Age-matched Wistar-Kyoto Research Articles

Upregulation of catalase and downregulation of glutathione peroxidase activity in the kidney precede the development of hypertension in pre-hypertensive SHR

Although oxidative stress has been implicated in the pathogenesis of hypertension in spontaneously hypertensive rats (SHRs), there is little information on the levels of primary antioxidant enzymes status (AOEs) in pre-hypertensive SHR. This study therefore determined the activities of primary AOEs and their mRNA levels, levels of hydrogen peroxide (H2O2), malondialdehyde (MDA) and total antioxidant status (TAS) in whole kidneys of SHR and age-matched Wistar-Kyoto (WKY) rats aged between 2 and 16 weeks. Compared with age-matched WKY rats, catalase (CAT) activity was significantly higher from the age of 2 weeks (P<0.001) and glutathione peroxide (GPx) activity was lower from the age of 3 weeks (P<0.001) in SHR. CAT mRNA levels were significantly higher in SHR aged 2, 4, 6 and 12 weeks. GPx mRNA levels were significantly lower in SHR at 8 and 12 weeks. Superoxide dismutase activity or its mRNA levels were not different between the two strains. H2O2 levels were significantly lower in SHR from the age of 8 weeks (P<0.01). TAS was significantly higher in SHR from the age of 3 weeks (P<0.05). MDA levels were only significantly higher at 16 weeks of age in the SHR (P<0.05). The data suggest that altered renal CAT and GPx mRNA expression and activity precede the development of hypertension in SHR. The raised CAT activity perhaps contributes to the higher TAS and lower H2O2 levels in SHR. In view of these findings, the precise role of oxidative stress in the pathogenesis of hypertension in SHR needs to be investigated further.

Dipeptidyl peptidase IV inhibition upregulates GLUT4 translocation and expression in heart and skeletal muscle of spontaneously hypertensive rats

The purpose of the current study was to test the hypothesis that the dipeptidyl peptidase IV (DPPIV) inhibitor sitagliptin, which exerts anti-hyperglycemic and anti-hypertensive effects, upregulates GLUT4 translocation, protein levels, and/or mRNA expression in heart and skeletal muscle of spontaneously hypertensive rats (SHRs). Ten days of treatment with sitagliptin (40mg/kg twice daily) decreased plasma DPPIV activity in both young (Y, 5-week-old) and adult (A, 20-week-old) SHRs to similar extents (∼85%). However, DPPIV inhibition only lowered blood pressure in Y-SHRs (119±3 vs. 136±4mmHg). GLUT4 translocation, total protein levels and mRNA expression were decreased in the heart, soleus and gastrocnemius muscle of SHRs compared to age-matched Wistar Kyoto (WKY) normotensive rats. These differences were much more pronounced between A-SHRs and A-WKY rats than between Y-SHRs and Y-WKY rats. In Y-SHRs, sitagliptin normalized GLUT4 expression in the heart, soleus and gastrocnemius. In A-SHRs, sitagliptin increased GLUT4 expression to levels that were even higher than those of A-WKY rats. Sitagliptin enhanced the circulating levels of the DPPIV substrate glucagon-like peptide-1 (GLP-1) in SHRs. In addition, stimulation of the GLP-1 receptor in cardiomyocytes isolated from SHRs increased the protein level of GLUT4 by 154±13%. Collectively, these results indicate that DPPIV inhibition upregulates GLUT4 in heart and skeletal muscle of SHRs. The underlying mechanism of sitagliptin-induced upregulation of GLUT4 in SHRs may be, at least partially, attributed to GLP-1.

OLMESARTAN AMELIORATES LEFT VENTRICULAR DIASTOLIC DYSFUNCTION IN SPONTANEOUSLY HYPERTENSIVE RATS THROUGH INHIBITING CALCINEURIN

ObjectivesTo test whether treatment of olmesartan ameliorates cardiac diastolic dysfunction in spontaneously hypertensive rats (SHR) and whether this is calcineurin (CaN) involved.MethodsTwo groups of 6-month-old male SHR were treated with...

Abstract 325: Cardiac Hypertrophy in Spontaneous Hypertensive Rats (SHR) is Associated With Decreased Cardiac Expression of SIK1 and SIK3 Isoforms

Numerous reports have highlighted the importance of protein kinases and transcription factors in cardiogenesis and cardiac hypertrophy. The salt-inducible kinase 1 (SIK1), a protein kinase encoded by the snf1lk gene, is involved in early cardiogenesis in mice. Genotype-phenotype association studies in three Swedish and one Japanese population-based cohorts suggested that 15Ser-SIK1 variant (15Gly-Ser change, T allele), bestow augmented SIK1 activity, was associated with lower blood pressure and decreases in left ventricular (LV) mass. The present study aims to determine the potential role of SIK1 as a mediator of cardiac hypertrophic signaling pathway and to evaluate age-related changes on the cardiac expression of several elements of the SIK network associated with hypertension, in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). For this purpose, mRNA expression levels of skeletal actin (SkA), β-myosin heavy chain (β-MHC) and matrix metalloproteinase-9 (MMP-9) as markers for cardiac hypertrophy, SIK isoforms (SIK1, SIK2 and SIK3), and transcription factors (MEF2C, NFAT5c, KFL4, Snail1 and Snail2) were determined, using Taqman, in 13- , 52- and 91-week old SHR and their normotensive counterparts WKY. Systolic and diastolic blood pressures, determined by the tail-cuff method, were significantly higher in 3- , in 3- , 13- and 21-month old SHR than in age-matched WKY. In the SHR, increases in heart/tibia length ratio were associated to the overexpression of cardiac hypertrophic genes (SkA and β-MHC) and the underexpression of cardiac SIK3, at the age of 3- , 13- and 21-months. Ageing accompanied by a diminished mRNA expression of SIK1 in SHR, relative to WKY (both at 13- and 21-months of age). Whereas, Snail2, MFAT5c and KLF4 mRNA levels decreased with aged SHR, comparing to age-match WKY (both at 13- and 21-months of age). These results indicate that SIK1 and SIK3 isoforms might be upstream mediators of transcriptions factors implicated in the regulation of cardiac hypertrophic growth response. In conclusion, ageing in SHR is accompanied by cardiac alterations that include hypertrophy and dysfunction of the SIK signaling network. Supported by grant PIC/IC/83204/2007.

838 Activation of the aldosterone/MR system by aging in spontaneously hypertensive rats

In the present study we hypothesised that age-associated changes in the renal aldosterone/mineralocorticoid receptor (MR) system may differ between normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). In WKY, body mass index (BMI) significantly increased with age. Fat mass accumulation may operate as a confounding factor therefore WKY were submitted to 15% food restriction (WKY-FR) from 5 to 13 and 52 weeks of age. Renal oxidative stress was increased in aged WKY and SHR. Long-term FR significantly reduced the BMI and decreased renal oxidative stress when compared to age-matched WKY and SHR. Aged WKY and SHR had increased MR functionality, which correlated positively with increased plasma aldosterone levels, nuclear MR content and abundance of aldosterone effectors in the renal medulla. In contrast, when compared to aged WKY and SHR, aged WKY-FR had decreased plasma aldosterone levels and decreased activation of the aldosterone/MR system in the renal medulla. Increases in renal oxidative stress and plasma aldosterone in aged WKY, to levels observed in SHR, were not sufficient to result in sustained increases in blood pressure. In conclusion, activation of the aldosterone/MR system is intensified by aging in SHR whereas hyperaldosteronism in WKY is associated with increases in body fat mass.

Unprovoked atrial tachyarrhythmias in aging spontaneously hypertensive rats: the role of the autonomic nervous system

Experimental models of unprovoked atrial tachyarrhythmias (AT) in conscious, ambulatory animals are lacking. We hypothesized that the aging, spontaneously hypertensive rat (SHR) may provide such a model. Baseline ECG recordings were acquired with radiotelemetry in eight young (14-wk-old) and eight aging (55-wk-old) SHRs and in two groups of four age-matched Wistar-Kyoto (WKY) rats. Quantification of AT and heart rate variability (HRV) analysis were performed based on 24-h ECG recordings in unrestrained rats. All animals were submitted to an emotional stress protocol (air-jet). In SHRs, carbamylcholine injections were also performed. Spontaneous AT episodes were observed in all eight aging SHRs (median, 91.5; range, 4-444 episodes/24 h), but not in young SHRs or WKY rats. HRV analysis demonstrated significantly decreased low frequency components in aging SHRs compared with age-matched WKY rats (P < 0.01) and decreased low/high frequency ratios in both young (P < 0.01) and aging (P = 0.01) SHRs compared with normotensive controls. In aging SHRs, emotional stress significantly reduced the number of arrhythmic events, whereas carbamylcholine triggered AT and significantly increased atrial electrical instability. This study reports the occurrence of unprovoked episodes of atrial arrhythmia in hypertensive rats, and their increased incidence with aging. Our results suggest that autonomic imbalance with relative vagal hyperactivity may be responsible for the increased atrial arrhythmogenicity observed in this model. We also provide evidence that, in this model, the sympatho-vagal imbalance preceded the occurrence of arrhythmia. These results indicate that aging SHRs may provide valuable insight into the understanding of atrial arrhythmias.

Casein Kinase 2-mediated Synaptic GluN2A Up-regulation Increases N-Methyl-d-aspartate Receptor Activity and Excitability of Hypothalamic Neurons in Hypertension

Increased glutamatergic input, particularly N-methyl-D-aspartate receptor (NMDAR) activity, in the paraventricular nucleus (PVN) of the hypothalamus is closely associated with high sympathetic outflow in essential hypertension. The molecular mechanisms underlying augmented NMDAR activity in hypertension are unclear. GluN2 subunit composition at the synaptic site critically determines NMDAR functional properties. Here, we found that evoked NMDAR-excitatory postsynaptic currents (EPSCs) of retrogradely labeled spinally projecting PVN neurons displayed a larger amplitude and shorter decay time in spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rats. Blocking GluN2B caused a smaller decrease in NMDAR-EPSCs of PVN neurons in SHRs than in WKY rats. In contrast, GluN2A blockade resulted in a larger reduction in evoked NMDAR-EPSCs and puff NMDA-elicited currents of PVN neurons in SHRs than in WKY rats. Blocking presynaptic GluN2A, but not GluN2B, significantly reduced the frequency of miniature EPSCs and the firing activity of PVN neurons in SHRs. The mRNA and total protein levels of GluN2A and GluN2B in the PVN were greater in SHRs than in WKY rats. Furthermore, the GluN2B Ser(1480) phosphorylation level and the synaptosomal GluN2A protein level in the PVN were significantly higher in SHRs than in WKY rats. Inhibition of protein kinase CK2 normalized the GluN2B Ser(1480) phosphorylation level and the contribution of GluN2A to NMDAR-EPSCs and miniature EPSCs of PVN neurons in SHRs. Collectively, our findings suggest that CK2-mediated GluN2B phosphorylation contributes to increased synaptic GluN2A, which potentiates pre- and postsynaptic NMDAR activity and the excitability of PVN presympathetic neurons in hypertension.

Inhibition of dipeptidyl peptidase IV by sitagliptin increases GLUT4 expression in insulin‐sensitive tissues of SHR

The goal of the present study was to investigate whether the dipeptidyl peptidase IV (DPPIV) inhibitor sitagliptin, which has been shown to exert both anti‐hyperglycemic and anti‐hypertensive actions, modulates GLUT4 expression in skeletal and cardiac muscles of SHR. To this end, male SHR at 5 and 20 weeks of age were treated with either sitagliptin (40 mg/kg twice daily) or vehicle for the period of ten days. Age‐matched Wistar Kyoto (WKY) rats were used as normotensive controls. Sitagliptin treatment inhibited the activity of DPPIV in the plasma of both young and adult SHR and also decreased the circulating levels of the enzyme. Sitagliptin only suppressed blood pressure rising in Y‐SHR. Levels of plasma insulin were not significantly different between the strains at young age. In contrast, adult SHR displayed enhanced plasma insulin concentration with fasting normoglycemia. Inhibition of DPPIV by sitagliptin restored plasma insulin levels of A‐SHR. GLUT4 in the plasma membrane (PM) was lower in Y‐SHR than in Y‐WKY in the heart, the slow‐twich oxidative soleus, the fast‐twich glycolytic EDL and the fast‐twich glycolytic‐oxidative gastrocnemius. DPPIV inhibition normalized PM GLUT4 in myocytes from Y‐SHR. Reduction of GLUT4 PM expression in the skeletal and cardiac myocytes was more pronounced in A‐SHR vs. A‐WKY than in Y‐SHR vs. YWKY. Inhibition of DPPIV by sitagliptin increased GLUT4 PM, total protein and GLUT4‐mRNA expression in the heart, soleus, EDL and gastrocnemius of treated‐A‐SHR to levels higher than those found in the A‐WKY. Collectively, our data demonstrate that DPPIV inhibition upregulates GLUT4 in skeletal and cardiac muscles of SHR.

Sexual dimorphism of vascular mechanics in rats

The purpose of this study was to test the hypotheses that male and female rats would exhibit dimorphic mechanical properties of the hindlimb vasculature and that these differences would persist in hypertension. Vascular resistance (R), compliance (C), and tissue viscoelasticity (K) of the hindlimb vascular bed were quantified from concurrent femoral artery blood flow and pressure waveforms (lumped Windkessel model) in age‐matched Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR) male (n=10 each) and female (n=6 each). Female WKY rats displayed greater R (15.4±5.7 vs 7.7±3.3mmHg/mL/min; P&lt;0.05) and K (3.4±2.4 vs 1.1±2.7mmHg/mL/min; P&lt;0.05) with lower C (3.4E‐3±0.8E‐3 vs 4.9E‐3±0.9E‐3mL/mmHg/g; P&lt;0.05) compared to males, respectively. Similarly, female SHRs had higher R (31.5±8.5 vs 17.6±7.8mmHg/mL/min; P&lt;0.05) and K (7.3±2.5 vs 2.9±1.0mmHg/mL/min; P&lt;0.05) with lower C (4.8E‐3±2.0E‐3 vs 6.8E‐3±3.5E‐3mL/mmHg/g; P&lt;0.05) than their male counterparts. Main effects of strain and sex were observed for each of R, K and C variables (all P&lt;0.05). Therefore, sex differences are apparent in the indices of steady state (R) and oscillatory (K and C) hindlimb vascular mechanics and these persist in hypertension.Supported by CIHR.

Heart failure-induced skeletal myopathy in spontaneously hypertensive rats

BackgroundAlthough skeletal muscle atrophy and changes in myosin heavy chain (MyHC) isoforms have often been observed during heart failure, their pathophysiological mechanisms are not completely defined. In this study we tested the hypothesis that skeletal muscle phenotype changes are related to myogenic regulatory factors and myostatin/follistatin expression in spontaneously hypertensive rats (SHR) with heart failure. MethodsAfter developing tachypnea, SHR were subjected to transthoracic echocardiogram. Pathological evidence of heart failure was assessed during euthanasia. Age-matched Wistar–Kyoto (WKY) rats were used as controls. Soleus muscle morphometry was analyzed in histological sections, and MyHC isoforms evaluated by electrophoresis. Protein levels were assessed by Western blotting. Statistical analysis: Student'st test and Pearson correlation. ResultsAll SHR presented right ventricular hypertrophy and seven had pleuropericardial effusion. Echocardiographic evaluation showed dilation in the left chambers and left ventricular hypertrophy with systolic and diastolic dysfunction in SHR. Soleus weight and fiber cross sectional areas were lower (WKY 3615±412; SHR 2035±224μm2; P<0.001), and collagen fractional volume was higher in SHR. The relative amount of type I MyHC isoform was increased in SHR. Myogenin, myostatin, and follistatin expression was lower and MRF4 levels higher in SHR. Myogenin and follistatin expression positively correlated with fiber cross sectional areas and MRF4 levels positively correlated with I MyHC isoform. ConclusionReduced myogenin and follistatin expression seems to participate in muscle atrophy while increased MRF4 protein levels can modulate myosin heavy chain isoform shift in skeletal muscle of spontaneously hypertensive rats with heart failure.

Angiotensin-(1–7) upregulates central nitric oxide synthase in spontaneously hypertensive rats

Increased blood pressure in hypertension is hypothesized to be caused by high sympathetic nervous system (SNS) activity. Since Ang (1–7) exerts an inhibitory neuromodulatory effect on the SNS through a NO-mediated mechanism, we tested the hypothesis that Ang (1–7) alters centrally nitric oxide synthase (NOS) activity and expression in spontaneously hypertensive rats (SHR). Since NOS activity is altered in relation to the development of hypertension in rats, we evaluated the effect of Ang-(1–7) on hypothalamic NOS activity in two different ages in SHR, corresponding to a prehypertensive phase (3–4weeks) and a established hypertension (13–14weeks) and compared with age-matched Wistar-Kyoto (WKY) rats. NOS activity was measured by the conversion of [3H]l-arginine to citrulline . Ang-(1–7) caused an impairment in NOS activity in prehypertensive SHR (26±4% reduction), while it induced an increase in NOS activity at established hypertension (48±9% increase). In contrast, Ang-(1–7) did not modify NOS activity in age-matched WKY rats. In another set of experiments, Ang-(1–7) was injected into the anterior hypothalamic area, mean arterial blood pressure (MAP) was registered and after 30, 60 and 180min nNOS expression was evaluated by Western-blot. Ang-(1–7) decreased MAP after 10min of injection and this effect was blocked by a NOS inhibitor. nNOS expression increased after 180min of Ang-(1–7) intrahypothalamic injection in both WKY and SHR (WKY: 3.6-fold increase above basal; SHR: 1.85-fold increase above basal). Our results suggest that Ang-(1–7) upregulates hypothalamic NOS in a hypertensive state as a compensatory and protective mechanism to combat hypertension.

Differential baseline expression and angiotensin II stimulation of leukemia-associated RhoGEF in vascular smooth muscle cells of spontaneously hypertensive rats

PurposeStudies to explore angiotensin II (Ang II) and its downstream signaling pathways via Rho guanine nucleotide exchange factors (RhoGEFs) and RhoA signaling are crucial to understanding the mechanisms of smooth muscle contraction leading to hypertension. This study aimed to investigate the Ang II–induced expression of RhoGEFs in vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) and to identify the possible regulator associated with hypertension.MethodsCultured VSMCs of the aorta from SHRs and Wistar-Kyoto (WKY) rats were treated with or without Ang II or Ang II plus Ang II type 2 receptor antagonists. The expression levels of RhoGEF messenger RNA (mRNA) and protein were determined. To evaluate the changes of aortic ring contractile force in response to Ang II, a nonviral carrier system was adopted to deliver the leukemia-associated RhoGEF (LARG) small interfering RNA via nanoparticles into aortic rings.ResultsThe baseline mRNA levels of three RhoGEFs in cultured VSMCs of WKY rats did not increase with age, but they were significantly higher in 12-week-old SHRs than in 5-week-old SHRs. Expression levels of LARG mRNA were higher in SHRs than in age-matched WKY rats. The baseline LAGR protein of 12-week-old SHRs was about four times higher than that of WKY rats of the same age. After Ang II–stimulation, LAGR protein expression was significantly increased in 12-week-old WKY rats but remained unchanged in 12-week-old SHRs. LARG small interfering RNA was successfully delivered into aortic rings using nanoparticles. LARG knockdown resulted in 12-week-old SHRs showing the greatest reduction in aortic ring contraction.ConclusionThere were differences in age-related RhoGEF expression at baseline and in response to Ang II–stimulation between SHRs and WKY rats in this study. Nanotechnology can assist in studying the silencing of LARG in tissue culture. The findings of this study indicate that LARG gene expression may be associated with the genesis of hypertension in SHRs.

Anti-apoptotic and pro-survival effects of exercise training on hypertensive hearts

Activated cardiac apoptosis was found in hearts from hypertensive animals, but little information regarding the effects of exercise training on cardiac apoptosis in hypertension is available. The purpose of this study was to evaluate the anti-apoptotic and pro-survival effects of exercise training on hypertensive hearts. 28 spontaneously hypertensive rats were divided into sedentary group (SHR) or underwent running exercise on treadmill for 1 h/day, 5 sessions/wk, for 12 wk (SHR-EX). Fourteen age-matched Wistar Kyoto rats served as a sedentary normotensive group (WKY). After exercise training or sedentary status, the excised hearts were measured by hemotoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay, and Western blotting. Fewer TUNEL-positive apoptotic cells were in SHR-EX groups than those in SHR. Protein levels of Fas ligand, Fas death receptor, tumor necrosis factor (TNF)-α, TNF receptor 1, Fas-associated death domain (FADD), activated caspase-8, and activated caspase-3 (Fas-dependent apoptotic pathways), as well as Bid, t-Bid, Bad, p-Bad, Bak, cytochrome c, activated caspase 9, and activated caspase-3 (mitochondria-dependent apoptotic pathways) were decreased in the SHR-EX group compared with the SHR group. Protein levels of IGF-1, IGF-1R, p-PI3K, p-Akt, p-Bad, and Bcl2 (cardiac pro-survival pathway) become more activated in SHR-EX groups than SHR and WKY. Exercise training prevented hypertension-enhanced cardiac Fas-dependent and mitochondria-dependent apoptotic pathways and enhanced cardiac pro-survival pathway in rat models. Our findings demonstrate new therapeutic effects of exercise training on hypertensive hearts for preventing apoptosis and enhancing survival.

Effects of Amlodipine Orotate on Hypertension-related Complications in Spontaneously Hypertensive Rats

Hypertension is a common problem in elderly patients, which usually requires chronic therapy under various physiological conditions including low gastric acidity (hypo- or anacidity). This study investigated a new salt type of amlodipine (CAS 88150-42-9) on blood pressure and hypertension-related complications in stroke-prone spontaneously hypertensive rats (SHR-SP). Amlodipine orotate was prepared by reacting orotic acid and amlodipine to increase the dissolution rate at higher gastric pH conditions. Twelve-week-old SHR-SP were randomly divided into five groups to receive either amlodipine orotate or amlodipine besylate (CAS 111470-99-6) at the doses of 3 and 10 mg/kg/day orally for four weeks. The age-matched normotensive Wistar Kyoto rats (WKY) served as the normal positive control group. The systolic blood pressure was reduced in the amlodipine treated SHR-SP in a dose-dependent manner with a similar potency irrespective of the salt type. Both amlodipines also reduced the left ventricular hypertrophy at high doses and concentration-dependently inhibited the Ca2+ induced contraction with a similar potency. Furthermore, semi-quantitative analysis of a cerebral injury revealed that the two salts of amlodipine reduced the stroke-re-lated lesions to a similar degree. These results suggest that the amlodipine orotate is effective in terms of its effects on hypertension, cardiac hypertrophy and stroke-related cerebral damage in SHR-SP.

Role of AMP-Activated Protein Kinase in -Lipoic Acid-Induced Vasodilatation in Spontaneously Hypertensive Rats

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) has recently emerged as an attractive and novel target for the regulation of vascular smooth muscle contraction. The present study investigated the vasodilatory effects of α-lipoic acid (α-LA) and the possible mechanism of its action on aortic rings from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Aortae were removed from WKY and SHR, and contractile responses to acetylcholine and α-LA studied in organ chamber. Phosphorylated AMPK (pAMPK), phosphorylated Peutz-Jeghers syndrome kinase LKB1 (pLKB1) and calcium/calmodulin-dependent protein kinase (CaMKK) protein level were measured in SHR, WKY, and aortic smooth muscle (A10) cells. α-LA (1-500 µmol/l) produced a concentration-dependent relaxation of precontracted aortic rings from 8- and 16-week-old SHR, but not in those from WKY rats. This vasodilatory effect of α-LA did not change after preincubation with N(G)-nitro-L-arginine methyl ester (100 µmol/l), but significantly suppressed by an AMPK inhibitor, compound C (40 µmol/l). The expression of pAMPKα, pLKB1, and CaMKK were also significantly reduced in endothelium-denuded arteries from 16-week-old SHR compared with those from younger SHR or age-matched WKY rats. After incubation with α-LA (100 µmol/l), the expression of pAMPKα and pLKB1 was significantly increased in the endothelium-denuded aortas from 16-week-old SHR, the expression of CaMKK was more reduced in the endothelium-denuded aortas of 8-week-old SHR, but this was not observed in WKY rats. α-LA also activated AMPKα phosphorylation in A10 cells. The effects of α-LA on vascular relaxation in SHR result from the enhanced phosphorylation of LKB1-AMPK in aortic smooth muscle.

Mechanisms of reduced contractility in an animal model of hypertensive heart failure

1. Alterations in intracellular Ca(2+) homeostasis have frequently been implicated as underlying the contractile dysfunction of failing hearts. Contraction in cardiac muscle is due to a balance between sarcolemmal (SL) and sarcoplasmic reticulum (SR) Ca(2+) transport, which has been studied in single cells and small tissue samples. However, many studies have not used physiological temperatures and pacing rates, and this could be problematic given different temperature dependencies and kinetics for transport processes. 2. Spontaneously-hypertensive rats (SHR) and their age-matched Wistar Kyoto controls (WKY) provide an animal model of hypertensive failure with many features in common to heart failure in humans. Steady-state measurements of Ca(2+) and force showed that peak stress was reduced in trabeculae from failing SHR hearts in comparison to WKY, although the Ca(2+) transients were bigger and decayed more slowly. 3. Dynamic Ca(2+) cycling was investigated by determining the recirculation fraction (RF) of activator Ca(2+) through the SR between beats during recovery from experimental protocols that potentiated twitch force. No difference in RF between rat strains was found, although the RF was dependent on the potentiation protocol used. 4. Superfusion with 10 mmol/L caffeine and 0 mmol/L [Ca(2+)](o) was used to measure SL Ca(2+) extrusion. The caffeine-induced [Ca(2+)](i) transient decayed more slowly in SHR trabeculae, suggesting that SL Ca(2+) extrusion was slower in SHR. 5. An ultrastructural immunohistochemical analysis of left ventricular free wall sections using confocal microscopy showed that t-tubule organization was disrupted in myocytes from SHR, with reduced labelling of the SR Ca(2+) -ATPase and Na(+) -Ca(2+) exchanger in comparison to WKY, with the latter possibly related to a lower fraction of t-tubules per unit cell volume. 6. We suggest that although Ca(2+) transport is altered in the progression to heart failure, force development is not limited by the amplitude of the Ca(2+) transient. Despite slower SR Ca(2+) transport, the recirculation fraction and dynamic response to a change of inotropic state minimally altered changes in the SHR model because there was a similar slowing in Ca(2+) extrusion across the surface membrane.

Possible role of nitric oxide generated by leukocytes in the pathogenesis of hypertensive cerebral edema in stroke-prone spontaneously hypertensive rats

We investigated the mechanisms responsible for cerebral edema in stroke-prone spontaneously hypertensive rats (SHRSP), including leukocyte activation and nitric oxide (NO) generation, both in vivo and in vitro. We also investigated the effects of angiotensin II (AngII) on leukocyte function in relation to NO production. Leukocyte–endothelial cell adhesion in brain microvessels was investigated by electron tomography using ultra-high voltage electron microscopy. Electron tomography clearly showed that leukocytes had well-developed intracellular organelles and abundant microvilli that were tangled with the endothelial cells in brain microvessels. Under confocal microscopic examination, diaminofluorescein-2 (a NO indicator)-positive cells were closely localized to anti-granulocyte-positive cells. The fluorescence intensity was much stronger in SHRSP than in age-matched Wistar–Kyoto (WKY) rats, as a normotensive control. Mac-1 (leukocyte integrin, CD11b/CD18), angiotensin type 1 (AT1) receptor and inducible nitric oxide synthase (iNOS) expression was higher (or tended to be higher) in SHRSP leukocytes than in WKY leukocytes. The plasma NO metabolite content was also higher in SHRSP than in WKY rats. All of these factors were upregulated by AngII stimulation. Furthermore, NO release from leukocytes was enhanced by AngII or lipopolysaccharide through NF-κB activation, but was suppressed by an AT1 receptor blocker or s-methyl-isothiourea. The present study revealed that one of the causative factors for cerebral edema in SHRSP rats is the generation of NO radicals by iNOS activated via NF-κB in AngII-stimulated leukocytes. Thus, the pathogenesis of cerebral edema in SHRSP is likely to involve inflammatory processes mediated by AngII.

Ascorbic Acid Levels and Neutrophil Superoxide Production in Blood of Pre-, Early and Late Hypertensive Stroke-Prone Spontaneously Hypertensive Rats

We compared ascorbic acid (AA) levels in the blood and TPA- and fMLP-stimulated superoxide (O2•−) production in neutrophils of pre-, early, and late hypertensive stroke-prone spontaneously hypertensive rats (SHRSP) with those of age-matched Wistar Kyoto rats (WKY), or two other normotensive strains of rats. Plasma and lymphocyte AA levels were about two-fold higher in SHRSP as early as 4 weeks old compared to WKY, and also higher than those of Wistar and Sprague-Dawley (SD) rats. Levels of AA were high in the liver and adrenal glands of SHRSP, indicating congenitally high AA levels. The production of O2•− in neutrophils was about two-fold higher in SHRSP than in WKY even at 4 weeks of age, and increased with age in both strains. Among SHRSP, AA levels in lymphocytes decreased at the late hypertensive stages with a decrease in hepatic l-gulono-γ-lactone oxidase (GLO) activities. These data suggest that bi-phasic AA levels in the blood of SHRSP comprise congenitally high levels and a decrease after persistent hypertension due to enhanced O2•− production and a decrease in de novo AA synthesis through GLO.

Long-term exercise attenuates blood pressure responsiveness and modulates kidney angiotensin II signalling and urinary sodium excretion in SHR

Observations have been made regarding the effects of long-term exercise training on blood pressure, renal sodium handling and renal renin-angiotensin-aldosterone (RAS) intracellular pathways in conscious, trained Okamoto-Aoki spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKy) normotensive rats, compared with appropriate age-matched sedentary SHR and WKy. To evaluate the influence of exercise training on renal function and RAS, receptors and intracellular angiotensin II (AngII) pathway compounds were used respectively, and lithium clearance and western blot methods were utilised. The current study demonstrated that increased blood pressure in SHR was blunted and significantly reduced by long-term swim training between the ages of 6 and 16 weeks. Additionally, the investigators observed an increased fractional urinary sodium excretion in trained SHR (SHR(T)) rats, compared with sedentary SHR (SHR(S)), despite a significantly decreased creatinine clearance (C(Cr)). Furthermore, immunoblotting analysis demonstrated a decreased expression of AT1(R) in the entire kidney of T(SHR) rats, compared with S(SHR). Conversely, the expression of the AT2(R), in both sedentary and trained SHR, was unchanged. The present study may indicate that, in the kidney, long-term exercise exerts a modulating effect on AngII receptor expression. In fact, the present study indicates an association of increasing natriuresis, reciprocal changes in renal AngII receptors and intracellular pathway proteins with the fall in blood pressure levels observed in T(SHR) rats compared with age-matched S(SHR) rats.

α2-Adrenoceptor-Mediated Inhibition of Catecholamine Release from the Adrenal Medulla of Spontaneously Hypertensive Rats is Preserved in the Early Stages of Hypertension

In this study, we evaluated the effect of α(2) -adrenoceptor activation on catecholamine release from the adrenal medulla of pre-hypertensive (6-week-old) and hypertensive (16-week-old) spontaneously hypertensive rats (SHR) and of age-matched normotensive control Wistar Kyoto (WKY) rats. Catecholamine overflow from isolated adrenal medullae was evoked by the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) in the absence and presence of the α(2) -adrenoceptor agonist medetomidine (MED). The spontaneous outflow of adrenaline was similar between age-matched SHR and WKY rats. However, the spontaneous outflow of noradrenaline was significantly lower in SHR compared with age-matched WKY rats. DMPP (0.1-3 mM) increased the outflow of noradrenaline and adrenaline in a concentration-dependent manner. The E(max) values for adrenaline overflow were similar between strains, but the E(max) values for noradrenaline overflow were significantly lower in SHR. The EC(50) values for noradrenaline and adrenaline overflow were significantly higher in SHR compared with age-matched WKY rats. MED (0.1-300 nM) reduced the DMPP-evoked overflow (DMPP 500 μM) of noradrenaline and adrenaline in a concentration-dependent manner and was capable of totally inhibiting this effect. The inhibitory action of MED was similar between age-matched SHR and WKY rats. In the adrenals, the α(2A)- and α(2B)-adrenoceptor subtypes had the highest mRNA expression levels; the α(2C)-adrenoceptor subtype had the lowest mRNA expression levels. The mRNA levels for the three subtypes were similar between strains. In conclusion, in SHR during the development of hypertension, adrenal α(2) -adrenoceptor inhibitory function is conserved, accompanied by reduced noradrenaline release and unchanged adrenaline release.