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.     

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.     

Gonadectomy prevents endothelial dysfunction in fructose-fed male rats, a factor contributing to the development of hypertension

Insulin resistance has been shown to be associated with increased blood pressure (BP). The sex hormones estrogen and testosterone have opposing effects in the development of increased BP. Since testosterone has been implicated in increased BP following insulin resistance, we have tried to dissect out the effects of insulin resistance on endothelium-dependent vasorelaxation in the presence and absence of testosterone. Both gonadectomized and sham-operated male Wistar rats fed with a high-fructose diet developed insulin resistance, but BP increased only in the sham-operated rats. Reintroduction of testosterone in vivo restored the increase in BP, thereby abolishing the protective effects of gonadectomy. Fructose feeding did not affect plasma testosterone levels. Insulin resistance induced endothelial dysfunction in the mesenteric arteries of sham-operated rats, which was prevented by gonadectomy, thus suggesting a key role for testosterone in the pathogenesis of secondary vascular complications. Subsequent to blocking the actions of endothelium-dependent hyperpolarizing factor (EDHF), relaxation to acetylcholine (ACh) was lower in sham-operated fructose-fed rats compared with other groups, suggesting the involvement of nitric oxide (NO) in vasorelaxation. Inhibition of NO synthesis nearly abolished the ACh-evoked relaxation in both fructose-fed groups, thus suggesting a testosterone-independent impairment of EDHF-mediated relaxation. The improvement in endothelial function following gonadectomy could be ascribed to a NO component, although plasma nitrite and nitrate levels were unchanged. In summary, testosterone is essential in vivo for the development of endothelial dysfunction and hypertension secondary to insulin resistance, suggesting a facilitatory role for testosterone in increasing BP in fructose-fed male rats.     

Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress

Tempol catalyzes the formation of H(2)O(2) from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H(2)O(2) by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H(2)O(2). Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-46619, or norepinephrine. During U-46619, tempol caused a transient dilation (22 +/- 2%), whereas NBT was ineffective (2 +/- 1%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K(+) channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 +/- 7%. Tempol, but not NBT, increased H(2)O(2) in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (184 +/- 20%) of mouse cremaster arterioles exposed to angiotensin II for 30 min, which was not seen with NBT (9 +/- 4%). The vasodilation to tempol was reduced by 68 +/- 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-46619 or angiotensin II is mediated in part via production of H(2)O(2) and is largely independent of the endothelium and potassium channels.     

Contribution of EDRF and EDHF to restoration of endothelial function following dietary restrictions in hypercholesterolemic rats

The mechanisms underlying the impairment of endothelium-mediated vasorelaxation induced by dietary hypercholesterolemia and the mechanisms of restoration of endothelial function following reintroduction of low cholesterol diet were evaluated. Feeding rats with high cholesterol diet induced hypercholesterolemia and high blood pressure. This was associated with reduced vasorelaxation in response to acetylcholine, isoproterenol, and adenosine. At the same time, exaggerated contractile responses to serotonin and phenylephrine were observed. Reintroduction of a normal diet to cholesterol fed rats resulted in significant normalization of blood pressure, serum lipid profile, relaxation and contractile responses. The contributions of endothelial derived relaxing factors (EDRF), endothelial derived contractile factors (EDCFs)/prostanoids, and endothelial derived hyperpoalrising factor (EDHF) to the vasorelaxation in each group of animals were assessed. EDCFs constricting activity was increased in both cholesterol fed groups as compared to the control group. EDRF and EDHF were found to be the primary factors involved in the regulation of endothelium-mediated responsiveness. In control animals, EDRF was responsible for 70-90% of relaxation, depending on the agonist used. In cholesterol fed animals, EDRF was significantly reduced while EDHF was maintained or enhanced showing that EDHF had a significant role in maintaining the endothelial responses. Importantly, the restoration of vasorelaxation following reintroduction of normal diet was mediated not only by improvement of EDRF-dependent relaxation, but also to a significant extent by a further increase in EDHF-mediated relaxation. Taken together, the data showed that EDRF was attenuated during hypercholesterolemia and dietary interventions with low fat content restored these responses. However, EDHF-mediated responses were not reduced by hypercholesterolemia and subsequently improved their function after application of low cholesterol diet. The results implicate EDHF-mediated relaxation is also an important mechanism for restoration of endothelial function upon application of dietary restrictions for reduction of serum cholesterol level.     

Impact of α-lipoic acid on liver peroxisome proliferator-activated receptor-α, vascular remodeling, and oxidative stress in insulin-resistant rats

This study sought to determine the impact of α-lipoic acid (LA) on superoxide anion (O(2)(?-)) production and peroxisome proliferator-activated receptor-α (PPARα) expression in liver tissue, plasma free fatty acids (FFA), and aortic remodeling in a rat model of insulin resistance. Sprague-Dawley rats (50-75 g) were given either tap water or a drinking solution containing 10% D-glucose for 14 weeks, combined with a diet with or without LA supplement. O(2)(?-) production was measured by lucigenin chemiluminescence, and PPAR-α expression by Western blotting. Cross-sectional area (CSA) of the aortic media and lumen and number of smooth muscle cells (SMC) were determined histologically. Glucose increased systolic blood pressure (SBP), plasma levels of glucose and insulin, and insulin resistance (HOMA index). All of these effects were attenuated by LA. Whereas glucose had no effect on liver PPAR-α protein level, it decreased plasma FFA. LA decreased the aortic and liver O(2)(?-) production, body weight, and plasma FFA levels in control and glucose-treated rats. Liver PPAR-α protein levels were increased by LA, and negatively correlated with plasma FFA. Medial CSA was reduced in all glucose-treated rats, and positively correlated with plasma FFA but not with SBP or aortic O(2)(?-) production. Glucose also reduced aortic lumen area, so that the media-to-lumen ratio remained unchanged. The ability of LA to lower plasma FFA appears to be mediated, in part, by increased hepatic PPAR-α expression, which may positively affect insulin resistance. Glucose-fed rats may serve as a unique model of aortic atrophic remodeling in hypertension and early metabolic syndrome.     

SDF-1α对胶质瘤细胞U87过氧化氢损伤的保护作用

目的：探讨基质细胞衍生因子1α（SDF-1α）对过氧化氢（H2O2）损伤人脑胶质瘤细胞U87的保护作用及机制。方法：双抗体夹心酶联免疫吸附试验（ELISA）检测胶质瘤细胞U87自分泌SDF-1α;细胞增殖实验研究外源SDF-1α对U87细胞增殖的影响;SDF-1α作用U87 12小时后,0.7 mM H2O2处理6小时,流式细胞术检测细胞凋亡率;蛋白质免疫印记实验（western blot）检测SDF-1α对U87细胞中蛋白激酶B（Akt）和细胞外信号调节激酶1/2（ERK1/2）磷酸化的影响。结果：胶质瘤细胞U87自身几乎不分泌SDF-1α,24小时内外源性SDF-1α对U87细胞增殖无明显影响;H2O2损伤后,SDF-1α预处理组细胞存活率高于对照组,凋亡率和死亡率低于对照组,差异具有统计学意义;Western blot显示SDF-1α处理能够诱导U87细胞Akt和ERK1/2的快速磷酸化。结论：SDF-1α能够提高H2O2损伤的U87细胞存活率,降低凋亡率和死亡率,其机制可能与磷脂酰肌醇3激酶（PI3K）-Akt和丝裂原活化蛋白激酶（MAPK）-ERK1/2通路的激活有关。     

Effects of diabetes and evening primrose oil treatment on responses of aorta,corpus cavernosum and mesenteric vasculature in rats

Diabetes causes endothelial dysfunction, with deleterious effects on nitric oxide (NO) mediated vasodilatation. However, in many vessels other local vasodilators such as endothelium-derived hyperpolarizing factor (EDHF), prostacyclin, epoxides or endocannabinoids are also important. Several of these factors may be derived from omega-6 essential fatty acids via arachidonate metabolism. Diabetes inhibits this pathway, a defect that may be bypassed by diets enriched with omega-6 gamma-linolenic acid-containing oils such as evening primrose oil (EPO). The aim was to examine the effects of preventive EPO treatment on endothelium-dependent and neurally mediated vasorelaxation. Diabetes was induced by streptozotocin in rats; duration was 8 weeks. Vascular responses were examined in vitro on thoracic aorta, corpus cavernosum and perfused mesenteric bed preparations. Diabetes caused 25% and 35% deficits, respectively, in aorta and corpus cavernosum NO-mediated endothelium-dependent relaxation to acetylcholine that were largely unaffected by EPO treatment. Moreover, a 44% reduction in maximum corpus cavernosum vasorelaxation to nitrergic nerve stimulation was not prevented by EPO. However, for the mesenteric vascular bed, a 29% diminution of responses to acetylcholine, mediated by both NO and EDHF, was 84% attenuated by EPO treatment. When the EDHF component was isolated during NO synthase inhibition, a 76% diabetic deficit was noted. This was completely prevented by EPO treatment, which also caused supernormal EDHF responses in nondiabetic rats. EPO treatment prevented the development of deficits in endothelium-dependent relaxation in diabetic rats. Effects were particularly marked on the resistance vessel EDHF system, which may have potential therapeutic relevance for diabetic microvascular complications.     

Hydrogen peroxide inhibits insulin signaling in vascular smooth muscle cells

Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H2O2 and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H2O2 on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser473. Pretreatment with H2O2 concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 microM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H2O2 inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 microM), for 30 min did not block the inhibitory effects of H2O2 on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H2O2. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.     

Endothelial dysfunction and arterial pressure regulation during early diabetes in mice: roles for nitric oxide and endothelium-derived hyperpolarizing factor

We determined whether nitric oxide (NO) counters the development of hypertension at the onset of diabetes in mice, whether this is dependent on endothelial NO synthase (eNOS), and whether non-NO endothelium-dependent vasodilator mechanisms are altered in diabetes in mice. Male mice were instrumented for chronic measurement of mean arterial pressure (MAP). In wild-type mice, MAP was greater after 5 wk of N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 mg x kg(-1) x day(-1) in drinking water; 97 +/- 3 mmHg) than after vehicle treatment (88 +/- 3 mmHg). MAP was also elevated in eNOS null mice (113 +/- 4 mmHg). Seven days after streptozotocin treatment (200 mg/kg iv) MAP was further increased in L-NAME-treated mice (108 +/- 5 mmHg) but not in vehicle-treated mice (88 +/- 3 mmHg) nor eNOS null mice (104 +/- 3 mmHg). In wild-type mice, maximal vasorelaxation of mesenteric arteries to acetylcholine was not altered by chronic L-NAME or induction of diabetes but was reduced by 42 +/- 6% in L-NAME-treated diabetic mice. Furthermore, the relative roles of NO and endothelium-derived hyperpolarizing factor (EDHF) in acetylcholine-induced vasorelaxation were altered; the EDHF component was enhanced by L-NAME and blunted by diabetes. These data suggest that NO protects against the development of hypertension during early-stage diabetes in mice, even in the absence of eNOS. Furthermore, in mesenteric arteries, diabetes is associated with reduced EDHF function, with an apparent compensatory increase in NO function. Thus, prior inhibition of NOS results in endothelial dysfunction in early diabetes, since the diabetes-induced reduction in EDHF function cannot be compensated by increases in NO production.     

Chronic and acute effects of different antihypertensive drugs on femoral artery relaxation of L-NAME hypertensive rats

We aimed to compare the effects of chronic and acute administration of structurally different antihypertensives, diuretics - indapamide and hydrochlorothiazide, ACE inhibitor - captopril and indapamide+captopril combination on endothelium-dependent relaxation of femoral artery isolated from nitric oxide (NO)-deficient rats. In the chronic experiment, femoral artery was isolated from Wistar rats receiving L-NAME (40 mg/kg/day) solely or with indapamide (1 mg/kg/day), hydrochlorothiazide (10 mg/kg/day), captopril (10 mg/kg/day), and indapamide+captopril combination for seven weeks. In the acute in vitro experiment, the incubation medium with femoral artery isolated from L-NAME-hypertensive rats was supplemented with investigated antihypertensives in the same concentration 10(-4) mol/l. Interestingly, chronic L-NAME treatment did not cause a reduction of vasorelaxation. Indapamide+captopril elevated relaxation above the control level and completely prevented blood pressure increase induced by L-NAME. Acute incubation with captopril only or indapamide+captopril improved relaxation of femoral artery isolated from L-NAME-hypertensive rats, while the incubation with all antihypertensives increased vasorelaxation of femoral artery isolated from control Wistar rats. In conclusion, NO might be involved in the indapamide- and hydrochlorothiazide-induced improvement of vasorelaxation, while different vasorelaxing factors (prostacyclin, EDHF) contribute to the captopril-induced improvement of vasorelaxation. During the chronic treatment additive and synergic effects of indapamide and captopril may contribute to the prevention of hypertension and increase of vasorelaxation.     

野百合碱诱导大鼠肺动脉高压及肺源性心脏病的病理生理特征

目的:探讨野百合碱诱发肺动脉高压及肺源性心脏病模型的建立机制。方法:雄性Wistar大鼠20只,随机分为两组(n=10):正常组,模型组。模型组大鼠腹腔一次性注射野百合碱50 mg/kg,对照组注射同剂量的溶媒,28 d后测定大鼠血流动力学参数,硝酸盐还原酶法测定血清和肺组织中一氧化氮的含量;放射免疫法测定血浆中内皮素、脑钠素和肺组织中肿瘤坏死因子、内皮素的含量。结果:与对照组比较,右心室压力上升、心率和平均动脉压下降,血液和肺组织中肿瘤坏死因子、一氧化氮、内皮素-1、脑钠素含量上升,具有统计学意义。结论:野百合碱通过诱发肺血管和组织炎性损伤,升高体内肿瘤坏死因子、一氧化氮、内皮素-1的含量,建立肺动脉高压及肺源性心脏病模型。     

Regulator of G protein signaling 2 deficiency causes endothelial dysfunction and impaired endothelium-derived hyperpolarizing factor-mediated relaxation by dysregulating Gi/o signaling

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for G(q/11)α and G(i/o)α subunits. RGS2 deficiency is linked to hypertension in mice and humans, although causative mechanisms are not understood. Because endothelial dysfunction and increased peripheral resistance are hallmarks of hypertension, determining whether RGS2 regulates microvascular reactivity may reveal mechanisms relevant to cardiovascular disease. Here we have determined the effects of systemic versus endothelium- or vascular smooth muscle-specific deletion of RGS2 on microvascular contraction and relaxation. Contraction and relaxation of mesenteric resistance arteries were analyzed in response to phenylephrine, sodium nitroprusside, or acetylcholine with or without inhibitors of nitric oxide (NO) synthase or K(+) channels that mediate endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation. The results showed that deleting RGS2 in vascular smooth muscle had minor effects. Systemic or endothelium-specific deletion of RGS2 strikingly inhibited acetylcholine-evoked relaxation. Endothelium-specific deletion of RGS2 had little effect on NO-dependent relaxation but markedly impaired EDHF-dependent relaxation. Acute, inducible deletion of RGS2 in endothelium did not affect blood pressure significantly. Impaired EDHF-mediated vasodilatation was rescued by blocking G(i/o)α activation with pertussis toxin. These findings indicated that systemic or endothelium-specific RGS2 deficiency causes endothelial dysfunction resulting in impaired EDHF-dependent vasodilatation. RGS2 deficiency enables endothelial G(i/o) activity to inhibit EDHF-dependent relaxation, whereas RGS2 sufficiency facilitates EDHF-evoked relaxation by squelching endothelial G(i/o) activity. Mutation or down-regulation of RGS2 in hypertension patients therefore may contribute to endothelial dysfunction and defective EDHF-dependent relaxation. Blunting G(i/o) signaling might improve endothelial function in such patients.     

Cell-free oxygen carriers: scientific foundations, clinical development, and new directions

The most significant hurdle to the development of a safe and effective hemoglobin-based oxygen carrier ("blood substitute") is generally thought to be its propensity to cause vasoconstriction in the microcirculation and hypertension. Two theories for this effect are currently being studied: in one, scavenging NO by hemoglobin reduces vasorelaxation; in the other, cell-free hemoglobin oversupplies O2 (a known vasoconstrictor) to vascular walls by facilitated diffusion. While both mechanisms might lead to reduction of local NO concentration, the important distinction between the two is that if the NO scavenging theory is correct, it greatly diminishes the prospects to develop any solution based on free hemoglobin. However, if the O2-oversupply theory is correct, modifications to the hemoglobin molecule can be envisioned that can prevent oversupply and reduce toxicity. This review summarizes the development of Hemospan, a novel modification of human hemoglobin whose design is based on the O2-oversupply theory. Because of its low P50 and increased molecular size, the release of O2 in resistance vessels (arterioles) by Hemospan is restricted, and vasoconstriction is greatly reduced.     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

Hydrogen peroxide enhances the expression of Giα proteins in aortic vascular smooth cells: role of growth factor receptor transactivation

Oxidative stress has been shown to increase the expression of G(i)α proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats. The present study was undertaken to examine if H(2)O(2), which induces oxidative stress, could also enhance the expression of G(i)α proteins in VSMC and to further explore the underlying signaling pathways responsible for this response. Treatment of VSMC with H(2)O(2) increased the expression of G(i)α proteins and not of G(s)α protein in a concentration- and time-dependent manner. A maximal increase of ～40-50% was observed at 100 μM and 1 h and was restored to control levels by AG1295 and AG1478, inhibitors of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R), respectively, and PD98059 and U126, inhibitors of extracellular signal-regulated kinase (ERK1/2), and wortmannin and AKT inhibitor VIII, inhibitors of PKB/AKT, respectively. In addition, H(2)O(2) also increased the phosphorylation of EGF-R, PDGF-R, ERK1/2, and AKT, which was attenuated by the respective inhibitors, whereas the inhibitors of EGF-R and PDGE-R also inhibited the enhanced phosphorylation of ERK1/2 and AKT. Furthermore, transfection of cells with short interfering RNA of EGF-R and PDGF-R restored the H(2)O(2)-induced enhanced expression of G(i)α proteins to control levels. The increased expression of G(i)α proteins was reflected in enhanced G(i) functions as demonstrated by enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by a low concentration of GTPγS, whereas G(s)α-mediated stimulations of AC were significantly decreased. Furthermore, H(2)O(2)-induced enhanced proliferation of VSMC was attenuated by dibutyryl-cAMP. These results suggest that H(2)O(2) increases the expression of G(i)α proteins in VSMC through the transactivation of EGF-R/PDGF-R and ERK1/2 and phosphatidylinositol-3 kinase signaling pathways.     

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently identified that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in mice. The present study was designed to examine whether this is also the case in humans. Bradykinin elicited endothelium-dependent relaxations and hyperpolarizations in the presence of indomethacin and N(omega)-nitro-l-arginine, which thus were attributed to EDHF, in human mesenteric arteries. The EDHF-mediated relaxations were significantly inhibited by catalase, an enzyme that specifically decomposes H(2)O(2), whereas catalase did not affect endothelium-independent hyperpolarizations to levcromakalim. Exogenous H(2)O(2) elicited relaxations and hyperpolarizations in endothelium-stripped arteries. Gap junction inhibitor 18alpha-glycyrrhetinic acid partially inhibited, whereas inhibitors of cytochrome P450 did not affect the EDHF-mediated relaxations. These results indicate that H(2)O(2) is also a primary EDHF in human mesenteric arteries with some contribution of gap junctions.     

Effect of the Na-K-2Cl cotransporter NKCC1 on systemic blood pressure and smooth muscle tone

Studies in rat aorta have shown that the Na-K-2Cl cotransporter NKCC1 is activated by vasoconstrictors and inhibited by nitrovasodilators, contributes to smooth muscle tone in vitro, and is upregulated in hypertension. To determine the role of NKCC1 in systemic vascular resistance and hypertension, blood pressure was measured in rats before and after inhibition of NKCC1 with bumetanide. Intravenous infusion of bumetanide sufficient to yield a free plasma concentration above the IC(50) for NKCC1 produced an immediate drop in blood pressure of 5.2% (P < 0.001). The reduction was not prevented when the renal arteries were clamped, indicating that it was not due to a renal effect of bumetanide. Bumetanide did not alter blood pressure in NKCC1-null mice, demonstrating that it was acting specifically through NKCC1. In third-order mesenteric arteries, bumetanide-inhibitable efflux of (86)Rb was acutely stimulated 133% by phenylephrine, and bumetanide reduced the contractile response to phenylephrine, indicating that NKCC1 influences tone in resistance vessels. The hypotensive effect of bumetanide was proportionately greater in rats made hypertensive by a 7-day infusion of norepinephrine (12.7%, P < 0.001 vs. normotensive rats) but much less so when hypertension was produced by a fixed aortic coarctation (8.0%), again consistent with an effect of bumetanide on resistance vessels rather than other determinants of blood pressure. We conclude that NKCC1 influences blood pressure through effects on smooth muscle tone in resistance vessels and that this effect is augmented in hypertension.