Nicardipine normalizes elevated levels of antioxidant activity in response to xanthine oxidase-induced oxidative stress in hypertensive rat heart

It has been reported that the production of oxygen radicals mediated by xanthine oxidase (XO) is stimulated in hypertensive cardiovascular endothelium, suggesting involvement of oxidative stress in pathogenesis of hypertension. In this study we estimated the effect of nicardipine, a calcium blocker, on the oxidative stress and antioxidant activities in left ventricles from spontaneously hypertensive rat (SHR) and stroke-prone SHR (SHRSP). The activity of XO increased 3.5-fold in SHR and 6.2-fold in SHRSP compared to that in normal controls (WKY). Interestingly, the levels of glutathione (GSH) and the activity of its synthesizing enzyme (γ-glutamylcysteine synthetase, γ-GCS) elevated concomitantly in SHR and SHRSP: the level of GSH increased 1.2-fold in SHR and 1.3-fold in SHRSP. The activity of γ-GCS was elevated 1.5-fold in SHR and 2.4-fold in SHRSP, accompanying an increase in the expression of its mRNA. Treatment of these rats with nicardipine, for 4 weeks improved blood pressure, from 176 ± 10 to 140 ± 8 mmHg in SHR, and from 201 ± 11 to 167 ± 5 mmHg in SHRSP, respectively, and decreased wet weight of heart, levels of GSH, and the activities of XO and γ-GCS. Nicardipine reduced the expression of γ-GCS mRNA. Collectively, these results suggest that reactive oxygen species produced by XO in hypertensive rat heart cause induction of the expression of γ-GCS and nicardipine plays a role in reducing the oxidative stress in hypertensive heart.     

自发性高血压大鼠中枢血管紧张素Ⅱ的变化对中枢肾上腺素...

The content of norepinephrine (NE) and epinephrine (E) in the brain of spontaneously hypertensive rats has proved abnormal, but the cause remained unknown. It was shown in the recent work that NE content in pons, posterior hypothalamus, nucleus caudatus and E concentration in medulla oblongata, anterior and posterior hypothalamus of 12-week old stroke-prone spontaneously hypertensive rats (SHRSP) were much higher than those of age-matched Wister-Kyoto rats (WKY). SHRSP also showed higher levels of systolic blood pressure (SBP) and brain angiotensin II (A II) than WKY. Intracerebroventricular (icv) perfusion of angiotensin-converting enzyme inhibitor captopril (20 micrograms for each time and three times for each day for four weeks) inhibited the synthesis of brain A II and reduced SBP and NE, E contents in all examined brain areas in SHRSP and WKY. However, the effects of chronically perfused captopril on SBP and brain NE, E levels in SHRSP were much more significant than in WKY. The results indicate that the modulatory effects of central renin-angiotensin system (RAS) on central adrenergic and noradrenergic system might be overactivated in SHRSP, which might partially responsible for the abnormally high levels of NE, E in some of the brain areas of SHRSP.     

Effects of Oxidative Stress on Vascular Reactivity in the Offspring of Protein-Restricted Stroke-Prone Spontaneously Hypertensive Rats

Oxidative stress was induced in 12-week-old offspring of protein-restricted (9% protein) and control (20% protein) protein-restricted stroke-prone spontaneously hypertensive rats (SHRSP) by administering phorbol 12-myristate 13-acetate (PMA) for 4 weeks to determine the effects of oxidative stress on the vascular function of the SHRSP offspring. There was no significant difference in the blood pressure of offspring of the protein-restricted dams and control dams. The plasma diacron-reactive oxygen metabolite (dROM) level at 16 weeks of age was significantly higher in offspring of the protein-restricted dams, whereas the anti-oxidative enzyme activity was similar in both groups. Acetylcholine (Ach)-induced relaxation was significantly reduced in offspring of the protein-restricted dams. The expression of endothelial nitric oxide synthase (eNOS) was lower and the expression of soluble guanylic acid cyclase (sGC) was higher in offspring of the protein-restricted dams. These results indicate that SHRSP offspring of the protein-restricted dams were sensitive to oxidative stress, and displayed the vascular dysfunction.     

Measurement of oxidative stress in stroke-prone spontaneously hypertensive rat brain using in vivo electron spin resonance spectroscopy

A number of researchers have reported that free radicals generated in the brain are involved in various brain dysfunctions, including ischemia-reperfusion injury, brain tumors, and neurodegenerative diseases. It has been reported that the spin probe MC-PROXYL can penetrate the blood-brain barrier and can be useful for evaluating oxidative stress in the brain. Preliminary comparisons were made by ESR imaging of the heads of live mice and isolated rat brains using the spin probe MC-PROXYL and the blood-brain-barrier impermeable probe carbamoyl-PROXYL. The results showed that MC-PROXYL, but not carbamoyl-PROXYL, was widely distributed in the brain. These methods were also applied for the imaging of brains from stroke-prone spontaneously hypertensive rats (SHRSPs). The rapid decay of 2D ESR images of MC-PROXYL in isolated SHRSP-brain was observed, compared to Wistar-Kyoto rats (WKYs), using the ESR imaging system. Furthermore, we provide evidence, by using L-band ESR non-invasively, that the decay rate of MC-PROXYL in the head region is faster in live SHRSPs than in live WKYs. Taken together, the high oxidative stress sustained by oxygen radical generation in SHRSPs may cause the alteration of MC-PROXYL metabolism in the brain. Our results suggest that in vivo ESR could be applied to the assessment of antioxidant effects on oxidative stress in the brain in animal disease models, such as the SHRSP.     

Feeding blueberry diets inhibits angiotensin II-converting enzyme (ACE) activity in spontaneously hypertensive stroke-prone rats

Feeding flavonoid-rich blueberries to spontaneously hypertensive stroke-prone rats (SHRSP) lowers blood pressure. To determine whether this is due to inhibition of angiotensin-converting enzyme (ACE) activity, as seen with other flavanoid-rich foods, we fed blueberries to SHRSP and normotensive rats and analyzed ACE activity in blood and tissues. After 2 weeks on a control diet, the hypertensive rats showed 56% higher levels of ACE activity in blood as compared with the normotensive rats (p < 0.05). Feeding a 3% blueberry diet for 2 weeks lowered ACE activity in the SHRSP (p < 0.05) but not the normotensive rats. ACE activity in plasma of SHRSP was no longer elevated at weeks 4 and 6, but blueberry feeding inhibited ACE in SHRSP after 6 weeks. Blueberry diets had no effect on ACE activity in lung, testis, kidney, or aorta. Our results suggest that dietary blueberries may be effective in managing early stages of hypertension, partially due to an inhibition of soluble ACE activity.     

Dietary S-allyl-L-cysteine reduces mortality with decreased incidence of stroke and behavioral changes in stroke-prone spontaneously hypertensive rats

S-Allyl-L-cysteine (SAC), an active organosulfur compound derived from garlic, was found to reduce mortality with lesser incidence of stroke and also to lower the overall stroke-related behavioral score in stroke-prone spontaneously hypertensive (SHRSP) rats by dietary administration. Consequently, the anti-stroke effect of dietary SAC was demonstrated in SHRSP rats.     

Therapeutic effects of stem cell on hyperglycemia,hyperlipidemia, and oxidative stress in alloxan-treated rats

Diabetes mellitus is the most common endocrine disorder that affects more than 285 million people worldwide. The purpose of this study was to investigate the effect of mesenchymal stem cells (MSCs) from the bone marrow of albino rats, on hyperglycemia, hyperlipidemia, and oxidative stress induced by intraperitoneal injection (i.p.) of alloxan at a dose of 150 mg/kg in rats. Injection of alloxan into rats resulted in a significant increase in serum glucose, total cholesterol, triglyceride, low density lipoprotein cholesterol, and sialic acid level and a significant decrease in serum insulin, high density lipoprotein-cholesterol, vitamin E, and liver glycogen as compared to their corresponding controls. Also, oxidative stress was noticed in pancreatic tissue as evidenced by a significant decrease in glutathione level, superoxide dismutase, glutathione-S-transferase activities, also a significant increase in malondialdehyde and nitric oxide levels when compared to control group. Treatment of diabetic rats with MSCs stem cells significantly prevented these alterations and attenuated alloxan-induced oxidative stress. In conclusion, rat bone marrow harbors cells that have the capacity to differentiate into functional insulin-producing cells capable of controlling hyperglycemia, hyperlipidemia, and oxidative stress in diabetic rats. This may be helpful in the prevention of diabetic complications associated with oxidative stress.     

Effect of coenzyme Q10 on structural alterations in the renal membrane of stroke-prone spontaneously hypertensive rats

To test the hypothesis that structural abnormalities exist in the kidney membrane of spontaneously hypertensive rats, we examined the effect of long-term administration of coenzyme Q10 on membrane lipid alterations in the kidney of stroke-prone spontaneously hypertensive rats (SHRSP). As compared with normotensive Wistar-Kyoto rats, renal membrane phospholipids, especially phosphatidylcholine and phosphatidylethanolamine, decreased and renal phospholipase A2 activity was enhanced with age in untreated SHRSP. Treatment with coenzyme Q10 attenuated the elevation of blood pressure, the membranous phospholipid degradation, and the enhanced phospholipase A2 activity. These results suggest that one factor contributing to the progress of hypertension is a structural membrane abnormality that alters the physical and functional properties of the cell membrane, and coenzyme Q10 might protect the renal membrane from damage due to hypertension in SHRSP.     

Elevated production of 20-HETE in the cerebral vasculature contributes to severity of ischemic stroke and oxidative stress in spontaneously hypertensive rats

Hypertension is a major risk factor for stroke, but the factors that contribute to the increased incidence and severity of ischemic stroke in hypertension remain to be determined. 20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to be a potent constrictor of cerebral arteries, and inhibitors of 20-HETE formation reduce infarct size following cerebral ischemia. The present study examined whether elevated production of 20-HETE in the cerebral vasculature could contribute to the larger infarct size previously reported after transient middle cerebral artery occlusion (MCAO) in hypertensive strains of rat [spontaneously hypertensive rat (SHR) and spontaneously hypertensive stroke-prone rat (SHRSP)]. The synthesis of 20-HETE in the cerebral vasculature of SHRSP measured by liquid chromatography-tandem mass spectrometry was about twice that seen in Wistar-Kyoto (WKY) rats. This was associated with the elevated expression of cytochrome P-450 (CYP)4A protein and CYP4A1 and CYP4A8 mRNA. Infarct volume after transient MCAO was greater in SHRSP (36+/-4% of hemisphere volume) than in SHR (19+/-5%) or WKY rats (5+/-2%). This was associated with a significantly greater reduction in regional cerebral blood flow (rCBF) in SHR and SHRSP than in WKY rats during the ischemic period (78% vs. 62%). In WKY rats, rCBF returned to 75% of control following reperfusion. In contrast, SHR and SHRSP exhibited a large (166+/-18% of baseline) and sustained (1 h) postischemic hyperperfusion. Acute blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016; 1 mg/kg) reduced infarct size by 59% in SHR and 87% in SHRSP. HET0016 had no effect on the fall in rCBF during MCAO but eliminated the hyperemic response. HET0016 also attenuated vascular O2*- formation and restored endothelium-dependent dilation in cerebral arteries of SHRSP. These results indicate the production of 20-HETE is elevated in the cerebral vasculature of SHRSP and contributes to oxidative stress, endothelial dysfunction, and the enhanced sensitivity to ischemic stroke in this hypertensive model.     

Muscarinic cholinoceptors and choline acetyltransferase activity in the hypothalamus of spontaneously hypertensive rats

To study the role of central cholinergic mechanisms in hypertension, we have determined muscarinic receptors using [³H](-)quinuclidinyl benzilate (QNB) and choline acetyltransferase (ChAT) activity in the brain regions of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and renal hypertensive rats. The number of muscarinic receptors was significantly (33–38%) elevated in the hypothalamus of SHR and SHRSP at the ages of 16 and 24 weeks compared to that of Wistar-Kyoto rats (WKY). An increased density of muscarinic receptors was consistently observed in the prehypertensive (5 weeks) and developmental (10 weeks) stages of hypertension. In contrast, in the hypothalamus of rats with renal hypertension there was no muscarinic receptor alteration. The receptor alteration in the SHRSP hypothalamus was not abolished by a chronic hypotensive treatment which prevented the development of hypertension, suggesting that an enhancement of the muscarinic receptors in spontaneous hypertension does not occur secondarily to the elevation of blood pressure. The hypothalamus of SHR and SHRSP at the ages of 5 and 24 weeks showed significantly less activity of ChAT. These data demonstrate that there is a specific increase in muscarinic receptors and a decrease in cholinergic activity in the hypothalamus of SHR and SHRSP. Thus, the present study suggests an important role for hypothalamic cholinergic receptors in the pathogenesis of spontaneous hypertension.     

Increased production of angiotensin II in the adrenal gland of stroke-prone spontaneously hypertensive rats with malignant hypertension

Angiotensin(Ang) contents in the adrenal gland of stroke-prone spontaneously hypertensive rats(SHRSP) and age-matched Wistar Kyoto rats(WKY) were determined using reverse phase high performance liquid chromatography combined with a specific radioimmunoassay. In normotensive 5 wk-old SHRSP, the adrenal renin activity was about 3 times higher than that of age-matched WKY while the adrenal Ang I and Ang II concentrations did not differ from those of WKY. In the severely hypertensive 25 wk-old SHRSP, the adrenal Ang II and Ang I, and plasma aldosterone concentrations were about 5-fold, 2-fold and 4-fold, respectively, increased compared with levels in the WKY. In the 25 wk-old SHRSP 24 h after bilateral nephrectomy, the adrenal Ang II and plasma aldosterone levels were not decreased and were 10 and 3 times, respectively, higher than those of nephrectomized control WKY. Thus, the enhanced local generation of Ang II in the adrenal gland may contribute to the increased release of aldosterone in SHRSP with malignant hypertension.     

Morphological changes in cerebral vascular smooth muscle cells in stroke-prone spontaneously hypertensive rats (SHRSP). A scanning and transmission electron microscopic study

The ultrastructure of the vascular smooth muscle cells of the middle cerebral artery in 6-month-old male stroke-prone spontaneously hypertensive rats (SHRSP) was studied by scanning (SEM) and transmission electron microscopy (TEM) and compared with that of age-matched normotensive Wistar Kyoto rats (WKY). Although the smooth muscle cells of WKY rats by SEM had a typical spindle shape and smooth surface texture, those of SHRSP were structurally modified by numerous surface invaginations and projections, bearing some structural resemblance to the myotendinous junction of skeletal muscle. Structural modifications affected more than half the surface of medial smooth muscle cells in SHRSP, but less than 0.6% of the surface of these cells in WKY rats. About 10% of medial smooth muscle cells were necrotic in SHRSP, but no necrotic cells were identified in WKY rats. By TEM, smooth muscle cells in SHRSP were shown to be irregular in profile with deep indentations of the plasma membrane and were surrounded by many layers of basal lamina-like material. The present study suggests that most smooth muscle cells in the middle cerebral artery of SHRSP may be modified to adapt to chronic hypertension by increasing the junctional area between muscle cells and connective tissue and that some cells may undergo necrosis.     

Proteomic identification of haptoglobin as a stroke plasma biomarker in spontaneously hypertensive stroke-prone rats

AIMS: We investigated changes in the expression of plasma proteins in spontaneously hypertensive stroke-prone rats (SHRSP) to identify stroke biomarkers. MAIN METHODS AND KEY FINDINGS: The present analysis using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) demonstrated that three peaks at mass/charge ratios (m/z) of 9330, 9480 and 9700 decreased in intensity during the development and progression of hypertensive stroke in SHRSPs, but not in age-matched control SHR and Wistar rats. Administration of verapamil, an L-type calcium channel blocker which was effective for hypertension in SHRSP rats, prevented the decrease in plasma protein expression. A candidate biomarker protein (m/z 9330) was identified using LC-MS/MS as haptoglobin (Hp). Immunoblotting with anti-Hp antibody demonstrated the decreased expression of both Hpalpha and Hpbeta chains in SHRSP. In contrast, haptoglobin mRNA expression in the liver of SHRSPs slightly increased as compared with control rats. SIGNIFICANCE: These findings suggest that Hp is a biomarker candidate for discriminating pathogenic alterations of stroke.     

Morphological changes in cerebral vascular smooth muscle cells in stroke-prone spontaneously hypertensive rats (SHRSP)

The ultrastructure of the vascular smooth muscle cells of the middle cerebral artery in 6-month-old male stroke-prone spontaneously hypertensive rats (SHRSP) was studied by scanning (SEM) and transmission electron microscopy (TEM) and compared with that of age-matched normotensive Wistar Kyoto rats (WKY). Although the smooth muscle cells of WKY rats by SEM had a typical spindle shape and smooth surface texture, those of SHRSP were structurally modified by numerous surface invaginations and projections, bearing some structural resemblance to the myotendinous junction of skeletal muscle. Structural modifications affected more than half the surface of medial smooth muscle cells in SHRSP, but less than 0.6% of the surface of these cells in WKY rats. About 10% of medial smooth muscle cells were necrotic in SHRSP, but no necrotic cells were identified in WKY rats. By TEM, smooth muscle cells in SHRSP were shown to be irregular in profile with deep indentations of the plasma membrane and were surrounded by many layers of basal laminalike material. The present study suggests that most smooth muscle cells in the middle cerebral artery of SHRSP may be modified to adapt to chronic hypertension by increasing the junctional area between muscle cells and connective tissue and that some cells may undergo necrosis.     

Accumulation of 8-oxo-deoxyguanosine in cardiovascular tissues with the development of hypertension

Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA is associated with mutagenesis and cell death. Little attention has been given to the biological significance of 8-oxo-dG accumulation in cardiovascular tissues during the different stage of hypertension and its prevention. We thus investigated the levels and localization of both 8-oxo-dG accumulation and expression of MTH1, which hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA, in the thoracic aorta prepared from stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wister-Kyoto rats (WKY), aged 5-32 weeks. HPLC-MS/MS analysis revealed that the levels of nuclear 8-oxo-dG in the aorta increased significantly in SHRSP, but not WKY, with aging. Immunohistochemical study revealed that both TUNEL reactivity and 8-oxo-dG immunoreactivity were increased in smooth muscle cells (SMC) and endothelial cells (EC) of the aorta with aging, and they exhibited similar distributions in serial sections. The number of 8-oxo-dG and TUNEL positive cells in EC, but not in SMC, was significantly higher in SHRSP than WKY at 32 weeks of age. In contrast, the expression levels of Mth1mRNA and MTH1 protein in the aorta were similarly decreased both in SHRSP and WKY with aging. However, the number of MTH1 expressing EC was remarkably increased in the older SHRSP compared to the younger ones or age-matched WKY. Hypertension significantly increased not only 8-oxo-dG accumulation but also the expression of MTH1 in EC of the aorta during aging. While accumulation of 8-oxo-dG in SMC of the aorta was slightly increased, the expression of MTH1 protein in SMC was rather decreased by hypertension. We thus suggest that MTH1 may protect EC in the aorta from the oxidative damage increased by hypertension.     

Sympathetic hyperreactivity to air-jet stress in the chromosome 1 blood pressure quantitative trait locus congenic rats

A chromosome 1 blood pressure quantitative trait locus (QTL) was introgressed from the stroke-prone spontaneously hypertensive rats (SHRSP) to Wistar-Kyoto (WKY) rats. This congenic strain (WKYpch1.0) showed an exaggerated pressor response to both restraint and cold stress. In this study, we evaluated cardiovascular and sympathetic response to an air-jet stress and also examined the role of the brain renin-angiotensin system (RAS) in the stress response of WKYpch1.0. We measured mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) responses to air-jet stress in WKYpch1.0, WKY, and SHRSP. We also examined effects of intracerebroventricular administration of candesartan, an ANG II type 1 receptor blocker, on MAP and HR responses to air-jet stress. Baseline MAP in the WKYpch1.0 and WKY rats were comparable, while it was lower than that in SHRSP rats. Baseline HR did not differ among the strains. In WKYpch1.0, air-jet stress caused greater increase in MAP and RSNA than in WKY. The increase in RSNA was as large as that in SHRSP, whereas the increase in MAP was smaller than in SHRSP. Intracerebroventricular injection of a nondepressor dose of candesartan inhibited the stress-induced pressor response to a greater extent in WKYpch1.0 than in WKY. Intravenous injection of phenylephrine caused a presser effect comparable between WKYpch1.0 and WKY. These results suggest that the chromosome 1 blood pressure QTL congenic rat has a sympathetic hyperreactivity to an air-jet stress, which causes exaggerated pressor responses. The exaggerated response is at least partly mediated by the brain RAS.