11,12-EET Stimulates the Association of BK Channel α and β1 Subunits in Mitochondria to Induce Pulmonary Vasoconstriction

In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca²⁺-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKβ₁ subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKβ₁ ^−/− mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKβ₁ ^−/− cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and β₁ subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and β₁ subunits of mitochondrial BK channels. The 11,12-EET-induced activation of BK channels results in loss of the mitochondrial membrane potential and depolarization of the pulmonary artery smooth muscle cells.     

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.     

Rat mesenteric arterial dilator response to 11,12-epoxyeicosatrienoic acid is mediated by activating heme oxygenase

11,12-Epoxyeicosatrienoic acid (11,12-EET), a potent vasodilator produced by the endothelium, acts on calcium-activated potassium channels and shares biological activities with the heme oxygenase/carbon monoxide (HO/CO) system. We examined whether activation of HO mediates the dilator action of 11,12-EET, and that of the other EETs, on rat mesenteric arteries. Dose-response curves (10(-9) to 10(-6) M) to 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and ACh (10(-9) to 10(-4) M) were evaluated in preconstricted (10(-6) mol/l phenylephrine) mesenteric arteries (<350 microm diameter) in the presence or absence of 1) the cyclooxygenase inhibitor indomethacin (2.8 microM), 2) the HO inhibitor chromium mesoporphyrin (CrMP) (15 microM), 3) the soluble guanylyl cyclase (GC) inhibitor ODQ (10 microM), and 4) the calcium-activated potassium channel inhibitor iberiotoxin (25 nM). The vasodilator response to 11,12-EET was abolished by CrMP and iberiotoxin, whereas indomethacin and ODQ had no effect. In contrast, the effect of ACh was attenuated by ODQ but not by CrMP. The vasodilator effect of 8,9-EET, like that of 11,12-EET, was greatly attenuated by HO inhibition. In contrast, the mesenteric vasodilator response to 5,6-EET was independent of both HO and GC, whereas that to 14,15-EET demonstrated two components, an HO and a GC, of equal magnitude. Incubation of mesenteric microvessels with 11,12-EET caused a 30% increase in CO release, an effect abolished by inhibition of HO. We conclude that the rat mesenteric vasodilator action of 11,12-EET is mediated via an increase in HO activity and an activation of calcium-activated potassium channels.     

Arachidonic acid inhibits epithelial Na channel via cytochrome P450 (CYP) epoxygenase-dependent metabolic pathways

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 microM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose-response curve of the AA effect on ENaC shows that 2 microM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) omega-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase-dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase-dependent metabolite, 11,12-EET.     

EET homologs potently dilate coronary microvessels and activate BK(Ca) channels

Epoxyeicosatrienoic acids (EETs) are released from endothelial cells and potently dilate small arteries by hyperpolarizing vascular myocytes. In the present study, we investigated the structural specificity of EETs in dilating canine and porcine coronary microvessels (50-140 microm ID) and activating large-conductance Ca2+-activated K+ (BK(Ca)) channels. The potencies and efficacies of EET regioisomers and enantiomers were compared with those of two EET homologs: epoxyeicosaquatraenoic acids (EEQs), which are made from eicosapentaenoic acid by the same cytochrome P-450 epoxygenase that generates EETs from arachidonic acid, and epoxydocosatetraenoic acids (EDTs), which are EETs that are two carbons longer. With EC50 values of 3-120 pM but without regio- or stereoselectivity, EETs potently dilated canine and porcine microvessels. Surprisingly, the EEQs and EDTs had comparable potencies and efficacies in dilating microvessels. Moreover, 50 nM 13,14-EDT activated the BK(Ca) channels with the same efficacy as either 11,12-EET enantiomer at 50 nM. We conclude that coronary microvessels and BK(Ca) channels possess low structural specificity for EETs and suggest that EEQs and EDTs may thereby also be endothelium-derived hyperpolarizing factors.     

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.     

Effects of tensile stress on the α1 nicotinic acetylcholine receptor expression in maxillofacial skeletal myocytes

The present study was to test the hypothesis that 11,12-epoxyeicosatrienoic acid (11,12-EET), a metabolic product of arachidonic acid by cytochrome P450 epoxygenase, regulates nitric oxide (NO) generation of the l-arginine/NO synthase (NOS) pathway in human platelets. Human platelets were incubated in the presence or absence of different concentrations of 11,12-EET for 2 h at 37°C, followed by measurements of activities of the l-arginine/NOS pathway. Incubation with 11,12-EET increased the platelet NOS activity, nitrite production, cGMP content, and the platelet uptake of l-[³H]arginine in a concentration-dependent manner. In addition, 11,12-EET attenuated intracellular free Ca²⁺ accumulation stimulated by collagen, which was at least partly mediated by EET-activated l-arginine/NOS pathway. It is suggested that 11,12-EET regulates platelet function through up-regulating the activity of the l-arginine/NOS/NO pathway.     

11,12-epoxyeicosatrienoic acid attenuates synthesis of prostaglandin E2 in rat monocytes stimulated with lipopolysaccharide

Cytochrome P-450 monooxygenase (epoxygenase)-derived arachidonic acid (AA) metabolites, including 11,12-epoxyeicosatrienoic acid (11,12-EET), possess anti-inflammatory and antipyretic properties. Prostaglandin E2 (PGE2), a cyclooxygenase (COX)-derived metabolite of AA, is a well-defined mediator of fever and inflammation. We have tested the hypothesis that 11,12-EET attenuates synthesis of PGE2 in monocytes, which are the cells that are indispensable for induction of fever and initiation of inflammation. Monocytes isolated from freshly collected rat blood were stimulated with lipopolysaccharide (LPS; 100 ng/2 x 10(5) cells) to induce COX-2 and stimulate generation of PGE2. SKF-525A, an inhibitor of epoxygenases, significantly augmented the lipopolysaccharide-provoked synthesis of PGE2 in cell culture in a concentration-dependent manner. It did not affect, however, elevation of the expression of COX-2 protein in monocytes stimulated with LPS. 11,12-EET also did not affect the induction of COX-2 in monocytes incubated with lipopolysaccharide. However, 11,12-EET suppressed, in a concentration-dependent fashion, the generation of PGE2 in incubates. Preincubation of a murine COX-2 preparation for 0-5 min with three concentrations of 11,12-EET (1, 5, and 10 microM) inhibited the oxygenation of [14C]-labeled AA by the enzyme. The inhibitory effect of 11,12-EET on COX-2 was time-and-concentration-dependent, suggesting a mechanism-based inhibition. Based on these data, we conclude that 11,12-EET suppresses generation of PGE2 in monocytes via modulating the activity of COX-2. These data support the hypothesis that epoxygenase-derived AA metabolites constitute a negative feedback on the enhanced synthesis of prostaglandins upon inflammation.     

11,12,20-Trihydroxy-eicosa-8(Z)-enoic acid: a selective inhibitor of 11,12-EET-induced relaxations of bovine coronary and rat mesenteric arteries

Arachidonic acid is metabolized to four regioisomeric epoxyeicosatrienoic acids (EETs) by cytochrome P-450. 5,6-, 8,9-, 11,12-, and 14,15-EET are equipotent in relaxing bovine coronary arteries (BCAs). Vasorelaxant effects of EETs are nonselectively antagonized by 14,15-epoxyeicosa-5(Z)-enoic acid. The 11,12-EET analogs, 20-hydroxy-11,12-epoxyeicosa-8(Z)-enoic acid (20-H-11,12-EE8ZE) and 11,12,20-trihydroxyeicosa-8(Z)-enoic acid (11,12,20-THE8ZE) were synthesized and tested for antagonist activity against EET-induced relaxations in BCAs. In U-46619-preconstricted arterial rings, 5,6-, 8,9-, 11,12-, and 14,15-EET caused concentration-dependent relaxations with maximal relaxations ranging from 80 to 96%. Preincubation of arteries with 20-H-11,12-EE8ZE (10(-5) M) inhibited relaxations to 14,15- and 11,12-EET, but not 5,6- and 8,9-EET; however, greatest inhibitory effect was against 11,12-EET (maximal relaxation = 80.6 ± 4.6 vs. 26.7 ± 7.4% without and with 20-H-11,12-EE8ZE, respectively). Preincubation with the soluble epoxide hydrolase inhibitor (tAUCB, 10(-6) M) significantly enhanced the antagonist effect of 20-H-11,12-EE8ZE against 14,15-EET-induced relaxations (maximal relaxation = 86.6 ± 4.4 vs. 27.8 ± 3.3%, without and with 20-H-11,12-EE8ZE and tAUCB) without any change in its effect against 11,12-EET-induced relaxations. In contrast to the parent compound, the metabolite, 11,12,20-THE8ZE (10(-5) M), significantly inhibited relaxations to 11,12-EET and was without effect on other EET regioisomers. Mass spectrometric analysis revealed conversion of 20-H-11,12-EE8ZE to 11,12,20-THE8ZE by incubation with BCA. The conversion was blocked by tAUCB. 14,15-Dihydroxy-eicosa-5Z-enoic acid (a 14,15-EET antagonist), but not 11,12,20-THE8ZE (an 11,12-EET antagonist), inhibited BCA relaxations to arachidonic acid and flow-induced dilation in rat mesenteric arteries. These results indicate that 11,12,20-THE8ZE is a selective antagonist of 11,12-EET relaxations and a useful pharmacological tool to elucidate the function of 11,12-EET in the cardiovascular system.     

Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (K(ATP)); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 +/- 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 +/- 1.9%, 11,12-EET; and 8.4 +/- 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 +/- 2.1%, 11,12-EET; and 9.7 +/- 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 +/- 4.6%) by the K(ATP) channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 +/- 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP omega-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 +/- 1.2%, DDMS; and 22.2 +/- 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP omega-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP omega-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac K(ATP) channels.     

Mechanism of rat mesenteric arterial KATP channel activation by 14,15-epoxyeicosatrienoic acid

Recently, we reported that 11,12-epoxyeicosatrienoic acid (11,12-EET) potently activates rat mesenteric arterial ATP-sensitive K(+) (K(ATP)) channels and produces significant vasodilation through protein kinase A-dependent mechanisms. In this study, we tried to further delineate the signaling steps involved in the activation of vascular K(ATP) channels by EETs. Whole cell patch-clamp recordings [0.1 mM ATP in the pipette, holding potential (HP) = 0 mV and testing potential (TP) = -100 mV] in freshly isolated rat mesenteric smooth muscle cells showed small glibenclamide-sensitive K(ATP) currents (19.0 +/- 7.9 pA, n = 5) that increased 6.9-fold on exposure to 5 microM 14,15-EET (132.0 +/- 29.0 pA, n = 7, P < 0.05 vs. control). With 1 mM ATP in the pipette solution, K(ATP) currents (HP = 0 mV and TP = -100 mV) were increased 3.5-fold on exposure to 1 microM 14,15-EET (57.5 +/- 14.3 pA, n = 9, P < 0.05 vs. baseline). In the presence of 100 nM iberiotoxin, 1 microM 14,15-EET hyperpolarized the membrane potential from -20.5 +/- 0.9 mV at baseline to -27.1 +/- 3.0 mV (n = 6 for both, P < 0.05 vs. baseline), and the EET effects were significantly reversed by 10 microM glibenclamide (-21.8 +/- 1.4 mV, n = 6, P < 0.05 vs. EET). Incubation with 5 microM 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), a 14,15-EET antagonist, abolished the 14,15-EET effects (31.0 +/- 11.8 pA, n = 5, P < 0.05 vs. 14,15-EET, P = not significant vs. control). The 14,15-EET effects were inhibited by inclusion of anti-G(s)alpha antibody (1:500 dilution) but not by control IgG in the pipette solution. The effects of 14,15-EET were mimicked by cholera toxin (100 ng/ml), an exogenous ADP-ribosyltransferase. Treatment with the ADP-ribosyltransferase inhibitors 3-aminobenzamide (1 mM) or m-iodobenzylguanidine (100 microM) abrogated the effects of 14,15-EET on K(ATP) currents. These results were corroborated by vasodilation studies. 14,15-EET dose-dependently dilated isolated small mesenteric arteries, and this was significantly attenuated by treatment with 14,15-EEZE or 3-aminobenzamide. These results suggest that 14,15-EET activates vascular K(ATP) channels through ADP-ribosylation of G(s)alpha.     

Effects of nitric oxide on mitochondrial permeability transition pore and thiol-mediated responses in cardiac myocytes

Nitric oxide (NO) alters the opening of mitochondrial permeability transition pore (mPTP). However, the signaling pathways of NO on mPTP remain elusive. We aimed to clarify the contribution of thiol-mediated responses to the effects of NO on mPTP in permeabilized myocytes. We found that (1) a high concentration of spermine NONOate (an NO donor; 500 μM) opened mPTP and depolarized ΔΨ(m). (2) A low concentration of NONOate (5 μM) prevented atractyloside (an mPTP opener)-induced mPTP opening. (3) Mn(III) tetrakis (4-benzoic acid) porphyrin (Mn-TBAP, ONOO(-) scavenger) attenuated the effect of high-concentration NONOate on mPTP opening, but did not inhibited the preventive effects of low-concentration NONOate. (4) When the interaction of NO with thiol was inhibited by N-ethylmaleimide, the opening (by high-concentration NONOate) and preventive effects (by low-concentration NONOate) of NONOate on mPTP were blocked. (5) Dithiothreitol (an inhibitor of disulfide bonds formation) prevented high-concentration NONOate-induced mPTP opening. (6) Ascorbic acid (an inhibitor of S-nitrosylation) prevented the preventive effects of low-concentration NONOate on mPTP. We conclude that opening of mPTP by high-concentration NO is related to disulfide bonds formation and oxidizing effects of ONOO(-). In contrast, the inhibitory effect of physiological concentrations of NO on mPTP is related to S-nitrosylation.     

Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids

Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K(+)(K(ATP)) channels. EETs activate K(ATP) channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to K(ATP) channel protein. A fluorescent ATP analog, 2,4,6-trinitrophenyl (TNP)-ATP, which increases its fluorescence emission significantly upon binding with proteins, was used for binding studies with glutathione-S-transferase (GST) Kir6.2 fusion proteins. TNP-ATP bound to GST fusion protein containing the C-terminus of Kir6.2 (GST-Kir6.2C), but not to the N-terminus of Kir6.2, or to GST alone. 11,12-EET (5 muM) did not change TNP-ATP binding K(D) to GST-Kir6.2C, but B(max) was reduced by half. The effect of 11,12-EET was dose-dependent, and 8,9- and 14,15-EETs were as effective as 11,12-EET in inhibiting TNP-ATP binding to GST-Kir6.2C. AA and 11,12-dihydroxyeicosatrienoic acid (11,12-DHET), the parent compound and metabolite of 11,12-EET, respectively, were not effective inhibitors of TNP-ATP binding to GST-Kir6.2C, whereas the methyl ester of 11,12-EET was. These findings suggest that the epoxide group in EETs is important for modulation of ATP binding to Kir6.2. We conclude that EETs bind to the C-terminus of K(ATP) channels, inhibiting binding of ATP to the channel.     

Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso-N-acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 microM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NOS activity was assessed using a fibroblast-reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 14.5 +/- 2.8 microM); but A23187 (3 microM)-stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 +/- 2.5; NONOate= 29.8 +/- 7.7; control = 14.5 +/- 2.5fmol cGMP/microg per 10(6) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2- to 3-fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive-feedback regulatory action of exogenous NO.     

Activation of rat mesenteric arterial KATP channels by 11,12-epoxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs), the cytochrome P-450 epoxygenase metabolites of arachidonic acid, are candidates of endothelium-derived hyperpolarizing factors. We have previously reported that EETs are potent activators of cardiac ATP-sensitive K(+) (K(ATP)) channels, but their effects on the vascular K(ATP) channels are unknown. With the use of whole cell patch-clamp techniques with 0.1 mM ATP in the pipette and holding at -60 mV, freshly isolated smooth muscle cells from rat mesenteric arteries had small glibenclamide-sensitive currents at baseline (13.1 +/- 3.9 pA, n = 5) that showed a 7.2-fold activation by 10 microM pinacidil (94.1 +/- 21.9 pA, n = 7, P < 0.05). 11,12-EET dose dependently activated the K(ATP) current with an apparent EC(50) of 87 nM. Activation of the K(ATP) channels by 500 nM 11,12-EET was inhibited by inclusion of the PKA inhibitor peptide (5 microM) but not by the inclusion of the PKC inhibitor peptide (100 microM) in the pipette solution. These results were corroborated by vasoreactivity studies. 11,12-EET produced dose-dependent vasorelaxation in isolated small mesenteric arteries, and this effect was reduced by 50% with glibenclamide (1 microM) preincubation. The 11,12-EET effects on vasorelaxation were also significantly attenuated by preincubation with cell-permeant PKA inhibitor myristoylated PKI(14-22), and, in the presence of PKA inhibitor, glibenclamide had no additional effects. These results suggest that 11,12-EET is a potent activator of the vascular K(ATP) channels, and its effects are dependent on PKA activities.     

Agonist-dependent variablity of contributions of nitric oxide and prostaglandins in human skeletal muscle.

The relative contributions of endothelium-dependent dilators [nitric oxide (NO), prostaglandins (PGs), and endothelium-derived hyperpolarizing factor (EDHF)] in human limbs are poorly understood. We tested the hypothesis that relative contributions of NO and PGs differ between endothelial agonists acetylcholine (ACh; 1, 2, and 4 microg.dl(-1).min(-1)) and bradykinin (BK; 6.25, 25, and 50 ng.dl(-1).min(-1)). We measured forearm blood flow (FBF) using venous occlusion plethysmography in 50 healthy volunteers (27 +/- 1 yr) in response to brachial artery infusion of ACh or BK in the absence and presence of inhibitors of NO synthase [NOS; with NG-monomethyl-L-arginine (L-NMMA)] and cyclooxygenase (COX; with ketorolac). Furthermore, we tested the idea that the NOS + COX-independent dilation (in the presence of L-NMMA + ketorolac, presumably EDHF) could be inhibited by exogenous NO administration, as reported in animal studies. FBF increased approximately 10-fold in the ACh control; L-NMMA reduced baseline FBF and ACh dilation, whereas addition of ketorolac had no further effect. Ketorolac alone did not alter ACh dilation, but addition of L-NMMA reduced ACh dilation significantly. For BK infusion, FBF increased approximately 10-fold in the control condition; L-NMMA tended to reduce BK dilation (P < 0.1), and addition of ketorolac significantly reduced BK dilation. Similar to ACh, ketorolac alone did not alter BK dilation, but addition of L-NMMA reduced BK dilation. To test the idea that NO can inhibit the NOS + COX-independent portion of dilation, we infused a dose of sodium nitroprusside (NO-clamp technique) during ACh or BK that restored the reduction in baseline blood flow due to L-NMMA. Regardless of treatment order, the NO clamp restored baseline FBF but did not reduce the NOS + COX-independent dilation to ACh or BK. We conclude that the contribution of NO and PGs differs between ACh and BK, with ACh being more dependent on NO and BK being mostly dependent on a NOS + COX-independent mechanism (EDHF) in healthy young adults. The NOS + COX-independent dilation does not appear sensitive to feedback inhibition from NO in the human forearm.     

Role of ADP-ribose in 11,12-EET-induced activation of K(Ca) channels in coronary arterial smooth muscle cells

We recently reported that cADP-ribose (cADPR) and ADP-ribose (ADPR) play an important role in the regulation of the Ca(2+)-activated K(+) (K(Ca)) channel activity in coronary arterial smooth muscle cells (CASMCs). The present study determined whether these novel signaling nucleotides participate in 11,12-epoxyeicosatrienoic acid (11,12-EET)-induced activation of the K(Ca) channels in CASMCs. HPLC analysis has shown that 11,12-EET increased the production of ADPR but not the formation of cADPR. The increase in ADPR production was due to activation of NAD glycohydrolase as measured by a conversion rate of NAD into ADPR. The maximal conversion rate of NAD into ADPR in coronary homogenate was increased from 2.5 +/- 0.2 to 3.4 +/- 0.3 nmol*(-1) *mg protein(-1) by 11,12-EET. The regioisomers of 8,9-EET, 11,12-EET, and 14,15-EET also significantly increased ADPR production from NAD. Western blot analysis and immunoprecipitation demonstrated the presence of NAD glycohydrolase, which mediated 11,12-EET-activated production of ADPR. In cell-attached patches, 11,12-EET (100 nM) increases K(Ca) channel activity by 5.6-fold. The NAD glycohydrolase inhibitor cibacron blue 3GA (3GA, 100 microM) significantly attenuated 11,12-EET-induced increase in the K(Ca) channel activity in CASMCs. However, 3GA had no effect on the K(Ca) channels activity in inside-out patches. 11,12-EET produced a concentration-dependent relaxation of precontracted coronary arteries. This 11,12-EET-induced vasodilation was substantially attenuated by 3GA (30 microM) with maximal inhibition of 57%. These results indicate that 11,12-EET stimulates the production of ADPR and that intracellular ADPR is an important signaling molecule mediating 11,12-EET-induced activation of the K(Ca) channels in CASMCs and consequently results in vasodilation of coronary artery.     

Reactive oxygen species mediate arachidonic acid-induced dilation in porcine coronary microvessels

Reactive oxygen species (ROS) have been proposed to mediate vasodilation in the microcirculation. We investigated the role of ROS in arachidonic acid (AA)-induced coronary microvascular dilation. Porcine epicardial coronary arterioles (110 +/- 4 microm diameter) were mounted onto pipettes in oxygenated Krebs buffer. Vessels were incubated with vehicle or 1 mM Tiron (a nonselective ROS scavenger), 250 U/ml polyethylene-glycolated (PEG)-superoxide dismutase (SOD; an O2- scavenger), 250 U/ml PEG-catalase (a H2O2 scavenger), or the cyclooxygenase (COX) inhibitors indomethacin (10 microM) or diclofenac (10 microM) for 30 min. After endothelin constriction (30-60% of resting diameter), cumulative concentrations of AA (10(-10)-10(-5)M) were added and internal diameters measured by video microscopy. AA (10-7 M) produced 37 +/- 6% dilation, which was eliminated by the administration of indomethacin (4 +/- 7%, P < 0.05) or diclofenac (-8 +/- 8%, P < 0.05), as well as by Tiron (-4 +/- 5%, P < 0.05), PEG-SOD (-10 +/- 6%, P < 0.05), or PEG-catalase (1 +/- 4%, P < 0.05). Incubation of small coronary arteries with [3H]AA resulted in the formation of prostaglandins, which was blocked by indomethacin. In separate studies in microvessels, AA induced concentration-dependent increases in fluorescence of the oxidant-sensitive probe dichlorodihydrofluorescein diacetate, which was inhibited by pretreatment with indomethacin or by SOD + catalase. We conclude that in porcine coronary microvessels, COX-derived ROS contribute to AA-induced vasodilation.     

Cytochrome P-450 metabolite of arachidonic acid mediates bradykinin-induced negative inotropic effect

This study focused on the mechanisms of the negative inotropic response to bradykinin (BK) in isolated rat hearts perfused at constant flow. BK (100 nM) significantly reduced developed left ventricular pressure (LVP) and the maximal derivative of systolic LVP by 20-22%. The cytochrome P-450 (CYP) inhibitors 1-aminobenzotriazole (1 mM and 100 microM) or proadifen (5 microM) abolished the cardiodepression by BK, which was not affected by nitric oxide and cyclooxygenase inhibitors (35 microM NG-nitro-L-arginine methyl ester and 10 microM indomethacin, respectively). The CYP metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET; 50 ng/ml) produced effects similar to those of BK in terms of the reduction in contractility. After the coronary endothelium was made dysfunctional by Triton X-100 (0.5 microl), the BK-induced negative inotropic effect was completely abolished, whereas the 14,15-EET-induced cardiodepression was not affected. In hearts with normal endothelium, after recovery from 14,15-EET effects, BK reduced developed LVP to a 35% greater extent than BK in the control. In conclusion, CYP inhibition or endothelial dysfunction prevents BK from causing cardiodepression, suggesting that, in the rat heart, endothelial CYP products mediate the negative inotropic effect of BK. One of these mediators appears to be 14,15-EET.     

NO inhibits signal transduction pathway for ATP release from erythrocytes via its action on heterotrimeric G protein Gi

The release of ATP from erythrocytes involves a signal transduction pathway of which cystic fibrosis transmembrane conductance regulator, PKA, adenylyl cyclase, and the heterotrimeric G proteins G(s) and G(i) are components. In the pulmonary circulation, ATP released from the erythrocyte stimulates nitric oxide (NO) synthesis, thereby regulating vascular resistance. We reported that NO liberated from an NO donor inhibited ATP release from erythrocytes in response to decreased Po(2) or mechanical deformation. Here, we investigated the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of G(i). Washed rabbit erythrocytes were incubated in the presence or absence of the NO donor N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate; 100 nM, 20 min), followed by treatment with agents that activate specific components of the signal transduction pathway promoting ATP release. Neither ATP release nor cAMP accumulation induced by either forskolin (100 microM, n = 7) or iloprost (100 nM, n = 6) was inhibited by spermine NONOate. These experiments suggest that the inhibitory action of NO is not the result of inactivation of adenylyl cyclase or G(s), respectively. However, spermine NONOate completely inhibited ATP release in response to mastoparan (10 microm, P < 0.05, n = 5), a specific activator of G(i). Spermine (100 nM, 20 min), the polyamine remaining after liberation of NO from spermine NONOate, had no affect on mastoparan-induced ATP release (n = 4). These results support the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of the heterotrimeric G protein G(i).