Acetylcholine causes endothelium-dependent contraction of mouse arteries

The goal of this study was to determine whether acetylcholine evokes endothelium-dependent contraction in mouse arteries and to define the mechanisms involved in regulating this response. Arterial rings isolated from wild-type (WT) and endothelial nitric oxide (NO) synthase knockout (eNOS(-/-)) mice were suspended for isometric tension recording. In abdominal aorta from WT mice contracted with phenylephrine, acetylcholine caused a relaxation that reversed at the concentration of 0.3-3 microM. After inhibition of NO synthase [with N(omega)-nitro-l-arginine methyl ester (l-NAME), 1 mM], acetylcholine (0.1-10 microM) caused contraction under basal conditions or during constriction to phenylephrine, which was abolished by endothelial denudation. This contraction was inhibited by the cyclooxygenase inhibitor indomethacin (1 muM) or by a thromboxane A(2) (TxA(2)) and/or prostaglandin H(2) receptor antagonist SQ-29548 (1 microM) and was associated with endothelium-dependent generation of the TxA(2) metabolite TxB(2.) Also, SQ-29548 (1 microM) abolished the reversal in relaxation evoked by 0.3-3 microM acetylcholine and subsequently enhanced the relaxation to the agonist. The magnitude of the endothelium-dependent contraction to acetylcholine (0.1-10 microM) was similar in aortas from WT mice treated in vitro with l-NAME and from eNOS(-/-) mice. In addition, we found that acetylcholine (10 microM) also caused endothelium-dependent contraction in carotid and femoral arteries of eNOS(-/-) mice. These results suggest that acetylcholine initiates two competing responses in mouse arteries: endothelium-dependent relaxation mediated predominantly by NO and endothelium-dependent contraction mediated most likely by TxA(2).     

Nitric oxide-mediated vasorelaxation by Rhizoma Ligustici wallichii in isolated rat thoracic aorta.

The vasorelaxant effect of Rhizoma Ligustici wallichii and its possible mechanism of action on the vasomotor tone of the rat thoracic aortic rings were examined in an organ bath. Chloroform extracts of Rhizoma Ligustici wallichii (Ch1LW) elicited a dose-dependent, transient, relaxing response in endothelium-intact rat aorta contracted with norepinephrine (NE). This relaxant effect was abolished by removal of the endothelium and also by pretreatment with nitric oxide synthase inhibitors. Neither a muscarinic receptor antagonist nor a cyclooxygenase inhibitor altered the Ch1LW-induced relaxation. Tetramethylpyrazine, derived from Rhizoma Ligustici wallichii as a potent vasodilating component, induced a complete relaxation in both endothelium-intact and denuded rat aortas contracted by NE, but nitric oxide synthase inhibitors did not affect the relaxation. Ch1LW-induced endothelium-dependent relaxation was mediated by nitric oxide released from the endothelium, and could be caused by component(s) other than tetramethylpyrazine.     

Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.     

Involvement of Na(+)/Ca(2+) exchanger in endothelial NO production and endothelium-dependent relaxation

Endothelial nitric oxide (NO) synthase (eNOS) is controlled by Ca(2+)/calmodulin and caveolin-1 in caveolae. It has been recently suggested that Na(+)/Ca(2+) exchanger (NCX), also expressed in endothelial caveolae, is involved in eNOS activation. To investigate the role played by NCX in NO synthesis, we assessed the effects of Na(+) loading (induced by monensin) on rat aortic rings and cultured porcine aortic endothelial cells. Effect of monensin was evaluated by endothelium-dependent relaxation of rat aortic rings in response to acetylcholine and by real-time measurement of NO release from cultured endothelial cells stimulated by A-23187 and bradykinin. Na(+) loading shifted the acetylcholine concentration-response curve to the left. These effects were prevented by pretreatment with the NCX inhibitors benzamil and KB-R7943. Monensin potentiated Ca(2+)-dependent NO release in cultured cells, whereas benzamil and KB-R7943 totally blocked Na(+) loading-induced NO release. These findings confirm the key role of NCX in reverse mode on Ca(2+)-dependent NO production and endothelium-dependent relaxation.     

Selected contribution: Effects of sex and ovariectomy on responses to platelets in porcine femoral veins.

Estrogen replacement increases risk of venous thrombosis. In this study, we determined responses in vitro to platelets and platelet products in veins from adult male and intact and ovariectomized female pigs. When contracted with prostaglandin F(2alpha), platelets (25,000 platelets/microl) caused relaxation in veins with endothelium. Higher numbers of platelets caused contraction in veins with and without endothelium. In veins without endothelium, contractions were greater in veins from male than in veins from female pigs, and contractions in intact female pig veins were greater than in ovariectomized females pig veins. Platelet products 5-hydroxytryptamine and thromboxane (analog U-46619) caused comparable contractions in all veins; contractions to prostacyclin were less in veins from intact female pigs. ADP caused comparable endothelium-dependent relaxations in all groups. These relaxations were increased by indomethacin in veins from intact males and females; with inhibition of nitric oxide, relaxations were comparable in all groups. These results suggest that venous responses to platelets vary with sex and presence of ovaries in female pigs. These variations reflect differences in type and quantity of substances released from platelets as well as the sensitivity of the smooth muscle to some vasoactive substances. In addition, products of cyclooxygenase may reduce endothelium-dependent relaxations in veins.     

The EGCg-induced redox-sensitive activation of endothelial nitric oxide synthase and relaxation are critically dependent on hydroxyl moieties

Several rich sources of polyphenols stimulate the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase (eNOS). The present study examined the molecular mechanism underlying the stimulatory effect of epicatechins on eNOS. NO-mediated relaxation was assessed using porcine coronary artery rings in the presence of indomethacin, and charybdotoxin plus apamin, inhibitors of cyclooxygenases and EDHF-mediated responses, respectively. The phosphorylation level of Akt and eNOS was assessed in cultured coronary artery endothelial cells by Western blot, and ROS formation using dihydroethidine. (−)-Epigallocatechin-3-O-gallate (EGCg) caused endothelium-dependent relaxations in coronary artery rings and the phosphorylation of Akt and eNOS in endothelial cells. These responses were inhibited by membrane-permeant analogues of superoxide dismutase and catalase, whereas native superoxide dismutase, catalase and inhibitors of major enzymatic sources of reactive oxygen species including NADPH oxidase, xanthine oxidase, cytochrome P450 and the mitochondrial respiration chain were without effect. The EGCg derivative with all hydroxyl functions methylated induced neither relaxations nor the intracellular formation of ROS, whereas both responses were observed when the hydroxyl functions on the gallate moiety were present. In conclusion, EGCg causes endothelium-dependent NO-mediated relaxations of coronary artery rings through the Akt-dependent activation of eNOS in endothelial cells. This response is initiated by the intracellular formation of superoxide anions and hydrogen peroxide, and is critically dependent on the gallate moiety and on the presence of hydroxyl functions possibly through intracellular auto-oxidation.     

Effects of recombinant eNOS gene expression on reactivity of small cerebral arteries

Resistance arteries are an important target for vascular gene therapy because they play a key role in the regulation of tissue blood flow. The present study was designed to determine the effects of recombinant endothelial (e) nitric oxide synthase (NOS) gene expression on vasomotor reactivity of small brain stem arteries (internal diameter, 253 +/- 2.5 microm). Arterial rings were exposed ex vivo to an adenoviral vector (10(9) and 10(10) plaque-forming units/ml) encoding eNOS gene or beta-galactosidase gene. Twenty-four hours after transduction, vascular function was examined by isometric force studies. Transgene expression was evident mainly in adventitia. In arteries with endothelium transduced with eNOS gene but not with control beta-galactosidase gene, relaxations to bradykinin and substance P were significantly augmented. Removal of endothelium abolished relaxations to bradykinin and substance P in control and beta-galactosidase arteries. However, in endothelium-denuded arteries transduced with recombinant eNOS, bradykinin and substance P caused relaxations that were abolished in the presence of the NOS inhibitor N(G)-nitro-L-arginine methyl ester. In control arteries, endothelium removal augmented relaxations to the nitric oxide donors sodium nitroprusside and diethylamine NONOate. This augmentation was absent in eNOS gene-transduced arteries without endothelium. Our results suggest that, in small brain stem arteries, expression of recombinant eNOS increases biosynthesis of nitric oxide. Adventitia of small arteries is a good target for expression of recombinant eNOS. Genetically engineered adventitial cells may serve as a substitute source of nitric oxide in cerebral arteries with dysfunctional endothelium.     

Inhibitory effect of valves on endothelium-dependent relaxations to calcium ionophore in canine saphenous vein

The present study was designed to evaluate endothelium-dependent relaxation to the calcium ionophore A-23187 in isolated canine saphenous veins. Isometric force recordings and cGMP measurements using isolated veins with and without valves were performed. During contractions to U-46619 (3 x 10(-7) M), endothelium-dependent relaxations to A-23187 (10(-9)-10(-6) M) were significantly reduced in rings with valves compared with rings without valves. Endothelial removal abolished A-23187-induced relaxation. Relaxations to forskolin (FK; 10(-8)-10(-5) M) and diethylaminodiazen-1-ium-1,2-dionate; DEA-NONOate, 10(-9)-10(-5) M) were identical in rings with and without valves. In rings without valves, a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M), and a cyclooxygenase inhibitor, indomethacin (10(-5) M), partially reduced A-23187-induced relaxation. However, in rings with valves, L-NAME had no effect, whereas indomethacin abolished the relaxation to A-23187. A selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 3x10(-6) M), had no effect on the relaxation to A-23187 in either group. In contrast, ODQ abolished the A-23187-induced increase in cGMP levels, suggesting that relaxation to nitric oxide released by A-23187 is independent of increases in cGMP. These results demonstrate that endothelium-dependent relaxation to A-23187 is reduced in regions of veins with valves compared with relaxation in the nonvalvular venous wall. Lower production of nitric oxide in endothelial cells of valvular segments appears to be a mechanism responsible for reduced reactivity to A-23187.     

Guanosine 5'-O-(3-thiotriphosphate) causes endothelium-dependent, pertussis toxin-sensitive relaxations in porcine coronary arteries.

To determine whether direct stimulation of endothelial G-proteins causes relaxations of the underlying vascular smooth muscle, the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and sodium fluoride were studied in porcine coronary arteries and endothelial cells. Isometric tension was measured in coronary rings contracted with prostaglandin F2 alpha. GTP gamma S (in the presence of saponin) and sodium fluoride (in the presence of AlCl3) relaxed rings with, but not those without endothelium. The responses were inhibited by nitro-L-arginine and pertussis toxin. In membrane fractions of coronary endothelial cells, GTP gamma S and sodium fluoride inhibited the ADP-ribosylation of G-proteins catalyzed with [32P]-NAD and pertussis toxin. These data suggest that direct stimulation of G-proteins in endothelial cells by GTP gamma S and sodium fluoride causes a pertussis toxin-sensitive relaxation which may be attributed to the release of nitric oxide.     

The role of nitric oxide in mediating nonadrenergic, noncholinergic relaxation in rat pulmonary artery.

Experiments were undertaken to investigate the existence of inhibitory nonadrenergic, noncholinergic (i-NANC) nerve activity by using in vitro functional and immunohistochemical techniques in rat main pulmonary arterial rings. Vessels precontracted with phenylephrine (3 microM) relaxed in response to electrical field stimulation (EFS) (50 V, 0.2 ms, 0.1-10 Hz for 5 s) in the presence of atropine (1 microM) and guanethidine (1 microM). Tetrodotoxin (0.3 microM) abolished this response, indicating that it is neuronal in origin. l-NAME (30 microM), methylene blue (10 microM), and removal of endothelium significantly reduced the EFS-induced relaxations. The inhibitory action of l-NAME was completely reversed by l-arginine (1 mM) but not by d-arginine (1 mM). Moreover l-arginine alone potentiated the magnitude of the relaxations elicited by EFS. On the other hand, immunohistochemical work clearly demonstrated the existence of neuronal nitric oxide synthase in the pulmonary artery vessel wall. All these results are consistent with the suggestion that nitric oxide is the likely mediator of this vasodilatation. However, the incomplete blockade of the responses by l-NAME gives evidence of an additional inhibitory NANC neurotransmitter(s) mediating the residual relaxation, which requires further experiments to clarify its nature.     

Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.     

双环醇减轻超氧阴离子诱导的大鼠胸主动脉舒张功能损伤

目的：探讨双环醇（bicyclol）对超氧阴离子（O2）诱导的血管舒张功能损伤的影响。方法：采用离体器官灌流技术,观察bicyclol对离体大鼠胸主动脉环张力的影响。采用焦酚（O2的供体）建立O2损伤模型,观察bicyclol预孵育对氧化应激损伤后血管内皮依赖性舒张功能的改善作用。结果：bicyclol（10-8~10-5mol/L）对由苯肾上腺素预收缩的内皮完整主动脉环产生舒张作用,该作用可被NO合酶抑制剂L-NAME和环氧化酶抑制剂吲哚美辛阻断。500μmol/L焦酚可引起乙酰胆碱诱导的主动脉环内皮依赖性舒张反应减弱,bicyclol（10-5mol/L）预孵育45 min可减轻焦酚的损伤作用。对于吲哚美辛处理的主动脉环,bicyclol（10-5mol/L）可抑制焦酚所致的血管舒张反应降低,但这一效应未见于L-NAME处理的主动脉环。结论：bicyclol具有内皮依赖性舒血管作用,并能对抗O2引起的血管舒张功能损伤,该作用通过NO途径介导。     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

Chronic exercise training improves ACh-induced vasorelaxation in pulmonary arteries of pigs.

We hypothesized that exercise training would lead to enhanced endothelium-dependent vasodilation in porcine pulmonary arteries. Pulmonary artery rings (2- to 3-mm OD) were obtained from female Yucatan miniature swine with surgically induced coronary artery occlusion (ameroid occluder). Exercise training was performed for 16 wk, and vasomotor responses were studied by using standard isometric techniques. Contractile responses to 80 mM KCl, isosmotic KCl (10-100 mM), and norepinephrine (10(-8) to 10(-4) M) did not differ between sedentary (Sed) and exercise-trained (Ex) pigs. Relaxation was assessed to endothelium-dependent and endothelium-independent vasodilators after norepinephrine contraction. Pulmonary arteries of Ex pigs exhibited greater maximal relaxation to ACh (61.9 +/- 3.5%) than did those of Sed pigs (52.3 +/- 3.9%; P < 0.05). Endothelium-independent relaxation to sodium nitroprusside did not differ. Inhibition of nitric oxide synthase significantly decreased acetylcholine-induced relaxation, with greater inhibition in arteries from Ex pigs (P < 0.05). Inhibition of cyclooxygenase enhanced relaxation to acetylcholine in arteries from Sed pigs. We conclude that exercise training enhances endothelium-dependent (ACh-mediated) vasorelaxation in pulmonary arteries by mechanisms of increased reliance on nitric oxide and reduced production of a prostanoid constrictor.     

EDHF mediates the relaxation of stretched canine femoral arteries to acetylcholine.

To test the hypothesis that mechanically stretched arteries relax to endothelium-derived vasodilators, we challenged endothelium-intact dog femoral artery rings stretched from 1 to 16 g total initial tension (active force and passive elastic) with 10(-6) M acetylcholine (ACh), an endothelium-dependent dilator. The relaxation to 10(-6) M sodium nitroprusside (SNP), an endothelium-independent dilator, increased with the total initial tension. The relaxation to ACh averaged approximately 65% of the relaxation to SNP at total initial tensions of 4 to 16 g. To determine the nature of the endothelial-derived products involved, we compared the ACh-induced relaxation of stretched rings (6.5 +/- 0.2 g total initial tension) with rings chemically contracted with phenylephrine (Phe, 10(-7) to 10(-5) M) (6.5 +/- 0.3 g total initial tension). ACh-induced relaxation was evaluated before and after the inhibition of the synthesis of eicosanoids [cyclooxygenase (10(-5) M indomethacin) and lipoxygenase (10(-5) M nordihydroguariaretic acid)] and nitric oxide [nitric oxide synthase (10(-5) M Nw-nitro-L-arginine)]. The contribution of endothelium-derived hyperpolarizing factor (EDHF) was identified by blocking calcium-activated potassium channels (10(-8) M iberiotoxin). SNP (10(-6) M) relaxed stretched rings by 1.7 +/- 0.1 g and chemically-activated rings by 4.8 +/- 0.2 g. ACh relaxed stretched rings to 73 +/- 3% of the SNP relaxation and this was only attenuated in the presence of iberiotoxin. ACh relaxed Phe-activated rings to 60 +/- 3% of the SNP relaxation. This relaxation was attenuated by inhibition of the synthesis of nitric oxide and (or) eicosanoids. Therefore, ACh relaxed stretched rings through the release of EDHF whereas the relaxation of chemically activated rings to ACh involved multiple endothelium-derived vasodilators.     

Effects of hsp90 binding inhibitors on sGC-mediated vascular relaxation

Vascular soluble guanylate cyclase (sGC) exists in multimeric complexes with endothelial nitric oxide (NO) synthase (eNOS) and heat shock protein 90 (hsp90). Whereas disruption of hsp90-eNOS complexes clearly attenuates eNOS-dependent vascular relaxation, the contribution of sGC-hsp90 complexes to eNOS- or NO donor-dependent relaxations remains unclear. Isolated rat thoracic aortic rings were preincubated with structurally diverse hsp90 binding inhibitors, radicicol (RA) or geldanamycin (GA), or vehicle for 0.5, 1, or 15 h. Preconstricted vessels were exposed to ACh, 8-bromo-cGMP (8-BrcGMP), forskolin, or one of three NO donors: nitroglycerin (NTG), sodium nitroprusside, or spermine NONOate (SNN). Both RA and GA inhibited endothelium-dependent relaxations dose dependently. Indomethacin or the antioxidant tiron did not affect the inhibition of ACh-induced relaxations by GA. Long-term (15 h) exposure to RA inhibited all NO donor-induced relaxations; however, GA inhibited SNN-induced relaxation only. The effects of GA and RA appeared to be selective because 15-h treatment with either agent did not affect forskolin-induced relaxations and only slightly decreased 8-BrcGMP-induced relaxations. Similarly to their effects on NO-donor-induced relaxation, 15-h exposure to RA, but not to GA, decreased hsp90-bound sGC protein expression and NTG-stimulated cGMP formation in aortic rings, whereas RA more than GA reduced SNN-stimulated cGMP formation. We conclude that RA, much more so than GA, selectively inhibits sGC-dependent relaxations of aortic rings by reducing sGC expression, disrupting sGC-hsp90 complex formation and decreasing cGMP formation. These studies suggest that hsp90 regulates both eNOS- and sGC-dependent relaxations.     

(±)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis

Praeruptorin A is a coumarin compound naturally occurring in the roots of Peucedanum praeruptorum Dunn., a commonly used traditional Chinese medicine for the treatment of certain respiratory diseases and hypertension. Although previous studies indicated the relaxant effects of (±)-praeruptorin A on tracheal and arterial preparations, little is known about the functional characteristics of the enantiomers. In the present study, the two enantiomers were successfully isolated and identified by using a preparative Daicel Chiralpak AD-H column, and their relaxant effects on aorta rings were observed and compared. (+)-Praeruptorin A showed more potent relaxation than (?)-praeruptorin A against KCl- and phenylephrine-induced contraction of rat isolated aortic rings with intact endothelium. Removal of the endothelium remarkably reduced the relaxant effect of (+)-praeruptorin A but not that of (?)-praeruptorin A. Pretreatment of aortic rings with N^ω-nitro-l-arginine methyl ester (l-NAME, an inhibitor of nitric oxide synthase) or methylene blue (MB, a soluble guanylyl cyclase inhibitor) resulted in similar changes of the relaxant effects of the two enantiomers to endothelium removal. Molecular docking studies also demonstrated that (+)-praeruptorin A was in more agreement to nitric oxide synthase pharmacophores than (?)-praeruptorin A. On the other hand, the two enantiomers of praeruptorin A could slightly attenuate the contraction of rat aortic rings induced by internal Ca²⁺ release from sarcoplasmic reticulum (SR). These findings indicated that (+)-praeruptorin A and (?)-praeruptorin A exerted distinct relaxant effects on isolated rat aorta rings, which might be mainly attributed to nitric oxide synthesis catalyzed by endothelial nitric oxide synthase.     

11,12,15-Trihydroxyeicosatrienoic acid mediates ACh-induced relaxations in rabbit aorta

Rabbit aortic endothelium metabolizes arachidonic acid (AA) by the 15-lipoxygenase pathway to vasodilatory eicosanoids, hydroxyepoxyeicosatrienoic acids (HEETAs), and trihydroxyeicosatrienoic acids (THETAs). The present study determined the chemical identity of the vasoactive THETA and investigated its role in ACh-induced relaxation in the rabbit aorta. AA caused endothelium-dependent, concentration-related relaxations of the rabbit aorta. Increasing the extracellular KCl concentration from 4.8 to 20 mM inhibited the relaxations to AA by approximately 60%, thereby implicating K+-channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from -39.6 +/- 2.7 to -56.1 +/- 3.4 mV. In rabbit aortic rings, [14C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its 14C-labeled products. Gas chromatography/mass spectrometry identified the metabolites as isomers of 11,12,15-THETA and 11,14,15-THETA. The 11,12,15-THETA relaxed and hyperpolarized the rabbit aorta, whereas 11,14,15-THETA had no vasoactive effect. The relaxations to 11,12,15-THETA were blocked by 20 mM KCl. In aortic rings pretreated with inhibitors of nitric oxide and prostaglandin synthesis, ACh caused a concentration-related relaxation that was completely blocked by 20 mM KCl. Pretreatment with the phospholipase A2 inhibitors mepacrine and 7,7-dimethyl-5,8-eicosadienoic acid, the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, nordihydroguaiaretic acid, and ebselen, or the hydroperoxide isomerase inhibitors miconazole and clotrimazole also blocked ACh-induced relaxations. ACh caused a threefold increase in THETA release. These studies indicate that AA is metabolized by endothelial cells to 11,12,15-THETA, which activates K+ channels to hyperpolarize the aortic smooth muscle membrane and induce relaxation. Additionally, this lipoxygenase pathway mediates the nonnitric oxide, nonprostaglandin relaxations to ACh in the rabbit aorta by acting as a source of an endothelium-derived hyperpolarizing factor.     

Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1

Aims

Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA:DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation.

Methods and Results

EPA:DHA 6∶1 and 9∶1 caused significantly greater endothelium-dependent relaxations in porcine coronary artery rings than EPA:DHA 3∶1, 1∶1, 1∶3, 1∶6, 1∶9, EPA and DHA alone, and EPA:DHA 6∶1 with a reduced EPA + DHA amount, which were inhibited by an eNOS inhibitor. Relaxations to EPA:DHA 6∶1 were insensitive to cyclooxygenase inhibition, and reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. EPA:DHA 6∶1 induced phosphorylation of Src, Akt, p38 MAPK, ERK, JNK and eNOS; these effects were inhibited by MnTMPyP. EPA:DHA 6∶1 induced the endothelial formation of ROS in coronary artery sections as assessed by dihydroethidium, and of superoxide anions and hydrogen peroxide in cultured endothelial cells as assessed by electron spin resonance with the spin probe CMH, and the Amplex Red based assay, respectively.

Conclusion

Omega-3 fatty acids cause endothelium-dependent NO-mediated relaxations in coronary artery rings, which are dependent on the EPA:DHA ratio and amount, and involve an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways to activate eNOS.     

Is hydrogen peroxide an EDHF in human radial arteries?

In human radial arteries, a nitric oxide/prostanoid-independent mechanism that has the pharmacological characteristics of an EDHF contributes to endothelium-dependent relaxation. H2O2 can act as an EDHF in some vascular beds. We examined the hypothesis that endogenously produced H2O2 mediated the nitric oxide/prostanoid-independent relaxation to carbachol in radial arteries obtained from patients undergoing coronary artery bypass surgery. Superoxide levels, measured by chemiluminescence, were similar in radial and internal mammary arteries, but immunohistochemical staining for Cu/Zn superoxide dismutase (SOD) was lower in endothelium from radial arteries. In organ chamber studies, neither addition of catalase nor addition of SOD to the bathing fluid modified nitric oxide/prostanoid-independent relaxations to carbachol in radial arteries. However, nitric oxide-dependent vasorelaxation was enhanced in the presence of SOD. Thus the nitric oxide/prostanoid-independent relaxation to carbachol is not due to H2O2 and, unlike nitric oxide-mediated vasorelaxation, is not attenuated by superoxide. Blood vessels showing EDHF-mediated relaxations resistant to oxidative stress may provide favorable outcomes in revascularization surgery.