Enhanced nitric oxide bioavailability in coronary arteries prevents the onset of heart failure in rats with myocardial infarction

AimThe endothelium, mainly via nitric oxide (NO) release, adjusts the coronary flow. Cardiac function is closely linked to blood flow; thus, we tested the hypothesis that NO modulation in coronary arteries could be differentially adjusted after myocardial infarction (MI) in the presence or absence of heart failure (HF).Methods and resultsFour weeks after coronary occlusion, the infarcted rats were subdivided into rats without (MI) or with HF signs according to haemodynamic parameters. The septal coronary arteries were subsequently used to perform functional and molecular experiments. Acetylcholine (ACh)-induced relaxation was decreased in the coronary arteries following HF, whereas it was enhanced in the arteries of the MI compared with those of SHAM-operated (SO) rats. The relaxation induced by the NO donor was similar among the groups. NO production, which was evaluated by 4,5-diaminofluorescein diacetate, was reduced in the coronary arteries of the HF group and increased in the arteries with MI after ACh-induced stimulation. HF coronary arteries exhibited oxidative stress, which was evaluated via ethidium bromide-positive nuclei, whereas it was decreased in MI. To evaluate the mechanisms involved in the enhanced ACh-induced relaxation in the arteries following MI, certain septal coronary arteries were pre-incubated with L-NAME (a nonselective NO synthase (NOS) inhibitor), 7-NI (a selective neuronal NOS (nNOS) inhibitor) or LY294002 (a PI3-kinase inhibitor). L-NAME and LY294002 reduced ACh-induced relaxation in the MI and SO rats; however, these effects were greater in the MI arteries. 7-NI reduced only the ACh-relaxation in MI. In addition, the eNOS, nNOS, Akt, and superoxide dismutase isoform protein expressions were greater in the coronary arteries of the MI than in those of the SO groups.ConclusionOur data suggested that endothelial function was closely related to cardiac function after coronary occlusion. The coronary arteries from the HF rats exhibited reduced NO bioavailability, whereas the MI rats exhibited increased NO bioavailability because of increased eNOS/nNOS/PI3-kinase/Akt pathway and a reduction in ROS generation. These results suggest that enhanced NO modulation can prevent the onset of HF.     

Vascular NAD(P)H oxidase mediates endothelial dysfunction in basilar arteries from Otsuka Long-Evans Tokushima Fatty (OLETF) rats

We examined the responses of basilar arteries taken from Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type 2 diabetes model. Both the nitric oxide (NO)-mediated relaxation and the cyclic 3',5'-guanosine monophosphate (cGMP) production elicited by acetylcholine (ACh) were much weaker in OLETF rats than in age-matched control Long Evans Tokushima Otsuka (LETO) rats. The contraction induced by an NO synthase (NOS) inhibitor [N(G)-nitro-L-arginine (L-NNA)] was weaker in the OLETF group. In that group, application of apocynin, an NAD(P)H oxidase inhibitor, normalized (i) ACh-induced relaxation, (ii) L-NNA-induced contraction, and (iii) ACh-induced cGMP production to the LETO levels. Superoxide anion production was greater in basilar arteries from OLETF rats than in those from LETO rats. The protein expression of gp91(phox), an NAD(P)H oxidase subunit, was upregulated in the OLETF arteries (versus LETO ones). These results suggest that the existence of endothelial dysfunction in basilar arteries in type 2 diabetes is related to increased oxidative stress mediated via NAD(P)H oxidase. Possibly, an impairment of NO-dependent relaxation responses and a basal impairment of NO signaling may be responsible for the increased risk of adverse cerebrovascular events in type 2 diabetes.     

Effect of chronic insulin treatment on NO production and endothelium-dependent relaxation in aortae from established STZ-induced diabetic rats

The hypothesis that the impaired endothelial function seen in streptozotocin (STZ)-induced diabetic rats may result from an increased nitric oxide (NO) metabolism was tested. Acetylcholine (ACh) increased the nitrite NO(2-) and nitrate (NO(3-)) levels in the perfusates from both control and diabetic aortic strips, although the level of NO(2-) was significantly lower in diabetic rats while the NO(3-) level was significantly higher. Both effects (decrease in NO(2-) and increase in NO(3-)) were ameliorated by chronic administration of insulin to diabetic rats but NOx (NO(2-) plus NO(3-)) was increased. The expression of endothelial nitric oxide synthase (eNOS) was significantly increased by chronic administration of insulin to diabetic rats. A decrease in NO(2-) and an increase in NO(3-) occurred following treatment of control aortae with hypoxanthine/xanthine oxidase. Incubating diabetic aortic strips with superoxide dismutase (SOD) normalized the production of both NO(2-) and NO(3-). Both the basal and the ACh-stimulated production of O(2)(-) were significantly higher in diabetic rats than in controls. These results demonstrate that the ACh-induced relaxation of aortic strips was significantly impaired in diabetic rats and that this impairment may be due to an abnormal oxidative metabolism of NO, rather than to a decrease in NOS mRNA and NO production.     

cGMP-dependent protein kinase mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo

cGMP and cGMP-dependent protein kinase type I (cGKI) mediate the dilation of large vessels in response to NO and acetylcholine (ACh). However, the physiological significance of the NO/cGMP/cGKI pathway in resistance vessels is controversial. Here, we analyzed NO- and ACh-induced dilations of arterioles in cGKI-deficient (cGKI-/-) or endothelial NO synthase-deficient (eNOS-/-) mice. Mean arterial pressure was similar in cGKI-/- and wild-type mice (105 mm Hg). Pressure drops in response to intracarotid bolus application of the NO donor sodium nitroprusside (SNP) were almost abolished in cGKI-/- mice, whereas ACh-induced pressure decreases remained intact in cGKI-/- and eNOS-/- mice. The direct observation of arterioles in the cremaster muscle by intravital microscopy showed impaired SNP-induced dilations in cGKI-/- mice (by 80%) and normal ACh-induced dilations in cGKI-/- and eNOS-/- mice. ACh-induced dilations in eNOS-/- mice were attenuated by iberiotoxin (by 50%), indicating that they were mediated in part by Ca2+-activated K+ channels, but not by inhibitors of cyclooxygenase or p450-monooxygenases. We conclude that cGMP and cGKI are the major effectors of NO to induce acute dilations of murine resistance vessels. However, the NO/cGMP/cGKI pathway is not essential for ACh-induced dilation of arterioles and for basal blood pressure regulation in mice.     

大黄素对糖尿病KKAy小鼠PI3-K信号转导通路的影响

目的 观察大黄素对2型糖尿病模型KKAy小鼠血糖、胰岛素水平及磷脂酰肌醇3-激酶(PI3-K)信号转导通路的影响.方法 随机血糖均≥13.9 mmol/L的SPF级KKAy小鼠16只,随机分为模型组和治疗组各8只,另选8只C57BL/6J小鼠为正常组.正常组和模型组灌服20 mL/(d·kg)无菌水,治疗组予50 mg/kg大黄素灌胃.8周后测定各组小鼠空腹血糖(FPG)、空腹胰岛素(HNS)并计算胰岛素敏感指数(ISI);用Western blot法测定三组小鼠骨骼肌、脂肪组织中胰岛素受体底物-1( IRS-1)、PI3-K水平及Akt丝氨酸(Ser)473磷酸化水平.结果 模型组小鼠较正常组FPG、FINS明显升高,ISI明显降低,而治疗组小鼠的FPG、FINS较模型组明显降低,ISI明显升高(P均＜0.05).模型组IRS-1、PL3 -K表达水平及胰岛素刺激后Akt Ser473磷酸化升高倍数低于正常组,治疗组IRS-1、PI3-K表达水平及Akt Ser473磷酸化升高倍数高于模型组(P均＜0.05).结论 大黄素灌胃可降低2型糖尿病模型KKAy小鼠血糖、胰岛素水平,并增强胰岛素敏感度,提高小鼠骨骼肌及脂肪组织中的IRS-1、PI3 -K水平及Akt Ser473磷酸化水平.     

Lovastatin maintains nitric oxide—but not EDHF-mediated endothelium-dependent relaxation in the hypercholesterolemic rabbit carotid artery

The endothelium contributes to the regulation of vascular tone by producing nitric oxide (NO) and the endothelium-derived hyperpolarising factor (EDHF). In hypercholesterolemia, endothelium-dependent relaxation is impaired but can be restored by treatment with lovastatin (LOVAS). We investigated the effects of LOVAS on NO and EDHF-mediated relaxation. Rabbits were fed 1% cholesterol diet for 4 weeks and 0.5% cholesterol for the following 12 weeks (CHOL-group). The LOVAS group additionally received 10 mg of lovastatin over the last 12-week period. Experiments were performed in carotid artery rings. Relaxant responses to acetylcholine (ACh) were recorded in the presence of indomethacin. Nitro-l-arginine (NOARG, 100 μM) and potassium chloride (KCl, 35 mM) were used to differentiate between NO- and EDHF-mediated relaxations. Cholesterol impaired ACh-induced relaxations and this effect was prevented by LOVAS (control 100±1%, CHOL 81±6%, LOVAS 98±1%). In the presence of NOARG, relaxations to ACh were not different between the LOVAS and CHOL groups (control 78±4%, CHOL 64±6%, LOVAS 64±5%). When KCl was used, ACh-induced relaxations were similar in the LOVAS and control group (control 75±5%, CHOL 49±6%, LOVAS 76±2%). In arteries treated with NOARG and KCl together, no relaxations were observed. Relaxations of arteries from the control group were not affected by 18 h preincubation with lovastatin (10μM). Lovastatin selectively maintains nitric oxide-mediated endothelium-dependent relaxation in hypercholesterolemic rabbit carotid arteries.     

Novel nitric oxide synthase--dependent mechanism of vasorelaxation in small arteries from hypertensive rats

To determine the mechanism(s) involved in vasorelaxation of small arteries from hypertensive rats, normotensive (NORM), angiotensin II-infused (ANG), high-salt (HS), ANG high-salt (ANG/HS), placebo, and deoxycorticosterone acetate-salt rats were studied. Third-order mesenteric arteries from ANG or ANG/HS displayed decreased sensitivity to acetylcholine (ACh)-induced vasorelaxation compared with NORM or HS, respectively. Maximal relaxations were comparable between groups. Blockade of Ca(2+)-activated K(+) channels had no effect on ANG versus blunting relaxation in NORM (log EC(50): -6.8+/-0.1 versus -7.2+/-0.1 mol/L). NO synthase (NOS) inhibition abolished ACh-mediated relaxation in small arteries from ANG, ANG/HS, and deoxycorticosterone acetate-salt versus blunting relaxation in NORM, HS, and placebo (% maximal relaxation: ANG: 2.7+/-1.8; ANG/HS: 7.2+/-3.2; NORM: 91+/-3.1; HS: 82.1+/-13.3; deoxycorticosterone acetate-salt: 35.2+/-17.7; placebo: 79.3+/-10.3), indicating that NOS is the primary vasorelaxation pathway in these arteries from hypertensive rats. We hypothesized that NO/cGMP signaling and NOS-dependent H(2)O(2) maintains vasorelaxation in small arteries from ANG. ACh increased NOS-dependent cGMP production, indicating that NO/cGMP signaling is present in small arteries from ANG (55.7+/-6.9 versus 30.5+/-5.1 pmol/mg), and ACh stimulated NOS-dependent H(2)O(2) production (ACh: 2.8+/-0.2 micromol/mg; N(omega)-nitro-l-arginine methyl ester hydrochloride+ACh: 1.8+/-0.1 micromol/mg) in small arteries from ANG. H(2)O(2) induced vasorelaxation and catalase blunted ACh-mediated vasorelaxation. In conclusion, Ca(2+)-activated K(+) channel-mediated relaxation is dysfunctional in small mesenteric arteries from hypertensive rats, and the NOS pathway compensates to maintain vasorelaxation in these arteries through NOS-mediated cGMP and H(2)O(2) production.     

促红细胞生成素抑制血管紧张素Ⅱ诱导的大鼠心脏成纤维细胞中转化生长因子β1和胶原的表达

目的 探讨促红细胞生成素(EPO)对血管紧张素Ⅱ(AngⅡ)诱导的心脏成纤维细胞(CF)中转化生长因子(TGF)-β1蛋白表达和胶原生成的影响,以及磷脂酰肌醇-3-激酶(PD-K)/Akt信号途径和一氧化氮合酶(NOS)在其中的作用.方法 应用胰酶和胶原酶双酶法分离培养新生大鼠CF细胞,应用EPO、Ang Ⅱ、PI3-K抑制剂LY294002、NOS抑制剂L-NAME等不同因素干预.ELISA法检测CF中Ⅰ型和Ⅲ型胶原的浓度.化学酶法检测CF培养液中的NO浓度以及NOS总的活性及其亚型的活性.Western blot检测Akt、p-Akt、内皮型一氧化氮合酶(eNOS)、iNOS和TGF-β1蛋白的表达.结果 EPO剂量依赖性的抑制Ang Ⅱ诱导的CF培养液中Ⅰ型和Ⅲ型胶原表达以及提高NO的浓度.10 U/ml的EPO对Ⅰ型和Ⅲ型胶原浓度的抑制分别达到了28%和46%,同时NO浓度则提高了154%.EPO也显著抑制了Ang Ⅱ促CF中TGF-β1蛋白的表达,同时Akt的磷酸化水平显著提高,并促进eNOS蛋白的表达.应用LY294002使eNOS蛋白表达水平明显下降,培养液中的NO浓度也随之下降.L-NAME不能降低eNOS蛋白表达,但抑制了NO的生成.EPO抑制Ang Ⅱ诱导的CF中TGF-β1蛋白的表达以及Ⅰ型和Ⅲ型胶原合成作用均能被二者阻断.结论 EPO可抑制Ang Ⅱ诱导的新生大鼠CF中TGF-β1的表达以及Ⅰ型和Ⅲ型胶原表达,可能是通过激活PI3-K/Akt信号途径促使CF中eNOS表达,从而促进NO的表达来实现.     

Phosphatidylinositol 3-kinase may mediate isoproterenol-induced vascular relaxation in part through nitric oxide production

Phosphatidylinositol 3-kinase (PI3-K) has been shown to mediate insulin and insulin-like growth factor-1 (IGF-1)-induced nitric oxide (NO) generation and, thus, vascular tone. A role for PI3-K in G-protein-coupled receptor signal transduction has also been reported. As beta2 -adrenergic vascular actions are partly dependent on NO, this study the role of PI3-K on in vitro isoproterenol (Iso)-induced endothelial cell (EC) nitric oxide synthase (NOS) activation and rat aortic vascular relaxation. Cell lysates of rat aortic EC (RAEC), exposed to Iso (10 micromol/L) for 5 minutes, were immunoprecipitated with an antiphosphotyrosine antibody prior to assay for Western blot for the p85-kd regulatory subunit of PI3-K. Endothelial NOS activity was determined by measuring nitrite production. Endothelium-intact aortic rings from male Wistar rats were preincubated with the PI3-K inhibitors, wortmannin (WT), or LY294002 (LY), precontracted with phenylepinephrine (PE), and relaxation to graded doses of Iso was measured. NO contribution to vascular relaxation was assessed by L-N(G)-nitroarginine methyl ester (L-NAME), a NOS inhibitor. Both Iso and IGF-1 induced an increase in p85 subunit phosphorylation as demonstrated by Western analysis, effects inhibited by preincubation with WT. Iso also enhanced association of p85 with the Triton X-100-insoluble fraction of RAEC, reflecting translocation of this enzyme to a cytoskeletal fraction. In addition, Iso as well as IGF-1 significantly increased eNOS activity measured by nitrite production. Both WT and LY markedly inhibited relaxation to Iso, while L-NAME nearly abolished this beta-adrenergic-mediated vasorelaxation. These data indicate that both Iso and IGF-1 activate the EC PI3-K pathway which mediates, in part, the release of NO and subsequent vasorelaxation in response to this beta-agonist Iso as well as to IGF-1.     

Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection

In the penis, nitric oxide (NO) can be formed by both neuronal NO synthase and endothelial NOS (eNOS). eNOS is activated by viscous drag/shear stress in blood vessels to produce NO continuously, a process mediated by the phosphatidylinositol 3-kinase (PI3kinase)/Akt pathway. Here we show that PI3-kinase/Akt physiologically mediates erection. Both electrical stimulation of the cavernous nerve and direct intracavernosal injection of the vasorelaxant drug papaverine cause rapid increases in phosphorylated (activated) Akt and eNOS. Phosphorylation is diminished by wortmannin and LY294002, inhibitors of PI3-kinase, the upstream activator of Akt. The two drugs also reduce erection. Penile erection elicited by papaverine is reduced profoundly in mice with targeted deletion of eNOS. Our findings support a model in which rapid, brief activation of neuronal NOS initiates the erectile process, whereas PI3-kinase/Akt-dependent phosphorylation and activation of eNOS leads to sustained NO production and maximal erection.     

体外研究胰岛素及磷脂酰肌醇3-激酶途径对一氧化氮生成的影响

目的探讨胰岛素及磷脂酰肌醇3-激酶(PI3-K)途径对NO生成的影响。方法检测胰岛素、葡萄糖以及PI3-K活性不可逆的抑制剂(Wortmannin)对培养的人脐静脉内皮细胞(HUVECs)PI3-K表达以及NO、超氧阴离子(O_2~-)产生和内皮型一氧化氮合酶(eNOS)活性的影响。实验分为对照组、10 mU/L胰岛素组、100 mU/L胰岛素组、甘露醇组、5 mmol/L葡萄糖+10 mU/L胰岛素组(5 mmol/L G1组)、25 mmol/L葡萄糖+100 mU/L胰岛素组(25 mmol/L G2组)、50 nmol/L Wortmannin组(50 nmol/L W组)、50 nmol/L Wortmannin+10 mU/L胰岛素组(50 nmol/L W1组)和50 nmol/L Wortmannin+100 mU/L胰岛素组(50 nmol/L W2组)。结果与对照组比较,不同浓度胰岛素组eNOS活性及NO水平显著升高(P<0.01);25 mmol/L G2组、50 nmol/L W组、50 nmol/LW1组和50 nmol/L W2组eNOS活性及NO水平均显著降低,O_2~-生成明显增加(P<0.01);与对照组比较,不同浓度胰岛组、50 nmol/L W组、50 nmol/L W1组和50 nmol/L W2组PI3-K蛋白表达显著升高(P<0.05,P<0.01)。结论 PI3-K信号途径对于促进NO产生、维持血管内皮细胞的正常功能具有重要作用,在高糖、高胰岛素状态下该条途径受损并由此引发内皮功能障碍。     

Endothelial dysfunction and infarct-size relate to impaired EDHF response in rat experimental chronic heart failure

BACKGROUND: The rat coronary ligation model of chronic heart failure has been extensively used to investigate its pathophysiology including the role of endothelial dysfunction. Inconsistent results have been obtained concerning the role of endothelial dilative mediators nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). AIMS: Our aim was to investigate involvement of NO and EDHF in aortic endothelial dysfunction in this model and the influence of individual infarct sizes. Furthermore, we investigated whether it is justified to regard rats that failed to develop large infarct sizes as SHAM controls. METHODS: We performed coronary ligations and SHAM operations and studied acetylcholine (ACh)-induced relaxations and underlying endothelial mediators in isolated aortic rings 12 weeks after infarction. By then, cardiac and hemodynamic parameters were deteriorated in animals with large myocardial infarctions (large-MI, 35+/-3%), but not those with small myocardial infarctions (small-MI, 5+/-2%). RESULTS: Large-MI showed decreased ACh-induced relaxation compared to SHAM due to decreased contribution of EDHF which was inversely correlated with individual infarct-size. Interestingly, small-MI showed significantly increased ACh-induced relaxation compared to SHAM due to increased NO contribution. CONCLUSIONS: Our results suggest that impaired aortic endothelial dilatory function in large-MI is mainly due to an impaired EDHF response and strongly depends on individual infarct-size. In addition, endothelium-dependent relaxation of small-MI rats differed from SHAM, indicating that both groups may not be pooled to serve as controls. These results emphasize the importance of infarct-size and choice of the control group, and may explain inconsistencies in previous studies.     

Protein kinase C activation increases endothelial nitric oxide release in mesenteric arteries from orchidectomized rats

The aim of the present study was to assess the effect of endogenous male sex hormones on endothelial nitric oxide synthase (eNOS) expression, release and function of the endothelial nitric oxide (NO), as well as to assess the regulatory action of protein kinase C (PKC) on acetylcholine (ACh)-induced endothelial NO release. For this purpose, superior mesenteric arteries from control and orchidectomized male Sprague-Dawley rats were used. eNOS expression and basal-and ACh-induced NO release were similar in arteries from both groups of rats. Orchidectomy decreased the vasodilator effect induced by ACh but did not alter that induced by sodium nitroprusside (SNP). The superoxide anion scavenger, superoxide dismutase (SOD), or the membrane-permeable mimetic of SOD, tempol, only enhanced ACh-induced relaxation in arteries from orchidectomized rats. ACh-induced TXA(2) formation was higher in arteries from orchidectomized than from control rats. Neither the PKC activator, phorbol 12,13-dibutyrate (PDBu), nor the non-selective PKC inhibitor, calphostin C, modified basal- or ACh-induced NO release in arteries from control rats. In arteries from orchidectomized rats, basal- and ACh-induced endothelial NO release were increased by PDBu but decreased by calphostin C. Both G?6976, a PKC inhibitor that is partially selective for conventional PKC isoforms, as well as PKCzeta pseudosubstrate inhibitor (PKCzeta-PI) decreased both basal- and ACh-induced NO release in arteries from orchidectomized rats. Neither PDBu nor calphostin C modified the vasodilator response induced by ACh in arteries from control rats. In segments from orchidectomized rats, PDBu enhanced the ACh-induced response, but this response was not modified by calphostin C, G?6976 or PKCzeta-PI. The vasodilator response induced by SNP was not altered by the PKC activators or inhibitors in any artery from either group. These results show that endogenous male sex hormone deprivation does not affect the eNOS expression or the endothelial NO release induced by ACh, but does decrease the vasodilator action of ACh, by increasing NO metabolism and TXA(2) formation. In addition, PKC seems to modulate eNOS activity only in mesenteric arteries from orchidectomized rats, in which conventional and PKCzeta isoforms are involved in the positive regulation of eNOS.     

磷酸二酯酶抑制剂对乳鼠心肌细胞PI3K—Akt—eNOS信号通路的影响

目的：探讨西洛他唑（Cilostazol）对乳鼠心肌细胞PI3K-Akt-eNOS信号通路的影响。方法：观察西洛他唑对乳鼠心肌细胞NO影响的时效和量效关系，再用PI3K及eNOS抑制剂进行干预。检测NO的浓度，Western免疫印迹法检测总Akt、磷酸化Akt（p—Akt—ser473）及总eNOS、磷酸化eNOS（p-eNOS-Ser1177）表达水平。结果：西洛他唑升高心肌细胞NO浓度呈剂量和时间依赖性，不同浓度的西洛他唑均能升高Akt和eNOS磷酸化水平，但对Akt及eNOS总蛋白表达无明显影响。eNOS抑制剂L—NAME和P13K抑制剂Wortmannin均能抑制西洛他唑诱导的NO浓度升高，Wortmannin还能阻断西洛他唑诱导的Akt和eNOS的磷酸化。结论：西洛他唑可能激活乳鼠心肌细胞的PI3K—Akt—eNOS信号通路而促进NO的产生。     

Exercise training does not alter acetylcholine-induced responses in isolated pulmonary artery from rat.

In chronic exercise-trained animals, acetylcholine (ACh)-stimulated endothelial nitric oxide (NO) release is enhanced in the systemic circulation. The purpose of the present study was to determine whether chronic exercise training also enhances NO-mediated relaxation in rat pulmonary artery. Sprague-Dawley rats were randomly divided into groups of exercise-trained and sedentary control rats. The exercise-trained rats ran on a motor-driven treadmill at 30 m x min(-1) up a 15 degree incline 10-60 min x day(-1), 5 days per week for 10 weeks, and had less body weight, lower serum total cholesterol and triglyceride levels than sedentary rats. Contraction induced by potassium chloride and prostaglandin (PG)F2alpha were similar between isolated conduit pulmonary arterial rings from sedentary and exercise-trained rats. There were no differences between PGF2alpha-precontracted rings from sedentary and exercise trained rats in both ACh and sodium nitroprusside-induced relaxations. The NO synthase inhibitor, nitro-L-arginine, suppressed ACh-induced relaxation in both sedentary and exercise-trained rats. These results suggested chronic exercise training did not alter the acetylcholine-induced endothelial NO production and release and the sensitivity of vascular smooth muscle cell to NO in isolated conduit pulmonary artery of rat.     

Localizing extracellular signal–regulated kinase (ERK) in pharmacological preconditioning's trigger pathway

Acetylcholine (ACh) and opioid receptor agonists trigger the preconditioned phenotype through sequential activation of the epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3-K), Akt, and nitric oxide synthase (NOS), and opening of mitochondrial (mito) K(ATP) channels with the generation of reactive oxygen species (ROS). Although extracellular signal-regulated kinase (ERK) has recently been reported to be part of this pathway, its location has not been determined. To address this issue, we administered a 5-min pulse of ACh (550 microM) prior to 30 min of ischemia in isolated rabbit hearts. It reduced infarction from 30.4 +/- 2.2% of the risk zone in control hearts to 12.3 +/- 2.8% and co-administration of the MEK, and, therefore, downstream ERK inhibitor U0126 abolished protection (29.1 +/- 4.6% infarction) con.rming ERK's involvement. MitoK(ATP) opening was monitored in adult rabbit cardiomyocytes by measuring ROS production with MitoTracker Red. ROS production was increased by each of three G protein-coupled agonists: ACh (250 microM), bradykinin (BK) (500 nM), and the delta-opioid agonist DADLE (20 nM). Co-incubation with the MEK inhibitors U0126 (500 nM) or PD 98059 (10 microM) blocked the increased ROS production seen with all three agonists. Direct activation of its receptor by EGF increased ROS production and PD 98059 blocked that increase, thus placing ERK downstream of the EGF receptor. Desferoxamine (DFO) which opens mitoK(ATP) through direct activation of NOS also increased ROS. PD 98059 could not block DFO-induced ROS production, placing ERK upstream of NOS. In isolated hearts, ACh caused phosphorylation of both Akt and ERK. U0126 blocked phosphorylation of ERK but not of Akt. The PI3-K inhibitor wortmannin blocked both. Together these data indicate that ERK is located between Akt and NOS.     

Heterogeneity of endothelium-dependent vasorelaxation in conductance and resistance arteries from Lyon normotensive and hypertensive rats

OBJECTIVES: The nature of endothelial factors in response to acetylcholine (ACh) was investigated in conductance and resistance arteries from Lyon normotensive (LN) and Lyon hypertensive (LH) rats. Differences in endothelial function between the two strains were evaluated. METHODS AND DESIGN: Relaxations to ACh were studied in the aorta and small mesenteric arteries (SMA). The relative contribution of nitric oxide (NO), prostanoids and endothelial-derived hyperpolarizing factor (EDHF) was assessed using appropriate inhibitors. Western blot of endothelial NO synthase was achieved. The membrane potential of smooth muscle cells was assessed using microelectrodes. RESULTS: In LN rats, endothelium-dependent relaxation to ACh involved exclusively NO in the aorta, whereas both NO and EDHF were implicated in SMA. In the latter, relaxation was almost entirely prevented by blockade of either the NO or EDHF pathway, although ACh was still able to produce hyperpolarization in the presence of NO synthase and cyclooxygenase inhibitors. In LH rats, relaxation to ACh was unchanged in SMA but moderately depressed in the aorta, despite unchanged endothelial NO synthase protein expression and sensitivity to NO. In addition, indomethacin, but not a selective cyclooxygenase-2 inhibitor, significantly reduced ACh relaxations in the aorta from LH rats but not from LN rats. CONCLUSIONS: These results document differential endothelial function in a conductance and in resistance arteries from LN rats and LH rats. They show that simultaneous participation of NO and EDHF is required to promote relaxation in SMA from both strains, whereas NO alone accounts for relaxation in aorta from LN rats. In LH rats, aortic relaxation induced by ACh is slightly decreased despite the involvement of vasodilator products from cyclooxygenase-1.     

Melatonin restores endothelium-dependent relaxation in aortic rings of pancreatectomized rats

In rats turned hyperglycemic by a subtotal pancreatectomy, a decreased relaxation response of aortic rings to acetylcholine (ACh) was found; this effect was amplified by preincubation in a high glucose medium (44 mmol/L). The relaxation response to ACh did not occur in endothelium-denuded rings or after the aortic rings were exposed to l-nitro-arginine methyl ester [L-NAME, a nitric oxide (NO) synthase inhibitor]. Incubation with the NO donor sodium nitroprusside (SNP) restored the impaired relaxation response seen in endothelium-denuded or L-NAME-treated aortic rings. Pancreatectomy decreased the vasorelaxation of aortic rings caused by SNP. Only in pancreatectomized rats, incubation in a high glucose medium impaired the relaxation effect of SNP. To assess whether melatonin preincubation reversed the impaired relaxation response to ACh (intact endothelium aortic rings) or to SNP (endothelium-denuded or L-NAME-treated rings) in hyperglycemic rats, cumulative dose-response curves were performed in the presence of 10(-5) mol/L melatonin. Melatonin preincubation did not modify ACh-induced relaxation of aortic rings in a normal glucose concentration but was highly effective in preventing the impairment of relaxation caused by a high glucose solution. Melatonin was also effective in restoring the impaired SNP-induced vasorelaxation seen in endothelium-denuded or L-NAME-treated aortic rings from hyperglycemic rats. The results further support the improvement by melatonin of the endothelial-mediated relaxation in blood vessels of diabetic rats.     

DIDS对十字孢碱诱导心肌细胞凋亡中磷脂酰肌醇3激酶/蛋白激酶B信号转导的影响

目的探讨氯离子通道阻断剂DIDS对十字孢碱诱导心肌细胞凋亡与磷脂酰肌醇3激酶/蛋白激酶B信号及其下游分子一氧化氮合酶/一氧化氮的关系。方法实验分为对照组、十字孢碱组、DIDS组、LY294002(特异性磷脂酰肌醇3激酶抑制剂)组和L-NAME(非特异性一氧化氮合酶抑制剂)组。在十字孢碱诱导心肌细胞凋亡模型上,观察DIDS对心肌细胞存活率、凋亡和磷脂酰肌醇3激酶/蛋白激酶B及其下游分子一氧化氮合酶/一氧化氮的影响。结果与十字孢碱组比,DIDS明显改善了细胞存活率,提高了细胞磷酸化蛋白激酶B活性2.1倍(P<0.01),增加了一氧化氮合酶和磷酸化一氧化氮合酶的水平和一氧化氮水平(P<0.01);LY294002预处理完全抑制了磷酸化蛋白激酶B、一氧化氮合酶和磷酸化一氧化氮合酶水平的升高及升高的一氧化氮,完全阻断了DIDS的抗细胞凋亡作用;L-NAME预处理也使升高的一氧化氮水平下降,但仅部分阻断了DIDS的细胞保护作用。结论DIDS通过激活磷脂酰肌醇3激酶/蛋白激酶B信号通路发挥其抑制十字孢碱诱导的心肌细胞凋亡作用。