NADPH氧化酶对自发性高血压大鼠体内氧化应激的影响

目的考察NADPH氧化酶对自发性高血压大鼠体内氧化应激的影响。方法22wk龄自发性高血压大鼠(SHR)和正常血压WKY大鼠,采用尾套法测定血压,Greiss反应测定血清一氧化氮分泌量,ABTS和FRAP法进行血清总抗氧化能力测定,血管环舒缩测定来评价超氧阴离子清除剂超氧化物歧化酶(SOD)和NADPH氧化酶抑制剂夹竹桃麻素(Apo)对大鼠腹主动脉内皮依赖性舒张反应;采用RT-PCR考察内皮型一氧化氮合酶(eNOS)、NADPH氧化酶亚基p22phox以及NADPH氧化酶亚基gp91phox类似物nox4mRNA表达。结果与WKY大鼠相比,SHR血压升高,而血清总抗氧化水平及NO分泌量均降低。PCR显示SHR胸主动脉中eNOS及p22phoxmRNA表达与WKY大鼠相比差异无显著性,而nox4表达则升高。SHR腹主动脉内皮依赖性舒张反应与WKY相比降低,SOD或Apo均能明显逆转该变化。结论结果提示SHR体内氧化应激状态与NADPH氧化酶gp91phox类似物nox4mRNA过表达有关;NADPH氧化酶依赖性的氧化应激参与了SHR内皮功能障碍的发生发展;药理调节NADPH氧化酶功能或应用抗氧化治疗可明显改善SHR内皮依赖性舒张反应。     

Mitogen-activated protein kinase contributes to elevated basal tone in aortic smooth muscle from hypertensive rats

The role of mitogen-activated protein kinase (MAPK) in increased basal tone -spontaneous resistance in vascular muscle strips- was clarified in aortic smooth muscle from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The MAPK/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, PD098059 (2'-amino-3'-methoxyflavone), significantly inhibited basal tone in a dose-dependent manner. The basal level of ERK1/2 activation was inhibited by PD098059 and was significantly greater in hypertensive rats than in sham-operated rats. In contrast, inhibition with PD098059 was not observed in sham-operated rats. GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide), an inhibitor of protein kinase C (PKC), decreased both basal tone and ERK1/2 activity in the hypertensive rats. In contrast, Y27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide) and verapamil, inhibitors of Rho kinase and voltage-dependent Ca2+ channels, respectively, significantly inhibited basal tone but not ERK1/2 activity. Thus, basal vascular tone is elevated by the altered activation of MAPK in DOCA-salt hypertensive rats, and this is regulated by PKC, but not by Rho or intracellular Ca2+.     

Study of in vivo and in vitro resting vasodilator nitric oxide tone in normotensive and genetically hypertensive rats

The effects of N^G-nitro- -arginine ( -NNA) on mean arterial pressure and the effects of both -NNA and methylene blue on isolated aorta tone, were studied in order to elucidate potential alterations in vasodilator resting nitric oxide (NO) tone in genetic hypertension. -NNA produced a significantly greater increase of mean arterial pressure in spontaneously hypertensive rats (SHR) than in Wistar Kyoto (WKY) rats; in both cases, -arginine completely inhibited the -NNA hypertensive effect. Neither ganglion blockade with hexamethonium nor cyclooxygenase inhibition with indomethacin significantly modified the effect of -NNA in both rat strains. In intact aorta rings, after submaximally contraction with KCl (25 mM), both -NNA and methylene blue induced strong dose-dependent contractions. The maximum contractions were, however, significantly greater in WKY rats than in SHR. The mechanical elimination of endothelium markedly inhibited both -NNA and methylene blue maximum contractions. In intact rings, -arginine completely inhibited the -NNA effects in both rat strains; in rubbed rings, the -arginine inhibitory effects were strong in WKY rats but not important and erratic in SHR. -Arginine had no effect on the contractions induced only by KCl in any of the preparations. In WKY rat-rubbed rings, sodium nitroprusside was significantly more effective in relaxing the contractions in response to 25 mM KCl than the contractions in response to methylene blue. These results indicate that contractions induced by -NNA and methylene blue in isolated aorta are principally due to the inhibition of an important endothelial resting vasodilator NO tone. They also show that hypertension reduces the resting vasodilator NO tone in isolated rat aorta, in spite of enhancing the total vasodilator NO tone in anaesthetized rat.     

Increased hypothalamic noradrenergic activity in one-kidney, one-clip renovascular hypertensive rats

To further define central and peripheral sympathetic nerve activity in the one-kidney and two-kidney, one-clip models of renovascular hypertension, plasma catecholamines and regional brain norepinephrine of these models were compared with the activities of their brain biosynthetic enzymes: tyrosine hydroxylase (TYH) and dopamine-beta-hydroxylase (DBH). Findings in the groups of 20 one-kidney and 17 two-kidney female Wistar rats were compared with those in 10 sham-operated rats. Systolic blood pressure, measured indirectly, and the mean arterial pressure, measured directly from the femoral arteries, verified development of renovascular hypertension in both the one and two-kidney animals. Plasma norepinephrine increased from 248 +/- 46 to 401 +/- 66 pg/ml in the one-kidney group only (p < 0.001). Hypothalamic TYH and DBH activities of the one-kidney animals were 48 and 34% greater than those of the two-kidney animals and 28 and 39% greater than the sham-operated animals. The multiple t-test indicated significant differences between the mean hypothalamic THY of the one- and two-kidney groups and between the hypothalamic DBH of the one-kidney animals and those of the two-kidney and sham groups (alpha = 0.05). Moreover, the mean norepinephrine content of the hypothalamus in the one-kidney animals was 66% greater than that of the two-kidney and sham groups (p < 0.05). These findings suggest that central noradrenergic pathways of the hypothalamus may be involved in the genesis of one-kidney renovascular hypertension.     

NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats

Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC₅₀ = 0.057 μM. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in ‘Production of reactive oxygen species’ (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death.     

Role of oxidative stress and nitric oxide in regulation of spontaneous tone in aorta of DOCA-salt hypertensive rats

1. The roles of nitric oxide (NO), superoxide anion (O(2)(-)), and hydrogen peroxide (H(2)O(2)) in the modulation of spontaneous tone were investigated in isolated aorta from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. 2. Increases in preload from 1 to 5 g were accompanied by increases in spontaneous tone in aortic rings from DOCA-salt hypertensive rats but not from SHAM-normotensive rats. 3. Tone was higher in endothelium-denuded aortic rings than in endothelium-intact vessels. Inhibition of nitric oxide synthase (NOS) with 300 microM N(G)-nitro-L-arginine methyl ester (l-NAME) increased spontaneous tone. 4. Basal O(2)(-) generation was higher in aortic rings from DOCA-salt hypertensive rats than in those from SHAM-normotensive rats. Stretch increased O(2)(-) levels even further in the DOCA-salt group. In rings isolated from DOCA-salt hypertensive rats, administration of the O(2)(-) scavenger, superoxide dismutase (SOD, 150 U ml(-1)), or the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase inhibitor, apocynin (100 microM), completely abolished the development of spontaneous tone in endothelium-intact aortic rings but not in endothelium-denuded or in L-NAME-treated rings. SOD and apocynin decreased the generation of O(2)(-) in endothelium-intact, endothelium-denuded, and L-NAME-treated aortic rings. 5. Oral treatment of DOCA-salt hypertensive rats with the O(2)(-) scavengers, tempol or tiron, or with apocynin for 3 weeks prevented the development of hypertension and abolished the increases in O(2)(-) generation and spontaneous tone. 6. Administration of catalase (1000 U ml(-1)) to aortic rings increased spontaneous tone in vessels from DOCA-salt hypertensive rats. 7. Administration of the cyclooxygenase (COX) inhibitor, valeroyl salicylate, or the thromboxane/prostaglandin antagonist, SQ 29548, to aortic rings abolished tone. 8. The results suggest that NO plays a major role in preventing the generation of spontaneous tone in isolated aortic rings from DOCA-salt hypertensive rats. NADPH-oxidase-derived O(2)(-) enhanced spontaneous tone by inactivating NO. Endogenous H(2)O(2) appears to mitigate the increase in tone. In addition, a COX component may also contribute to spontaneous tone.     

Sesamin ameliorates arterial dysfunction in spontaneously hypertensive rats via downregulation of NADPH oxidase subunits and upregulation of eNOS expression

Aim:

Sesamin is one of the major lignans in sesame seeds with antihyperlipidemic, antioxidative and antihypertensive activities. The aim of this study was to examine the effects of sesamin on arterial function in spontaneously hypertensive rats (SHRs).

Methods:

SHRs were orally administered sesamin (40, 80 and 160 mg·kg⁻¹·d⁻¹) for 16 weeks. After the rats were killed, thoracic aortas were dissected out. The vasorelaxation responses of aortic rings to ACh and nitroprusside were measured. The expression of eNOS and NADPH oxidase subunits p47^phox and p22^phox in aortas were detected using Western blotting and immunohistochemistry. Aortic nitrotyrosine was measured with ELISA. The total antioxidant capacity (T-AOC) and MDA levels in aortas were also determined.

Results:

The aortic rings of SHRs showed significantly smaller ACh-induced and nitroprusside-induced relaxation than those of control rats. Treatment of SHRs with sesamin increased both the endothelium-dependent and endothelium-independent relaxation of aortic rings in a dose-dependent manner. In aortas of SHRs, the level of T-AOC and the expression of nitrotyrosine, p22^phox and p47^phox proteins were markedly increased, while the level of MDA and the expression of eNOS protein were significantly decreased. Treatment of SHRs with sesamin dose-dependently reversed these biochemical and molecular abnormalities in aortas.

Conclusion:

Long-term treatment with sesamin improves arterial function in SHR through the upregulation of eNOS expression and downregulation of p22^phox and p47^phox expression.     

Increased renal neuroleptic binding in spontaneously hypertensive rats

The present experiments demonstrate high affinity stereospecific binding of the dopaminergic ligand [3H]spiroperidol to rat renal membranes. The binding was saturable, regionally distributed, and showed the same specificity as the caudate [3H]spiroperidol site for displacement by dopaminergic antagonists. Since there is an association between hypertension and abnormalities of renal blood flow, which appears to be regulated in part by dopamine, [3H]spiroperidol binding was measured in kidneys of spontaneously hypertensive rats. In two independent experiments renal cortical [3H]spiroperidol stereospecific binding was increased in spontaneously hypertensive rats.     

Cardiovascular effects of prazosin in normotensive and genetically hypertensive rats

1. The cardiovascular effects of prazosin, a new antihypertensive drug, were studied in normotensive and genetically hypertensive rats. 2. Prazosin, infused intra-arterially, lowered vascular resistance in the blood-perfused rat hind limb. This effect was dependent on the presence of intact sympathetic innervation to the limb; no direct vasodilatation was demonstrated. In this preparation prazosin infusion reduced vasoconstrictor responses to noradrenaline. 3. In the saline-perfused rat mesenteric artery preparation prazosin reduced responses to noradrenaline and sympathetic nerve stimulation but not those to serotonin and vasopressin. Prazosin was more potent than phentolamine, on a molar basis, in reducing the vasoconstrictor effects of noradrenaline. 4. A comparison of the effects of prazosin injected intravenously and into a lateral cerebral ventricle failed to show any central action of the drug on blood pressure. Experiments using the donor blood-perfused, vascularly isolated rat hind limb preparation confirmed that the sympatholytic effect of prazosin occurred within the limb itself.     

Plasma lipids in genetically hypertensive rats of the Lyon strain

Of the four standardized strains of genetically hypertensive rats, only the Lyon hypertensive rats spontaneously exhibit a higher body weight than their normotensive controls. In order to determine if this increased body weight is associated with alterations in the lipid metabolism, plasma triglycerides (TG), phospholipids (PL), total cholesterol (TPC), and high-density lipoprotein cholesterol (CHDL) were followed in Lyon hypertensive (LH), normotensive (LN), and hypotensive (LL) male rats between 5 and 32 weeks of age. TG were stable with age in LL and LN but increased in LH rats. PL decreased in LL, remained stable in LN, but increased with age in LH rats. TPC and CHDL were stable in LL and LN and increased with age in LH rats. Plasma lipids were not related to the blood pressure level, but were positively related to the body weight in the hypertensive strain. Thus, starting at the age of 5 weeks, LH rats exhibit spontaneously a significant increase in blood pressure, body weight, and plasma lipid concentrations.     

Calcium/calmodulin-dependent kinase II mediates the phosphorylation and activation of NADPH oxidase 5

Excessive synthesis of reactive oxygen species contributes to the pathology of many human diseases and originates from changes in the expression and posttranslational regulation of the transmembrane NADPH oxidases (Noxes). Nox5 is a novel Nox isoform whose activity is regulated by intracellular calcium levels. We have reported that the activity and calcium-sensitivity of Nox5 can also be modulated by direct phosphorylation. However, the kinases that phosphorylate Nox5 have not been identified, and thus, the goal of this study was to determine whether calcium-activated kinases such as calcium/calmodulin-dependent kinase II (CAMKII) are involved. We found that Nox5 activity in bovine aortic endothelial cells was suppressed by two doses of the CAMKII inhibitor 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93). In cotransfected COS-7 cells, wild-type and constitutively active CAMKII, but not a dominant-negative, robustly increased basal Nox5 activity. The ability of CAMKII to increase Nox5 activity was also observed with fixed calcium concentrations in an isolated enzyme activity assay. CAMKII did not elevate intracellular calcium or activate other Nox enzymes. In vitro phosphorylation assays revealed that CAMKII can directly phosphorylate Nox5 on Thr494 and Ser498 as detected by phosphorylation state-specific antibodies. Mass spectrometry (MS) analysis revealed the phosphorylation of additional, novel sites at Ser475, Ser502, and Ser675. Of these phosphorylation sites, mutation of only Ser475 to alanine prevented CAMKII-induced increases in Nox5 activity. The ability of CAMKIIα to phosphorylate Ser475 in intact cells was supported by the binding of Nox5 to phosphoprotein-affinity columns and via MS/MS analysis. Together, these results suggest that CAMKII can positively regulate Nox5 activity via the phosphorylation of Ser475.     

Increased Cdk5/p35 activity in the dentate gyrus mediates depressive-like behaviour in rats

Depression is one of the most pervasive and debilitating psychiatric diseases, and the molecular mechanisms underlying the pathophysiology of depression have not been elucidated. Cyclin-dependent kinase 5 (Cdk5) has been implicated in synaptic plasticity underlying learning, memory, and neuropsychiatric disorders. However, whether Cdk5 participates in the development of depressive diseases has not been examined. Using the chronic mild stress (CMS) procedure, we examined the effects of Cdk5/p35 activity in the hippocampus on depressive-like behaviour in rats. We found that CMS increased Cdk5 activity in the hippocampus, accompanied by translocation of neuronal-specific activator p35 from the cytosol to the membrane in the dentate gyrus (DG) subregion. Inhibition of Cdk5 in DG but not in the cornu ammonis 1 (CA1) or CA3 hippocampal subregions inhibited the development of depressive-like symptoms. Overexpression of p35 in DG blocked the antidepressant-like effect of venlafaxine in the CMS model. Moreover, the antidepressants venlafaxine and mirtazapine, but not the antipsychotic aripiprazole, reduced Cdk5 activity through the redistribution of p35 from the membrane to the cytosol in DG. Our results showed that the development of depressive-like behaviour is associated with increased Cdk5 activity in the hippocampus and that the Cdk5/p35 complex plays a key role in the regulation of depressive-like behaviour and antidepressant actions.     

Administration of rosmarinic acid reduces cardiopathology and blood pressure through inhibition of p22phox NADPH oxidase in fructose-fed hypertensive rats

Rosmarinic acid (RA), a caffeic acid ester, has insulin-sensitizing and antioxidant effects in high fructose-fed model of insulin resistance (IR). This study investigated whether RA supplementation prevents cardiac abnormalities and hypertension in fructose-fed rats (FFR). Rats fed with fructose diet (60 g/100 g) for 60 days exhibited metabolic abnormalities and rise in plasma and cardiac lipids and whole body IR. The levels of cardiac antioxidants and plasma ferric reducing antioxidant power were significantly reduced in FFR concomitant with increased levels of lipid peroxidation and protein oxidation products. A significant rise in troponin T, creatine kinase-MB, aspartate transaminase, and lactate dehydrogenase in plasma of FFR was noted. RA supplementation to FFR (10 mg/kg from the 16th day) significantly improved insulin sensitivity, reduced lipid levels, oxidative damage, and the expression of p22phox subunit of nicotinamide adenine dinucleotide phosphate reduced oxidase, and prevented cardiac hypertrophy. Fructose-induced rise in blood pressure was also lowered by RA through decrease in endothelin-1 and angiotensin-converting enzyme activity and increase in nitric oxide levels. Histology revealed a reduction in myocardial damage in RA-supplemented FFR. These findings suggest that RA acts as a vasoactive substance and a cardioprotector through its antioxidant property. Thus, RA may be useful in reducing the cardiovascular risk associated with IR.     

Increased pressor responses to physostigmine in spontaneously hypertensive rats

Summary Intravenous injection of physostigmine evoked a pressor response in unanaesthetized rats. Spontaneously hypertensive rats (SHR) showed increased pressor responses, but the responses were within normal limits in renal hypertensive and DOCA-saline hypertensive rats. The pressor effect in SHR was abolished by the i.v. injection of atropine sulphate but not by the i.v. injection of atropine methyl bromide. After inhibition of the peripheral muscarinic receptors by atropine methyl bromide, oxotremorine also produced a pressor response in unanaesthetized rats. In contrast to the pressor effect of physostigmine, there was no difference between the oxotremorine-induced pressor response of SHR and that of normotensive Wistar-Kyoto rats. The pressor effect of oxotremorine in SHR was blocked by the i.v. injection of atropine sulphate.     

Vasopressin and oxytocin in genetically hypertensive rats of the Lyon strain

To investigate the role of arginine vasopressin (AVP) in genetic hypertension, we measured 24-h urinary volume, osmolality, and 24-h AVP excretion, as well as pituitary and pineal AVP and oxytocin levels, in genetically hypertensive (LH), normotensive (LN), and low blood pressure (LL) 5-and 45-week-old female rats of the Lyon strains. We also determined vascular sensitivity to AVP, norepinephrine, and angiotensin II in 6- and 21-week-old rats. AVP secretion was increased in both LH and LL rats compared with LN controls. Previous reports of increased AVP secretion in spontaneously hypertensive rats have suggested that AVP might play a role in high blood pressure. The existence of a similar increase in LL rats indicates that genetic hypertension of LH rats is not related directly to their increased AVP secretion. Furthermore, the vascular sensitivity to AVP was not specifically enhanced in 21-week-old LH rats compared with LN and LL controls. This study provides evidence against a major role of vasopressin in the genesis and maintenance of high blood pressure in this model of experimental hypertension, and emphasizes that the choice of controls in such investigations is of crucial importance.     

Decreased cAMP content of the hypothalamus of genetically hypertensive rats

Summary In genetically hypertensive rats an altered catecholamine content of hypothalamic structures has been reported. In the present study cAMP was estimated in the hypothalamus and cortex of genetically hypertensive rats and compared with normotensive controls of the same strain. It is shown, that the cAMP content of the hypothalamus of the hypertensive animals was decreased to about 60% of control values, whereas there was no difference of the cAMP content in the cortical regions of the same animals. These results indicate an alteration of the adenyl cyclase-cAMP-phosphodiesterase system in hypothalamic structures of genetically hypertensive rats.Supported by the Deutsche Forschungsgemeinschaft