14,15-Epoxyeicosa-5(Z)-enoic acid: a selective epoxyeicosatrienoic acid antagonist that inhibits endothelium-dependent hyperpolarization and relaxation in coronary arteries

Endothelium-dependent hyperpolarization and relaxation of vascular smooth muscle are mediated by endothelium-derived hyperpolarizing factors (EDHFs). EDHF candidates include cytochrome P-450 metabolites of arachidonic acid, K(+), hydrogen peroxide, or electrical coupling through gap junctions. In bovine coronary arteries, epoxyeicosatrienoic acids (EETs) appear to function as EDHFs. A 14,15-EET analogue, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) was synthesized and identified as an EET-specific antagonist. In bovine coronary arterial rings preconstricted with U46619, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET induced concentration-related relaxations. Preincubation of the arterial rings with 14,15-EEZE (10 micromol/L) inhibited the relaxations to 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET but was most effective in inhibiting 14,15-EET-induced relaxations. 14,15-EEZE also inhibited indomethacin-resistant relaxations to methacholine and arachidonic acid and indomethacin-resistant and L-nitroarginine-resistant relaxations to bradykinin. It did not alter relaxation responses to sodium nitroprusside, iloprost, or the K(+) channel activators (NS1619 and bimakalim). Additionally, in small bovine coronary arteries pretreated with indomethacin and L-nitroarginine and preconstricted with U46619, 14,15-EEZE (3 micromol/L) inhibited bradykinin (10 nmol/L)-induced smooth muscle hyperpolarizations and relaxations. In rat renal microsomes, 14,15-EEZE (10 micromol/L) did not decrease EET synthesis and did not alter 20-hydroxyeicosatetraenoic acid synthesis. This analogue acts as an EET antagonist by inhibiting the following: (1) EET-induced relaxations, (2) the EDHF component of methacholine-induced, bradykinin-induced, and arachidonic acid-induced relaxations, and (3) the smooth muscle hyperpolarization response to bradykinin. Thus, a distinct molecular structure is required for EET activity, and alteration of this structure modifies agonist and antagonist activity. These findings support a role of EETs as EDHFs.     

14,15-epoxyeicosatrienoic acid represents a transferable endothelium-dependent relaxing factor in bovine coronary arteries

Bradykinin causes arterial relaxation and hyperpolarization, which is mediated by a transferable endothelium-derived hyperpolarizing factor (EDHF). In coronary arteries, epoxyeicosatrienoic acids (EETs) are involved in the EDHF response. However, the role of EETs as transferable mediators of EDHF-dependent relaxation remains poorly defined. Two small bovine coronary arteries were cannulated and perfused in tandem in the presence of the nitric oxide synthase inhibitor, nitro-L-arginine (30 micromol/L), and the cyclooxygenase inhibitor, indomethacin (10 micromol/L). Luminal perfusate from donor arteries with intact endothelium perfused endothelium-denuded detector arteries. Detector arteries were constricted with U46619 and diameters were monitored. Bradykinin (10 nmol/L) added to detector arteries did not induce dilation (5+/-2%), whereas bradykinin addition to donor arteries dilated detector arteries by 26.5+/-7% (P<0.05). These dilations were blocked by donor artery endothelium removal and detector artery treatment with the EET-selective antagonist, 14,15-epoxyeicosa-5(Z)-monoenoic acid (14,15-EEZE; 10 micromol/L, -5+/-6%) but not 14,15-EEZE treatment of donor arteries (20+/-5%). 14,15-EET (0.1 to 10 micromol/L) added to detector arteries induced maximal dilations of 82+/-5% that were inhibited 50% by detector artery treatment with 14,15-EEZE (32+/-12%) but not donor artery treatment with 14,15-EEZE. Liquid chromatography-electrospray ionization mass spectrometry analysis verified the presence of 14,15-EET in the perfusate from an endothelium-intact but not denuded artery. These results show that bradykinin stimulates donor artery 14,15-EET release that dilates detector arteries. 14,15-EEZE blocked the donor artery, endothelium-dependent, bradykinin-induced relaxations, and attenuated relaxations to 14,15-EET. These results suggest that EETs are transferable EDHFs in coronary arteries.     

Stable 5,6-epoxyeicosatrienoic acid analog relaxes coronary arteries through potassium channel activation

5,6-epoxyeicosatrienoic acid (5,6-EET) is a cytochrome P450 epoxygenase metabolite of arachidonic acid that causes vasorelaxation. However, investigations of its role in biological systems have been limited by its chemical instability. We developed a stable agonist of 5,6-EET, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy)pentanoic acid (PTPA), in which the 5,6-epoxide was replaced with a 5-ether. PTPA obviates chemical and enzymatic hydrolysis. In bovine coronary artery rings precontracted with U46619, PTPA (1 nmol/L to 10 micromol/L) induced concentration-dependent relaxations, with maximal relaxation of 86+/-5% and EC50 of 1 micromol/L. The relaxations were inhibited by the cyclooxygenase inhibitor indomethacin (10 micromol/L; max relaxation 43+/-9%); the ATP-sensitive K+ channel inhibitor glybenclamide (10 micromol/L; max relaxation 49+/-6%); and the large conductance calcium-activated K+ channel inhibitor iberiotoxin (100 nmol/L; max relaxation 38+/-6%) and abolished by the combination of iberiotoxin with indomethacin or glybenclamide or increasing extracellular K+ to 20 mmol/L. Whole-cell outward K+ current was increased nearly 6-fold by PTPA (10 micromol/L), which was also blocked by iberiotoxin. Additionally, we synthesized 5-(pentadeca-6(Z),9(Z)-dienyloxy)pentanoic acid and 5-(pentadeca-3(Z),9(Z)-dienyloxy)pentanoic acid (PDPA), PTPA analogs that lack the 8,9 or 11,12 double bonds of arachidonic acid and therefore are not substrates for cyclooxygenase. The PDPAs caused concentration-dependent relaxations (max relaxations 46+/-13% and 52+/-7%, respectively; EC50 1micromol/L), which were not altered by glybenclamide but blocked by iberiotoxin. These studies suggested that PTPA induces relaxation through 2 mechanisms: (1) cyclooxygenase-dependent metabolism to 5-ether-containing prostaglandins that activate ATP-sensitive K+ channels and (2) activation of smooth muscle large conductance calcium-activated K+ channels. PDPAs only activate large conductance calcium-activated K+ channels.     

5,6-epoxyeicosatrienoic acid mediates the enhanced renal vasodilation to arachidonic acid in the SHR

We have shown a cytochrome P450-dependent renal vasodilator effect of arachidonic acid in response to inhibition of cyclooxygenase and elevation of perfusion pressure, which was enhanced in the spontaneously hypertensive rat (SHR) and linked to increased production of and/or responsiveness to epoxyeicosatrienoic acids (EETs). In the SHR, vasodilation elicited by low doses of arachidonic acid was attenuated by the nitric oxide synthase inhibitor Nw-nitro-L-arginine (50 micromol/L), whereas the responses to high doses were unaffected. Inhibition of epoxygenases with miconazole (0.3 micromol/L) in the presence of Nw-nitro-L-arginine greatly reduced the renal vasodilator response to all doses of arachidonic acid. Tetraethylammonium (10 mmol/L), a nonselective K+ channel blocker, abolished the nitric oxide-independent renal vasodilator effect of arachidonic acid as well as the vasodilator effect of 5,6-EET, confirming that EET-dependent vasodilation involves activation of K+ channels. Under conditions of elevated perfusion pressure (200 mm Hg) and cyclooxygenase inhibition, 5,6-EET, 8, 9-EET, and 11,12-EET caused renal vasodilatation in both SHR and Wistar-Kyoto rats (WKY), whereas 14,15-EET produced vasoconstriction. 5,6-EET was the most potent renal vasodilator of the EET regioisomers in the SHR by a factor of 4 or more. In the SHR, 5,6-EET- and 11,12-EET-induced renal vasodilatation was >2-fold greater than that registered in WKY. Thus, the augmented vasodilator responses to arachidonic acid in the SHR is through activation of K+ channels, and 5,6-EET is the most likely mediator.     

Epoxyeicosatrienoic acids increase intracellular calcium concentration in vascular smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid. They are potent endogenous vasodilator compounds produced by vascular cells, and EET-induced vasodilation has been attributed to activation of vascular smooth muscle cell (SMC) K(+) channels. However, in some cells, EETs activate Ca(2+) channels, resulting in Ca(2+) influx and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). We investigated whether EETs also can activate Ca(2+) channels in vascular SMC and whether the resultant Ca(2+) influx can influence vascular tone. The 4 EET regioisomers (1 micromol/L) increased porcine aortic SMC [Ca(2+)](i) by 52% to 81%, whereas arachidonic acid, dihydroxyeicosatrienoic acids, and 15-hydroxyeicosatetraenoic acid (1 micromol/L) produced little effect. The increases in [Ca(2+)](i) produced by 14,15-EET were abolished by removal of extracellular Ca(2+) and by pretreatment with verapamil (10 micromol/L), an inhibitor of voltage-dependent (L-type) Ca(2+) channels. 14,15-EET did not alter Ca(2+) signaling induced by norepinephrine and thapsigargin. When administered to porcine coronary artery rings precontracted with a thromboxane mimetic, 14,15-EET produced relaxation. However, when administered to rings precontracted with acetylcholine or KCl, 14,15-EET produced additional contractions. In rings exposed to 10 mmol/L KCl, a concentration that did not affect resting ring tension, 14,15-EET produced small contractions that were abolished by EGTA (3 mmol/L) or verapamil (10 micromol/L). These observations indicate that 14,15-EET enhances [Ca(2+)](i) influx in vascular SMC through voltage-dependent Ca(2+) channels. This 14,15-EET-induced increase in [Ca(i)(2+)] can produce vasoconstriction and therefore may act to modulate EET-induced vasorelaxation.     

Hydrogen peroxide inhibits cytochrome p450 epoxygenases: interaction between two endothelium-derived hyperpolarizing factors

The cytochrome P450 epoxygenase (CYP)-derived metabolites of arachidonic acid the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2) both function as endothelium-derived hyperpolarizing factors (EDHFs) in the human coronary microcirculation. However, the relative importance of and potential interactions between these 2 vasodilators remain unexplored. We identified a novel inhibitory interaction between CYPs and H2O2 in human coronary arterioles, where EDHF-mediated vasodilatory mechanisms are prominent. Bradykinin induced vascular superoxide and H2O2 production in an endothelium-dependent manner and elicited a concentration-dependent dilation that was reduced by catalase but not by 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE), 6-(2-propargyloxyphenyl)hexanoic acid, sulfaphenazole, or iberiotoxin. However, in the presence of catalase, an inhibitory effect of these compounds was unmasked. In a tandem-bioassay preparation, application of bradykinin to endothelium-intact donor vessels elicited dilation of downstream endothelium-denuded detectors that was partially inhibited by donor-applied catalase but not by detector-applied EEZE; however, EEZE significantly inhibited dilation in the presence of catalase. EET production by human recombinant CYP 2C9 and 2J2, 2 major epoxygenase isozymes expressed in human coronary arterioles, was directly inhibited in a concentration-dependent fashion by H2O2 in vitro, as observed by high-performance liquid chromatography (HPLC); however, EETs were not directly sensitive to oxidative modification. H2O2 inhibited dilation to arachidonic acid but not to 11,12-EET. These findings suggest that an inhibitory interaction exists between 2 EDHFs in the human coronary microcirculation. CYP epoxygenases are directly inhibited by H2O2, and this interaction may modulate vascular EET bioavailability.     

Mechanisms by which epoxyeicosatrienoic acids (EETs) elicit cardioprotection in rat hearts

Cytochrome P450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce reductions in infarct size in canine myocardium following ischemia-reperfusion injury via opening of either the sarcolemmal K(ATP) (sarcK(ATP)) or mitochondrial K(ATP) (mitoK(ATP)) channel. In the present study, we subjected intact rat hearts to 30 min of left coronary artery occlusion and 2 h of reperfusion followed by tetrazolium staining to determine infarct size as a percent of the area at risk (IS/AAR, %). The results demonstrate that the two major regioisomers of the CYP epoxygenase pathway, 11,12-EET (2.5 mg/kg, iv) and 14,15-EET (2.5 mg/kg, iv) significantly reduced myocardial infarct size (IS/AAR, %) in rats as compared with control (41.9+/-2.3%, 40.9+/-1.2% versus 61.5+/-1.6%, respectively), whereas, a third regioisomer, 8,9-EET (2.5 mg/kg, iv) had no effect (55.2+/-1.4). The protective effect of pretreatment with 11,12- and 14,15-EETs was completely abolished (61.9+/-0.7%, 58.6+/-3.1%, HMR; 63.3+/-1.2%, 63.2+/-2.5%, 5-HD) in the presence of the selective sarcK(ATP) channel antagonist, HMR 1098 (6 mg/kg, iv) or the selective mitoK(ATP) channel antagonist, 5-HD (10 mg/kg, iv) given 10 min after 11,12- or 14,15-EET administration but 5 min prior to index ischemia. Furthermore, concomitant pretreatment with 11,12- or 14,15-EET in combination with the free radical scavenger, 2-mercaptopropionyl glycine (2-MPG), at a dose (20 mg/kg, iv) that had no effect on IS/AAR (57.7+/-1.3%), completely abolished the cardioprotective effect of 11,12- and 14,15-EETs (58.2+/-1.6%, 61.4+/-1.0%), respectively. These data suggest that part of the cardioprotective effects of EETs in rat hearts against infarction is the result of an initial burst of reactive oxygen species (ROS) and subsequent activation of both the sarcK(ATP) and mitoK(ATP) channel.     

Epoxygenase products of arachidonic acid are endogenous constituents of the hypothalamus involved in D2 receptor-mediated, dopamine-induced release of somatostatin

The epoxyeicosatrienoic acids (EETs) were discovered as products of a cyclooxygenase/lipoxygenase-independent, cytochrome P-450 catalyzed metabolism of arachidonic acid (AA) termed the "epoxygenase" pathway. The rat hypothalamus is able to synthesize EETs from exogenous AA, and 5,6-EET has been found to release the neuropeptide somatostatin (SRIF) from hypothalamic nerve terminals of the median eminence (ME). In the present study, hypothalami from male rats were examined for the presence of endogenous EETs, using chemical, chromatographic, and mass spectral analysis procedures. The samples were initially separated in a C18 Sepralyte column, fractionated on TLC plates, and purified by reverse phase HPLC. Thereafter, they were esterified (pentafluorobenzyl esters) and subjected to negative ion chemical ionization/gas chromatography (GC)/mass spectral (MS) analysis. The GC retention time and the MS fragmentation patterns revealed the presence of a mixture of 8,9-, 11,12- and 14,15-EETs; instability of 5,6-EET during the isolation protocol precluded its identification. Total hypothalamic EET concentration was estimated to be 120 ng/g wet tissue. The 8,9-regiosomer released SRIF from ME nerve terminals with an ED50 of 5 x 10(-12) M; Dopamine (DA) and the D2 receptor agonist PPHT, but not the D1 receptor agonist SKF-38393, induced SRIF release from the ME. This effect was blocked by clotrimazole and ketoconazole, two inhibitors of microsomal cytochrome P-450 function and AA epoxygenase in particular. In contrast, the inhibitors failed to affect the increase in SRIF release induced by 8,9-EET. These results indicate that: 1) in addition to cyclooxygenase and lipoxygenase products, epoxygenase metabolites of AA are endogenous compounds of the hypothalamus, and 2) EETs may mediate the increase in SRIF release from hypothalamic neurons induced by the interaction of DA with D2 receptors.     

Endogenous biosynthesis of arachidonic acid epoxides in humans: increased formation in pregnancy-induced hypertension.

Arachidonic acid is metabolized by means of P450 isoenzyme(s) to form epoxyeicosatrienoic acids (EETs) and their corresponding dihydroxy derivatives (DHETs). In the present study, we established the presence in human urine of 8,9-, 11,12-, and 14,15-EETs and their corresponding DHETs by developing quantitative assays and using negative ion, chemical ionization GC/MS and octadeuterated internal standards. Urinary excretion of 8,9- and 11,12-DHET increased in healthy pregnant women compared with nonpregnant female volunteers. By contrast, excretion of 11,12-DHET and 14,15-DHET, but not the 8,9-DHET regioisomer, increased even further in patients with pregnancy-induced hypertension. Intravenous administration of [3H]14,15-EET to three dogs markedly increased its DHET in plasma. The terminal half-life ranged from 7.9-12.3 min and the volume of distribution (3.5-5.3 liters) suggested limited distribution outside the plasma compartment. Negligible radioactivity was detected in urine; this fact infers that under physiological circumstances, urinary DHETs largely derive from the kidney. That P450 metabolites of arachidonic acid are formed in humans supports the hypothesis that these metabolites contribute to the physiological response to normal pregnancy and the pathophysiology of pregnancy-induced hypertension.     

Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids

The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14, 15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22+/-4 mm Hg (P:<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N:, N:'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.     

Involvement of eicosanoids in release of oxytocin and vasopressin from the neural lobe of the rat pituitary

Arachidonic acid (AA) is oxidized via three pathways which result in several series of distinct metabolites. Cyclooxygenase produces prostaglandins (PGs), prostacyclins, and thromboxanes. Lipoxygenase produces hydroperoxy/hydroxyeicosatetraenoic acids (HPETE/HETEs) and leukotrienes. Epoxygenase, a recently uncovered pathway, results in epoxyeicosatrienoic acids (EETs). Based on reverse phase HPLC product analysis, this study establishes that all three pathways of AA metabolism are present in microsomal incubates of the neural lobe of the pituitary gland. Addition of PGE2 to incubated fragments of neural lobes of the rat pituitary stimulates secretion of both arginine vasopressin (AVP) and oxytocin in vitro. Inclusion of 5-HETE and 12-HETE in the incubation medium stimulates marginal release of AVP and oxytocin by 12-HETE only. The magnitude of AVP and oxytocin secretion stimulated by the epoxygenase metabolites 8,9-, 11,12-, and 14,15-EET is equal to that caused by PGE2. Maximal stimulation of secretion (3- to 4-fold) requires an EET concentration 10-15 times greater than that of PGE2. In contrast, 5,6-EET is inactive. These data suggest that oxygenated products of AA play a role in AVP and oxytocin secretion. Although PGs appear to be the dominant arachidonate metabolites involved in the release of AVP and oxytocin, the EETs probably have a contributing role.     

Metabolism of arachidonic acid by rat adrenal glomerulosa cells: synthesis of hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids

Metabolites of arachidonic acid have been implicated in the regulation of aldosterone release. To form a basis for further investigations in this area, the present study has isolated and identified the metabolites formed from exogenous arachidonic acid by adrenal zona glomerulosa cells and characterized the effects of several inhibitors on the synthesis of these eicosanoids. Rat adrenal glomerulosa cells metabolized exogenous [14C]arachidonic acid to products comigrating with the prostaglandins (PGs), hydroxyeicosatatraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs). The metabolites were found in the cells and the incubation media; however, none of the metabolites were found esterified to cellular lipids. The major metabolites were identified as 6-keto PGF1 alpha, PGE2, PGF2 alpha, PGD2, 12(S)-HETE, 15(S)-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. The identities of the HETEs and EETs were confirmed by gas chromatography/mass spectrometry. There was no evidence for the synthesis of leukotrienes. The cyclooxygenase inhibitor, indomethacin, the lipoxygenase inhibitors, nordihydroguaiaretic acid, baicalein and AA861, and the combined cyclooxygenase/lipoxygenase inhibitors, BW755C and eicosatetrayenoic acid, inhibited the formation of the [14C]PGs, the [14C]HETEs, and the [14C]EETs. Metyrapone and clotrimazole, inhibitors of cytochrome P450, increased the synthesis of [14C]PGs and [14C]HETEs and reduced the synthesis of [14C] EETs. Superoxide dismutase did not alter arachidonic acid metabolism. In contrast, arachidonic acid metabolism was increased in cells pretreated with catalase. These data indicate that adrenal glomerulosa cells metabolize exogenous arachidonic acid to a number of oxygenated metabolites including PGs, HETEs, and EETs. From studies with inhibitors, the EETs appear to be synthesized by a cytochrome P450 epoxygenase and the HETEs by lipoxygenases.     

Dynamic modulation of interendothelial gap junctional communication by 11,12-epoxyeicosatrienoic acid

Functional gap junctional communication between vascular cells has been implicated in ascending dilatation and the cytochrome P-450 (CYP) inhibitor-sensitive and NO- and prostacyclin-independent dilatation of many vascular beds. Here, we assessed the mechanisms by which the epoxyeicosatrienoic acids (EETs) generated by a CYP 2C enzyme control interendothelial gap junctional communication. In CYP 2C-expressing porcine coronary endothelial cells, bradykinin, which enhances EET formation, elicited a biphasic effect on the electrical coupling and transfer of Lucifer yellow between endothelial cells, consisting of a transient increase in coupling followed by a sustained uncoupling. The initial phase was sensitive to the CYP 2C9 inhibitor sulfaphenazole and the protein kinase A (PKA) inhibitors Rp-cAMPS and KT5720 and could be mimicked by forskolin and caged cAMP as well as by the PKA activators 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphorothioate sodium salt and Sp-cAMPS. Gap junction uncoupling in bradykinin-stimulated porcine coronary endothelial cells was prevented by inhibiting the activation of extracellular signal-regulated kinase (ERK)1/2. In human endothelial cells, which express little CYP 2C, bradykinin elicited only an ERK1/2-mediated inhibition of intercellular communication. The CYP 2C9 product, 11,12-EET, also exerted a dual effect on the electrical and dye coupling of human endothelial cells, which was sensitive to PKA inhibition. These results demonstrate that an agonist-activated CYP-dependent pathway as well as 11,12-EET can positively regulate interendothelial gap junctional communication, most probably via the activation of PKA, an effect that is curtailed by the subsequent activation of ERK1/2.     

Synthesis of lipoxygenase and epoxygenase products of arachidonic acid by normal and stenosed canine coronary arteries

Coronary vascular injury promotes blood cell-vessel wall interactions that influence arachidonic acid metabolism and coronary blood flow patterns. Since lipoxygenase and cytochrome P-450 epoxygenase metabolites of arachidonic acid are synthesized by vascular and inflammatory cells and have a variety of important biological actions, we investigated the metabolism of arachidonic acid by these pathways in normal and stenosed, endothelially injured canine coronary arteries. We found and confirmed by gas chromatography/mass spectrometry that primarily 12- and 15-hydroxyeicosatetraenoic acids (HETEs) are synthesized by both coronary artery segments. Lesser amounts of 11-, 9-, 8-, and 5-HETEs are also produced. 15-Ketoeicosatetraenoic acid is also synthesized. The synthesis of 14C-HETEs is fivefold to 10-fold greater by the stenosed than the normal coronary artery. Specific radioimmunoassays indicated that the stenosed coronary artery synthesized 93 +/- 14 and 1,102 +/- 154 ng/g of tissue of 15- and 12-HETE, respectively, while the normal coronary artery produced 17 +/- 3 and 162 +/- 68 ng/g of tissue of 15- and 12-HETE, respectively. Products comigrating with 14,15-; 11,12-; 8,9-; and 5,6-epoxyeicosatrienoic acids (EETs) and the corresponding dihydroxyeicosatrienoic acids (DHETs) were detected predominantly in stenosed coronary arteries by high-pressure liquid chromatography. The structures of the EETs were confirmed by GC/MS. The EETs and prostaglandin I2 produced endothelium-independent, concentration-related relaxations of dog coronary artery rings. These data indicate that normal and stenotic coronary arteries metabolize arachidonic acid to HETEs, DHETs, and EETs along with prostaglandins; however, the synthesis of these metabolites is greater in the stenosed, endothelially injured vessel. The EETs may be synthesized during the development of cyclic flow variations and counteract the vasoconstrictor effects of thromboxane A2.     

Apamin-sensitive K+ currents mediate arachidonic acid-induced relaxations of rabbit aorta

Arachidonic acid induces an endothelium-dependent relaxation of the rabbit aorta that is blocked by lipoxygenase inhibitors. The cellular vasodilatory mechanisms activated by arachidonic acid metabolites remain undefined. In rabbit thoracic aortic rings pretreated with indomethacin (10 micromol/L) and contracted with phenylephrine, arachidonic acid (0.1 to 100 micromol/L) induced concentration-dependent relaxations. Maximal relaxations averaged 45+/-3% and were inhibited by increasing extracellular K+ (30 mmol/L, 15+/-5%; P<0.001) or incubation with apamin (100 nmol/L, 26+/-7%; P<0.05) but not incubation with charybdotoxin (100 nmol/L, 41+/-5%). In aortic strips with an intact endothelium that were treated with phenylephrine, arachidonic acid (10 micromol/L) increased the membrane potential from -28.7+/-1.3 to -37.8+/-3.0 mV (P<0.01). Preincubation with apamin did not alter basal membrane potential but inhibited arachidonic acid-induced hyperpolarization (-31.5+/-1.5 mV). Incubation of rabbit aortic segments with apamin or charybdotoxin did not alter [14C]arachidonic acid metabolism. Whole-cell outward K+ currents from isolated rabbit aortic smooth muscle cells averaged 43.0+/-4.8 pA/pF at 60 mV and were significantly decreased to 35.7+/-4.2 pA/pF by apamin (P<0.001). Subsequent addition of charybdotoxin further decreased maximal currents to 14.4+/-2.3 pA/pF. Addition of 11,12,15-trihydroxyeicosatrienoic acid increased the outward whole-cell K+ current. In inside-out patches of aortic smooth muscle, apamin inhibited the calcium activation (100 to 300 nmol/L; P<0.001) of a small-conductance K+ channel (approximately 24 pS). These results suggest that arachidonic acid induces endothelium-dependent hyperpolarization and relaxation of rabbit aorta through activation of smooth muscle, apamin-sensitive K+ currents.     

TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels

Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) (5 micromol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 micromol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.     

Cytochrome P450 and arachidonic acid metabolites: role in myocardial ischemia/reperfusion injury revisited

Ischemia-reperfusion of the heart and other organs results in the accumulation of unesterified arachidonic acid (AA) via the action of membrane-bound phospholipases, primarily phospholipase A2. AA can be metabolized by the classical cyclooxygenase (COX) and lipoxygenase (LOX) pathways to well-characterized metabolites and their respective cardioprotective end products such as prostacyclin (PGI2) and 12-hydroxyeicosatetraenoic acid (12-HETE). However, it has only been recently recognized that another less well-characterized pathway of AA metabolism, the cytochrome P450 (CYP) pathway, may have important cardiovascular effects. Several lines of data support the possibility that certain CYP metabolites resulting from the hydroxylation of AA such as 20-hydroxyeicosatetraenoic acid (20-HETE) are potent vasoconstrictors and may produce detrimental effects in the heart during ischemia and pro-inflammatory effects during reperfusion. On the other hand, a group of regioisomers resulting from the epoxidation of AA, including 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acid (EETs), have been shown to reduce ischemic and/or reperfusion injury in the heart and vasculature. This review will discuss the detrimental and beneficial actions, including the potential cellular mechanisms responsible as a result of stimulating or inhibiting the two arms of this novel CYP pathway. The therapeutic potential of increasing EET concentrations and/or reducing 20-HETE concentrations will also be addressed.     

表氧化二十碳三烯酸对游离脂肪酸诱导胰岛β细胞凋亡的抑制作用

目的:研究11,12-表氧化二十碳三烯酸(11,12-epoxyeicosatrienoic acids,11,12-EETs)抑制游离脂肪酸(free fatty acids,FFAs)对原代小鼠胰岛β细胞凋亡的影响,探讨11,12-EETs保护胰岛β细胞是否通过抑制内质网应激相关转录因子ATF4与ATF6核转位来实现.方法:棕榈酸(palmitic acids,PA)诱导原代小鼠胰岛β细胞凋亡,与11,12-EETs共同孵育24h后,采用流式细胞术检测该细胞的线粒体膜电位的变化与细胞凋亡水平的改变,以观察11,12-EETs对原代小鼠胰岛β细胞的保护作用;用免疫印迹法检测内质网应激(endoplasmic reticulum stress,ERS)相关转录因子ATF4与ATF6在胞浆与胞核中的蛋白表达,以此观察二者的核转位改变.结果:100nmol/L11,12-EETs与400μmol/LPA共同孵育胰岛β细胞24h后,与FFAs对照组相比,FFAs+EET组可显著提升胰岛β细胞的存活率(62.1%±7.3%vs53.0%±6.1%),并明显降低胰岛β细胞线粒体的去极化水平(23.6%±3.4%vs35.2%±4.7%);11,12-EETs可有效抑制PA诱导的细胞凋亡(24.5%±4.2%vs40.1%±5.6%);同时,PA刺激的ATF4与ATF6的核转位受到11,12-EETs的影响,胞核ATF4与ATF6蛋白水平显著性降低,胞浆ATF4与ATF6蛋白水平明显上调.结论:11,12-EETs可以抑制FFAs诱导的凋亡,其分子机制可能是通过抑制FFAs引发的ATF4与ATF6转位导致内质网应激相关基因表达.     

Intestinal vasodilation by epoxyeicosatrienoic acids: arachidonic acid metabolites produced by a cytochrome P450 monooxygenase

Purified synthetic products from the cytochrome P450 pathway of arachidonate metabolism were applied to the intestinal serosa. Arteriolar blood flow was calculated using video microscopy. After a steady-state baseline, a bolus containing 10-60 micrograms 14,15-epoxyeicosatrienoic acid/ml (14,15-EET) had no detectable effect on blood flow. However, 25 +/- 3 micrograms 11,12-EET/ml and 36 +/- 2 micrograms 8,9-EET/ml caused increases (134 +/- 8% and 127 +/- 6%) that were similar to those elicited by 8 +/- 2 micrograms adenosine/ml (138 +/- 12%). Furthermore, the increases (275 +/- 38%) produced by 32 +/- 6 micrograms 5,6-EET/ml exceeded those elicited (160 +/- 10%) by a similar concentration (27 +/- 3 micrograms/ml) of adenosine. Thus, a structure-activity relationship is suggested. Nevertheless, these values probably underestimate the potency of the EETs because the vasoactivity was reduced by contact with water. The activity of the cyclooxygenase pathway seemed to limit the formation of vasoactive quantities of EETs, or other nonprostanoids, from exogenous arachidonate in the serosa but not the mucosa. A bolus (1.3 +/- 0.2 mg/ml) or continuous application (122 +/- 45 micrograms/ml) of arachidonate caused blood flow increases (236 +/- 14% or 229 +/- 27%) that were almost eliminated (129 +/- 5% or 121 +/- 9%) by a cyclooxygenase inhibitor; the residual response was abolished by a cytochrome P450 inhibitor. However, cytochrome P450 inhibitors alone did not attenuate the arachidonate response. In contrast, a continuous application of 194 micrograms arachidonate/ml to the mucosa caused a markedly smaller blood flow increase (119 +/- 8%) and cyclooxygenase inhibitors potentiated (132 +/- 8%), rather than reduced, this response. We conclude that EETs are a labile class of vasodilators with a potency comparable to adenosine in the intestinal microcirculation. Indirect evidence suggests regional differences in the formation of vasoactive quantities of arachidonate metabolites within the intestinal wall.