BK.     

Reno-protective mechanisms of epoxyeicosatrienoic acids in cardiovascular disease

Cardiovascular disease (CVD) is the leading cause of mortality worldwide, and it is well known that end-stage renal disease (ESRD) is a profound consequence of the progression of CVD. Present treatments only slow CVD progression to ESRD, and it is imperative that new therapeutic strategies are developed to prevent the incidence of ESRD. Because epoxyeicosatrienoic acids (EETs) have been shown to elicit reno-protective effects in hypertensive animal models, the current review will focus on addressing the reno-protective mechanisms of EETs in CVD. The cytochrome P-450 epoxygenase catalyzes the oxidation of arachidonic acid to EETs. EETs have been identified as endothelium-derived hyperpolarizing factors (EDHFs) with vasodilatory, anti-inflammatory, antihypertensive, and antiplatelet aggregation properties. EETs also have profound effects on vascular migration and proliferation and promote angiogenesis. The progression of CVD has been linked to decreased EETs levels, leading to the concept that EETs should be therapeutically targeted to prevent end-organ damage associated with CVD. However, EETs are quickly degraded by the enzyme soluble epoxide hydrolase (sEH) to their less active diols, dihydroxyeicosatrienoic acids (DHETs). As such, one way to increase EETs level is to inhibit their degradation to DHETs by using sEH inhibitors. Inhibition of sEH has been shown to effectively reduce blood pressure and organ damage in experimental models of CVD. Another approach to target EETs is to develop EET analogs with improved solubility and resistance to auto-oxidation and metabolism by sEH. For example, stable ether EET analogs dilate afferent arterioles and lower blood pressure in hypertensive rodent animal models. EET agonists also improve insulin signaling and vascular function in animal models of metabolic syndrome.     

Differences in positional esterification of 14,15-epoxyeicosatrienoic acid in phosphatidylcholine of porcine coronary artery endothelial and smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are readily incorporated into phospholipids of smooth muscle cells (SMC) and endothelial cells (EC). Incorporation of EETs into intact porcine coronary arteries potentiates EC-dependent relaxation, but not vasorelaxation induced by agents that act solely on SMC. To explore the potential mechanisms responsible for this difference, porcine coronary artery SMC and EC preloaded with [3H]14,15-EET were treated with calcium ionophore A23187. Although the amount of EET incorporated into EC and SMC was similar, A23187 stimulated a five-fold increase in release of radioactivity from EC, but only a 21% increase in release from SMC. Thin layer chromatography (TLC) examination of cell lipids demonstrated that > 70% of the incorporated radioactivity was present in phosphatidylcholine (PC) in both SMC and BC. After treatment of EC PC with PLA2, TLC analysis indicated that approximately equal to 75% of radioactivity was present as free EET, and 25% of radioactivity was present as lyso-PC. Therefore, most of the 14,15-EET was esterified into the sn-2 position of PC in EC. However, in SMC, approximately equal to 70% of radioactivity was present as lyso-PC after PLA2 treatment, indicating that the EET was predominately esterified into the sn-1 position. In contrast, all of the 14,15-EET was esterified into the sn-2 position of PI in both EC and SMC. These results suggest that the preferential incorporation of 14,15-EET into the sn-1 position of PC in SMC may help to explain the greater retention of the compound in SMC, while incorporation into the sn-2 position of PC in EC may facilitate agonist-induced 14,15-EET release and potentiation of EC-dependent porcine coronary artery relaxation.     

Modelling the electrophysiological endothelial cell response to bradykinin

The goal of the present study is to construct a biophysical model of the coronary artery endothelial cell response to bradykinin. This model takes into account intracellular Ca2+ dynamics, membrane potential, a non-selective cation channel, and two Ca(2+)-dependent K+ channels, as well as intra- and extracellular Ca2+ sources. The model reproduces the experimental data available, and predicts certain quantities which would be hard to obtain experimentally, like the individual K+ channel currents when the membrane potential is allowed to freely evolve, the implication of epoxyeicosatrienoic acids (EETs), and the total K+ released during stimulation. The main results are: (1) the large-conductance K+ channel participates only very little in the overall response; (2) EETs are required in order to explain the experimental current-potential relationships, but are not an essential component of the bradykinin response; and (3) the total K+ released during stimulation gives rise to a concentration in the intercellular space which is of millimolar order. This concentration change is compatible with the hypothesis that K+ contributes to the endothelium-derived hyperpolarizing factor phenomenon.