Determination of EETs using microbore liquid chromatography with fluorescence detection

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid involved in the regulation of vascular tone. The method of microbore column high-performance liquid chromatography with fluorescence detection was developed to determine 14,15-EET, 11, 12-EET, and the mixture of 8,9-EET and 5,6-EET. Tridecanoic acid (TA) was used as an internal standard. EETs were reacted with 2-(2, 3-naphthalimino)ethyl trifluoromethanesulfonate (NT) to form highly fluorescent derivatives. A C(18) microbore column and a water-acetonitrile mobile phase were used for separation. Samples were excited at 259 nm, and the fluorescence was detected at 395 nm. The overall recoveries were 88% for EETs and 40% for TA. EETs were detected in concentrations as low as 2 pg (signal-to-noise ratio = 3). The method was used to determine the EET production from endothelial cells (ECs). Bradykinin and methacholine (10(-6) M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for determination of EETs at low concentrations normally detected in complex biological samples.     

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.     

8,9-Epoxyeicosatrienoic acid inhibits antibody production of B lymphocytes in mice

Epoxyeicosatrienoic acids (EETs), synthesized from arachidonic acid by cytochrome P450 epoxygenases, are converted to dihydroxyeicosatrienoic acids by soluble epoxide hydrolase. EETs exert anti-inflammatory effects. However, the effect of EETs on humoral immunity is poorly understood. The present study is to investigate the potential role of EETs on B cell function and mechanisms. We examined the role of EETs on antibody production of splenic B cells from C57BL/6 and apolipoprotein E-deficient (ApoE-/-) mice by means of ELISA. Of the 4 EET regioisomers, 8,9-EET decreased basal and activation-induced B cell antibody secretion. As well, 8,9-EET significantly inhibited B-cell proliferation and survival, plasma cell differentiation and class-switch recombination. Western blot analysis revealed that lipopolysaccharide-induced nuclear translocation of NF-κB could be attenuated by 8,9-EET. Furthermore, germinal center formation was impaired by 8,9-EET in mice in vivo. 8,9-EET may inhibit B-cell function in vitro and in vivo, which suggests a new therapeutic strategy for diseases with excess B cell activation.     

Characterization of 5,6- and 8,9-epoxyeicosatrienoic acids (5,6- and 8,9-EET) as potent in vivo angiogenic lipids

The cytochrome P450 arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs) are powerful, nonregioselective, stimulators of cell proliferation. In this study we compared the ability of the four EETs (5,6-, 8,9-, 11,12-, and 14,15-EETs) to regulate endothelial cell proliferation in vitro and angiogenesis in vivo and determined the molecular mechanism by which EETs control these events. Inhibition of the epoxygenase blocked serum-induced endothelial cell proliferation, and exogenously added EETs rescued cell proliferation from epoxygenase inhibition. Studies with selective ERK, p38 MAPK, or PI3K inhibitors revealed that whereas activation of p38 MAPK is required for the proliferative responses to 8,9- and 11,12-EET, activation of PI3K is necessary for the cell proliferation induced by 5,6- and 14,15-EET. Among the four EETs, only 5,6- and 8,9-EET are capable of promoting endothelial cell migration and the formation of capillary-like structures, events that are dependent on EET-mediated activation of ERK and PI3K. Using subcutaneous sponge models, we showed that 5,6- and 8,9-EET are pro-angiogenic in mice and that their neo-vascularization effects are enhanced by the co-administration of an inhibitor of EET enzymatic hydration, presumably because of reduced EET metabolism and inactivation. These studies identify 5,6- and 8,9-EET as powerful and selective angiogenic lipids, provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity, and suggest a physiological role for them in angiogenesis and de novo vascularization.     

The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity peroxisome proliferator-activated receptor ligands

Cytochromes P450 of the CYP2C and CYP4A gene subfamilies metabolize arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) and to 19- and 20-hydroxyeicosatetraenoic acids (HETEs), respectively. Abundant functional studies indicate that EETs and HETEs display powerful and often opposing biological activities as mediators of ion channel activity and regulators of vascular tone and systemic blood pressures. Incubation of 8,9-, 11,12-, and 14,15-EETs with microsomal and purified forms of rat CYP4A isoforms led to rapid NADPH-dependent metabolism to the corresponding 19- and 20-hydroxylated EETs. Comparisons of reaction rates and catalytic efficiency with those of arachidonic and lauric acids showed that EETs are one of the best endogenous substrates so far described for rat CYP4A isoforms. CYP4A1 exhibited a preference for 8,9-EET, whereas CYP4A2, CYP4A3, and CYP4A8 preferred 11,12-EET. In general, the closer the oxido ring is to the carboxylic acid functionality, the higher the rate of EET metabolism and the lower the regiospecificity for the EET omega-carbon. Analysis of cis-parinaric acid displacement from the ligand-binding domain of the human peroxisome proliferator-activated receptor-alpha showed that omega-hydroxylated 14,15-EET bound to this receptor with high affinity (K(i) = 3 +/- 1 nm). Moreover, at 1 microm, the omega-alcohol of 14,15-EET or a 1:4 mixture of the omega-alcohols of 8,9- and 11,12-EETs activated human and mouse peroxisome proliferator-activated receptor-alpha in transient transfection assays, suggesting a role for them as endogenous ligands for these orphan nuclear receptors.     

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.     

Endothelial cell prostacyclin production induced by activated neutrophils

A bovine aortic endothelial cell (EC) line released prostacyclin (greater than 1 pmol/10(+5) EC cells) when incubated with fMet-Leu-Phe (FMLP)-stimulated rat and human neutrophils (PMNs). This prostaglandin (PG) I2 was shown to come from the ECs and not from the PMNs by radioactive, high-performance liquid chromatography, and immunochemical criteria. Both FMLP-stimulated rat peritoneal and human peripheral PMNs as well as their stimulated cell-free supernatants and unstimulated sonicates could elicit the release of PGI2 from ECs. Since phorbol myristate acetate stimulated PMN adherence but elicited little PGI2 release from ECs, the PGI2 stimulation in ECs is unrelated to PMN adhesion. The addition of catalase and superoxide dismutase to FMLP-stimulated PMNs enhanced rather than reduced PGI2 formation, indicating that activated oxygen products of the PMN are not responsible for the induction of PGI2. Incubation of ECs with leukotriene (LT) B4, LTC4, or LTD4 did not trigger PGI2 release nor did aspirin pretreatment of the PMNs reduce the PGI2 induction. These data suggest that arachidonic acid metabolites of the PMNs were not responsible for the PGI2 induction. Available data indicates that the PMN factor that stimulates PGI2 from ECs is either released concomitantly with the azurophilic granules or is closely related to this event.     

Mobilization of neutrophil sialidase activity desialylates the pulmonary vascular endothelial surface and increases resting neutrophil adhesion to and migration across the endothelium

The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to interact with other cells. PMN activation with various stimuli mobilizes intracellular sialidase to the plasma membrane, where it cleaves sialic acid from cell surfaces. Because enhanced PMN adherence, spreading, deformability, and motility each are associated with surface desialylation and are critical to PMN diapedesis, we studied the role of sialic acid on PMN adhesion to and migration across pulmonary vascular endothelial cell (EC) monolayers in vitro. Neuraminidase treatment of either PMN or EC increased adhesion and migration in a dose-dependent manner. Neuraminidase treatment of both PMNs and ECs increased PMN adhesion to EC more than treatment of either PMNs or ECs alone. Moreover, neuraminidase treatment of ECs did not change surface expression of adhesion molecules or release of IL-8 and IL-6. Inhibition of endogenous sialidase by either cross-protective antineuraminidase antibodies (45.5% inhibition) or competitive inhibition with pseudo-substrate (41.2% inhibition) decreased PMN adhesion to ECs; the inhibitable sialidase activity appeared to be associated with activated PMNs. Finally, EC monolayers preincubated with activated PMNs became hyperadhesive for subsequently added resting PMNs, and this hyperadhesive state was mediated through endogenous PMN sialidase activity. Blocking anti-E-selectin, anti-CD54 and anti-CD18 antibodies decreased PMN adhesion to tumor necrosis factor-activated ECs but not to PMN-treated ECs. These data implicate desialylation as a novel mechanism through which PMN-EC adhesion can be regulated independent of de novo protein synthesis or altered adhesion molecule expression. The ability of activated PMNs, through endogenous sialidase activity, to render the EC surface hyperadherent for unstimulated PMNs may provide for rapid amplification of the PMN-mediated host response.     

Protective effects of epoxyeicosatrienoic acids on human endothelial cells from the pulmonary and coronary vasculature

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 (CYP) metabolites synthesized from the essential fatty acid arachidonic acid to generate four regioisomers, 14,15-, 11,12-, 8,9-, and 5,6-EET. Cultured human coronary artery endothelial cells (HCAECs) contain endogenous EETs that are increased by stimulation with physiological agonists such as bradykinin. Because EETs are known to modulate a number of vascular functions, including angiogenesis, we tested each of the four regioisomers to characterize their effects on survival and apoptosis of HCAECs and cultured human lung microvascular endothelial cells (HLMVECs). A single application of physiologically relevant concentration of 14,15-, 11,12-, and 8,9-EET but not 5,6-EET (0.75-300 nM) promoted concentration-dependent increase in cell survival of HLMVECs and HCAECs after removal of serum. The lipids also protected the same cells from death via the intrinsic, as well as extrinsic, pathways of apoptosis. EETs did not increase intracellular calcium concentration ([Ca2+]i) or phosphorylate mitogen-activated protein kinase p44/42 when applied to these cells, and their protective action was attenuated by the phosphotidylinositol-3 kinase inhibitor wortmannin (10 microM) but not the cyclooxygenase inhibitor indomethacin (20 microM). Our results demonstrate for the first time the capacity of EETs to enhance human endothelial cell survival by inhibiting both the intrinsic, as well as extrinsic, pathways of apoptosis, an important underlying mechanism that may promote angiogenesis and endothelial survival during atherosclerosis and related cardiovascular ailments.     

Arachidonic acid epoxygenase: structural characterization and quantification of epoxyeicosatrienoates in plasma.

Gas chromatographic/mass spectroscopic and chiral analysis showed the presence of enzymatically derived 8,9-, 11,12- and 14,15-EET in rat plasma (2.8:1:3.4 molar ratio, respectively; 10.2 +/- 0.4 ng total EET/ml plasma). Greater than 90% of the plasma EETs was esterified to the phospholipids of circulating lipoproteins. The lipoprotein fraction with the highest EET concentration was LDL (8.1 +/- 0.9 ng/mg of protein) followed by HDL and VLDL (3.5 +/- 0.1 and 1.9 +/- 0.3 ng/mg of protein, respectively). In light of the biological activities of the EETs, these results suggest a potential systemic function for the cytochrome P-450 epoxygenase.     

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.     

Epoxyeicosatrienoic acids inhibit the activation of NLRP3 inflammasome in murine macrophages

Epoxyeicosatrienoic acids (EETs) derived from arachidonic acid exert anti-inflammation effects. We have reported that blocking the degradation of EETs with a soluble epoxide hydrolase (sEH) inhibitor protects mice from lipopolysaccharide (LPS)-induced acute lung injury (ALI). The underlying mechanisms remain essential questions. In this study, we investigated the effects of EETs on the activation of nucleotide-binding domain leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) inflammasome in murine macrophages. In an LPS-induced ALI murine model, we found that sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl), TPPU, profoundly attenuated the pathological injury and inhibited the activation of the NLRP3 inflammasome, characterized by the reduction of the protein expression of NLRP3, ASC, pro-caspase-1, interleukin precursor (pro-IL-1β), and IL-1β p17 in the lungs of LPS-treated mice. In vitro, primary peritoneal macrophages from C57BL/6 were primed with LPS and activated with exogenous adenosine triphosphate (ATP). TPPU treatment remarkably reduced the expression of NLRP3 inflammasome-related molecules and blocked the activation of NLRP3 inflammasome. Importantly, four EETs (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) inhibited the activation of NLRP3 inflammasome induced by LPS + ATP or LPS + nigericin in macrophages in various degree. While the inhibitory effect of 5,6-EET was the weakest. Mechanismly, EETs profoundly decreased the content of reactive oxygen species (ROS) and restored the calcium overload in macrophages receiving LPS + ATP stimulation. In conclusion, this study suggests that EETs inhibit the activation of the NLRP3 inflammasome by suppressing calcium overload and ROS production in macrophages, contributing to the therapeutic potency to ALI.     

Effects of autonomic blockers on linear and nonlinear indexes of blood pressure and heart rate in SHR

Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC(50) = 6 x 10(-7) M), bradykinin (EC(50) = 1 x 10(-9) M), sodium nitroprusside (SNP; EC(50) = 2 x 10(-7) M), and bimakalim (BMK; EC(50) = 1 x 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1-15 x 10(-3) M) inhibited [(3)H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 x 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E(m)) of isolated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 x 10(-5) and 1 x 10(-4) M) decreased E(m) (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E(m) (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced increase in E(m). In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability (NP(o)) of a calcium-activated potassium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NP(o) but blocked the 14,15-EET-induced increase in NP(o). These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.     

Effects of endotoxin on neutrophil-mediated I/R injury in isolated perfused rat hearts

With the use of a syngeneic model, we demonstrate that rat polymorphonuclear neutrophils (PMNs) exacerbate ischemia-reperfusion injury in the isolated rat heart. However, PMNs (19 x 10(6) cells) from lipopolysaccharide (LPS)-treated rats (LPS-PMNs; 100 mg/kg administered 7 h before exsanguination) induce less reperfusion injury in the isolated heart. Average recovery of left ventricular developed pressure after 20 min of ischemia and 60 min of reperfusion was 51 +/- 4% in hearts receiving PMNs from saline-treated control rats (saline-PMNs) versus 78 +/- 2% in hearts receiving LPS-PMNs. Ischemic hearts reperfused with LPS-PMNs recovered to the same extent as did hearts reperfused with Krebs buffer only. LPS-PMNs and saline-PMNs showed no difference in basal or phorbol ester-induced superoxide production. Whereas twice the number of LPS-PMNs was positive for nitroblue tetrazolium, the percent positive for L-selectin, a receptor integral in PMN-adhesion to endothelium, was 50% less in LPS-PMNs than in controls. After reperfusion, three-fourths of the saline-PMNs remained within the hearts, whereas only one-fourth of LPS-PMNs were trapped. These data suggest that PMNs from LPS-treated rats do not exacerbate ischemia-reperfusion injury as do control PMNs, possibly, due to impaired PMN adhesion to endothelium as a result of decreased L-selectin receptors.     

11,12-epoxyeicosatrienoic acid stimulates heme-oxygenase-1 in endothelial cells

As epoxyeicosatrienoic acids (EETs), particularly 11,12-EET, and the heme oxygenase/carbon monoxide (HO/CO) system share overlapping biological activities, we examined a possible link between 11,12-EET and HO activity in endothelial cells. Confocal microscopy analysis of immunostaining of HO-1 and HO-2 in cultured endothelial cells treated with 11,12-EET (1 microM) showed an increase in florescence of HO-1 protein in the various cellular compartments, but not of HO-2. Incubation of endothelial cells with 11,12-EET (1 microM) for 24 h increased the level of HO-1 protein by about three-fold. Similarly, incubation of endothelial cells with 8,9-EET and sodium nitroprussiate, a known inducer of HO-1, increased HO-1 protein without any effect on HO-2. Upregulation of HO-1 by 11,12-EET, as well as 8,9-EET, was associated with an increase in HO activity, which was inhibited by stannous mesoporphirin (10 microM). Incubation of rat aortas with 11,12-EET (1 microM for 60 min) increased HO activity. These findings identify a novel effect of EETs on endothelial HO-1 and indicate that the signaling pathway of EETs in endothelial cells is possibly via an increase in HO-1 expression and activity.     

Novel regulatory mechanism of cardiomyocyte contractility involving ICAM-1 and the cytoskeleton

ICAM-1 mediates interaction of cardiomyocytes with the extracellular matrix and leukocytes and may play a role in altering contractility. To investigate this possibility, rat ventricular cardiomyocytes were activated using TNF-alpha, IL-1beta, or LPS, washed, cultured with quiescent rat polymorphonuclear leukocytes (PMNs) for 4 h, and electrically stimulated to determine fractional shortening. PMNs cultured with activated cardiomyocytes reduced control fractional shortening of 20.5 +/- 0.7% by -2.8 +/- 0.3% per adherent PMN (P < 0.001). Fixing PMNs with paraformaldehyde or glutaraldehyde did not prevent PMN-mediated decreases in cardiomyocyte fractional shortening. However, PMN adherence and decreased fractional shortening were prevented by anti-ICAM-1 and anti-CD18 antibodies. Reduced fractional shortening was reproduced in the absence of PMNs by ICAM-1 binding using cross-linking antibodies (reduced by 36 +/- 3% from control, P < 0.01). Immunofluorescent staining demonstrated increased cortical cytoskeleton-associated focal adhesion kinase expression after ICAM-1 cross-linking, suggesting involvement of the actin cytoskeleton. Indeed, disruption of F-actin filament assembly using cytochalasin D or latrunculin A did not prevent PMN adherence but prevented decreased fractional shortening. Inhibition of the cytoskeleton-associated Rho-kinase pathway with HA-1077 prevented ICAM-1-mediated decreases in cardiomyocyte contractility, further suggesting a central role of the actin cytoskeleton. Importantly, ICAM-1 cross-linking did not alter the total intracellular Ca2+ transient during cardiomyocyte contraction but greatly increased heterogeneity of intracellular Ca2+ release. Thus we have identified a novel regulatory mechanism of cardiomyocyte contractility involving the actin cytoskeleton as a central regulator of the normally highly coordinated pattern of sarcoplasmic Ca2+ release. Cardiomyocyte ICAM-1 binding, by PMNs or other ligands, induces decreased cardiomyocyte contractility via this pathway.     

The effects of platelet activating factor and retinoic acid on the expression of ELAM-1 and ICAM-1 and the functions of neutrophils

Preincubation of pulmonary microvascular endothelial cells (PMVECs) with platelet-activating factor (PAF) for 3.5 h increased the adhesion rate of polymorphonuclear leukocytes (PMNs) to PMVECs from 57.3% to 72.8% (p < 0.01). Preincubation of PMNs with PAF also increased PMN-PMVEC adhesion rate. All-trans retinoic acid (RA) blocked the adherence of untreated PMNs to PAF-pretreated PMVECs but not the adherence of PAF-pretreated PMNs to untreated PMVECs. PAF increased the expression of intercellular adhesion molecule-1 (ICAM-1) and E-selection (ELAM-1) on PMVECs, PMN chemotaxis to zymosan-activated serum and histamine, and PMN aggregation and the release of acid phosphatase from PMNs. Co-incubation of RA inhibited PAF-induced PMN aggregation, the release of acid phosphatase from PMNs, and PMN chemotaxis to zymosan-activated serum and histamine while the expression of ICAM-1 and ELAM-1 did not change. Our results suggest that RA can be used to ameliorate PMN-mediated inflammation.     

Epoxyeicosatrienoic acids constrict isolated pressurized rabbit pulmonary arteries

Little information is available regarding the vasoactive effects of epoxyeicosatrienoic acids (EETs) in the lung. We demonstrate that 5, 6-, 8,9-, 11,12-, and 14,15-EETs contract pressurized rabbit pulmonary arteries in a concentration-dependent manner. Constriction to 5,6-EET methyl ester or 14,15-EET is blocked by indomethacin or ibuprofen (10(-5) M), SQ-29548, endothelial denuding, or submaximal preconstriction with the thromboxane mimetic U-46619. Constriction of pulmonary artery rings to phenylephrine is blunted by treatment with the epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide. Pulmonary arteries and peripheral lung microsomes metabolize arachidonate to products that comigrate on reverse-phrase HPLC with authentic regioisomers of 5,6-, 8,9-, 11,12-, and 14,15-EETs, but no cyclooxygenase products of EETs could be demonstrated. Proteins of the CYP2B, CYP2E, CYP2J, CYP1A, and CYP2C subfamilies are present in pulmonary artery and peripheral lung microsomes. Constriction of isolated rabbit pulmonary arteries to EETs is nonregioselective and depends on intact endothelium and cyclooxygenase, consistent with the formation of a pressor prostanoid compound. These data raise the possibility that EETs may contribute to regulation of pulmonary vascular tone.     

8,9-Epoxyeicosatrienoic acid analog protects pulmonary artery smooth muscle cells from apoptosis via ROCK pathway

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid (AA) catalyzed by cytochrome P450 (CYP), have many essential biologic roles in the cardiovascular system including inhibition of apoptosis in cardiomyocytes. In the present study, we tested the potential of 8,9-EET and derivatives to protect pulmonary artery smooth muscle cells (PASMCs) from starvation induced apoptosis. We found 8,9-epoxy-eicos-11(Z)-enoic acid (8,9-EET analog (214)), but not 8,9-EET, increased cell viability, decreased activation of caspase-3 and caspase-9, and decreased TUNEL-positive cells or nuclear condensation induced by serum deprivation (SD) in PASMCs. These effects were reversed after blocking the Rho-kinase (ROCK) pathway with Y-27632 or HA-1077. Therefore, 8,9-EET analog (214) protects PASMC from serum deprivation-induced apoptosis, mediated at least in part via the ROCK pathway. Serum deprivation of PASMCs resulted in mitochondrial membrane depolarization, decreased expression of Bcl-2 and enhanced expression of Bax, all effects were reversed by 8,9-EET analog (214) in a ROCK dependent manner. Because 8,9-EET and not the 8,9-EET analog (214) protects pulmonary artery endothelial cells (PAECs), these observations suggest the potential to differentially promote apoptosis or survival with 8,9-EET or analogs in pulmonary arteries.     

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.