Involvement of sex hormones, oxidative stress, ACE and ACE2 activity in the impairment of renal function and remodelling in SHR.

AIMS: Hypertension is a relevant sex and sex hormones-dependent risk factor where the cardiovascular and renal health of the population are concerned. Men experience greater losses of renal function (RF) than women, but the mechanisms remain somewhat unclear. Our goal was to evaluate the relationship between oxidative stress (OS), angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activities and RF in male and female SHR. MAIN METHODS: Twelve-week-old spontaneously hypertensive rats (SHR) were submitted to either castration or SHAM surgery and divided into 4 groups, SHAM or Castrated (CAST) males or females. After 51 days we evaluated RF (inulin and sodium para-aminohippurate), ACE and ACE2 activities (fluorimetry), OS (flow cytometry), collagen deposition (picrosirius red) and protein expression (western blot). KEY FINDINGS: Males presented lower RF than females and castration impaired this parameter in both groups. Sexual dimorphism was not observed regarding OS and inflammation; however, castration increased this parameter more severely in males than in females. SHAM males exhibited higher collagen deposition than females, though castration increased it in both sexes, eliminating the difference. We found sexual dimorphism regarding renal ACE and ACE2 activities, which were lower in males than in females. Although castration did not alter ACE activity, it reduced ACE2 activity in females and increased it in males. SIGNIFICANCE: These results indicate that sex hormones affect RF in SHR. As alterations in the oxidative system were capable of promoting podocyte injury, inflammation, and collagen deposition, we put forward that these effects are differently modulated by ACE and ACE2.

Anti-Renal Fibrotic Effect of Exercise Training in Hypertension.

The purpose of this study was to evaluate the effects of exercise training on renal fibrosis in hypertensive rats. Masson's trichrome staining and Western blotting were performed on the excised renal cortex from sixteen male spontaneously hypertensive rats (SHR), which were randomly divided into either a sedentary hypertensive group (SHR) or exercise hypertensive group (SHR-EX, running on an exercise treadmill for 60 min/day, 5 sessions/week, for 12 weeks), and from eight male Wistar-Kyoto rats which served as a sedentary normotensive group (WKY). The systolic blood pressure (SBP) and renal fibrosis in hypertensive rats improved after exercise training. The inflammatory-related protein levels of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), as well as the fibrotic-related protein levels of transforming growth factor-beta (TGF-ß), phospho-Smad2/3 (p-Smad2/3), connective tissue growth factor (CTGF), matrix metalloproteinase-9 (MMP-9), and matrix metalloproteinase-2 (MMP-2) were decreased in the SHR-EX group when compared with the SHR group. Exercise training suppressed the hypertension-induced renal cortical inflammatory and fibrotic pathways in hypertensive rat models. These findings might indicate a new therapeutic effect for exercise training to prevent renal fibrosis in hypertensive nephropathy.

Cytochrome P450 1B1 contributes to increased blood pressure and cardiovascular and renal dysfunction in spontaneously hypertensive rats.

PURPOSE: We investigated the contribution of cytochrome P450 (CYP) 1B1 to hypertension and its pathogenesis by examining the effect of its selective inhibitor, 2,4,3',5'-tetramethoxystilbene (TMS), in spontaneously hypertensive rats (SHR). METHODS: Blood pressure (BP) was measured bi-weekly. Starting at 8 weeks, TMS (600 µg/kg, i.p.) or its vehicle was injected daily. At 14 weeks, samples were collected for measurement. RESULTS: TMS reversed increased BP in SHR (207 ± 7 vs. 129 ± 2 mmHg) without altering BP in Wistar-Kyoto rats. Increased CYP1B1 activity in SHR was inhibited by TMS (RLU: aorta, 5.4 ± 0.7 vs. 3.7 ± 0.7; heart, 6.0 ± 0.8 vs. 3.4 ± 0.4; kidney, 411 ± 45 vs. 246 ± 10). Increased vascular reactivity, cardiovascular hypertrophy, endothelial and renal dysfunction, cardiac and renal fibrosis in SHR were minimized by TMS. Increased production of reactive oxygen species and NADPH oxidase activity in SHR, were diminished by TMS. In SHR, TMS reduced increased plasma levels of nitrite/nitrate (46.4 ± 5.0 vs. 28.1 ± 4.1 µM), hydrogen-peroxide (36.0 ± 3.7 vs. 14.1 ± 3.8 µM), and thiobarbituric acid reactive substances (6.9 ± 1.0 vs. 3.4 ± 1.5 µM). Increased plasma levels of pro-inflammatory cytokines and catecholamines, and cardiac activity of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, c-Src tyrosine kinase, and protein kinase B in SHR were also inhibited by TMS. CONCLUSIONS: These data suggests that increased oxidative stress generated by CYP1B1 contributes to hypertension, increased cytokine production and sympathetic activity, and associated pathophysiological changes in SHR. CYP1B1 could be a novel target for developing drugs to treat hypertension and its pathogenesis.

Development of novel rat model for high-fat and high-cholesterol diet-induced steatohepatitis and severe fibrosis progression in SHRSP5/Dmcr.

OBJECTIVES: Patients with nonalcoholic fatty liver disease are increasing worldwide, and preventive measures are an urgent need and primary concern today. AIM: This study aimed to develop and clarify the usefulness of the SHRSP5/Dmcr rat, derived from a stroke-prone spontaneously hypertensive rat, as a novel animal model for time-course analysis of steatohepatitis and the severe fibrosis progression often observed in the disease. METHODS: Ten-week-old male SHRSP5/Dmcr rats were divided into six groups: half were fed a high-fat and high-cholesterol-containing diet (HFC diet), and the others the control, stroke-prone (SP) diet for 2, 8, and 14 weeks. RESULTS: The HFC diet significantly increased serum transaminase and gamma glutamyl transpeptidase activities, tumor necrosis factor alpha levels, and serum and hepatic total cholesterol levels over time. In contrast, this diet decreased serum albumin, glucose, and adiponectin levels throughout or the later stage of the feeding period, but did not influence serum insulin levels. Histopathologically, the HFC diet increased microvesicular steatosis, and focal or spotty necrosis with lymphocyte infiltrations were observed in the liver at 2 weeks, macrovesicular steatosis, ballooned hepatocytes with Mallory-Denk body formation in some, and multilobular necrosis and fibrosis at 8 weeks. Interestingly, this fibrosis formed a honeycomb network at 14 weeks. These changes are very similar to those observed in patients with non-alcoholic steatohepatitis. CONCLUSIONS: SHRSP5/Dmcr rats appear to be a useful model for analyzing the time-dependent changes of HFC diet-induced steatohepatitis and fibrosis progression.

Trabecular bone area and bone healing in spontaneously hypertensive rats: a histometric study

Clinical and experimental studies have demonstrated some negative effect of hypertension on bone mineral density. The aim of this study was to evaluate bone healing and trabecular bone area (TBA) in spontaneously hypertensive rats (SHR), a well-established model of essential hypertension, when compared to normotensive rats (NTR). A critical-size defect was surgically created in the right tibia of SHR (n = 12) and normotensive rats (NTR; n = 12), while the contralateral tibia was left intact. Eight days later, the animals were sacrificed and the specimens processed in order to obtain decalcified sections. The area of newly-formed bone (NB) within the defect of the right tibia and the TBA in the left tibia were histometrically evaluated. At 8 days post-operative, SHR presented a significantly smaller area of NB when compared to NTR (p < 0.05). In addition, SHR demonstrated a lower TBA than NTR group. In conclusion, the present study demonstrated that SHR rats presented a disturbed bone healing and reduced TBA.

Perfil nutricional e cardiovascular de ratos normotensos e hipertensos sob dieta hiperlipídica / Nutritional and cardiovascular profiles of normotensive and hypertensive rats kept on a high fat diet / Perfil nutricional y cardiovascular de ratas normotensas e hipertensos bajo dieta hiperlipídica

FUNDAMENTO: Embora dietas hiperlipídicas (DH) promovam distúrbios nutricionais e cardíacos, poucos estudos avaliaram sua influência em ratos normotensos Wistar-Kyoto (WKY) e espontaneamente hipertensos (SHR). OBJETIVO: Avaliar e comparar o perfil nutricional e cardiovascular de WKY e SHR tratados com DH. MÉTODOS: 20 WKY e 20 SHR foram distribuídos em quatro grupos: WKY-controle (WKY-C), WKY-DH, SHR-controle (SHR- C) e SHR-DH. Os grupos C e DH receberam, respectivamente, dieta normocalórica e DH durante 20 semanas. Foram avaliados: peso corporal (PC), adiposidade, glicemia, lípides séricos, com dosagens de colesterol total e triacilglicerol, insulina e leptina. O estudo cardiovascular contemplou a pressão arterial sistólica (PAS), avaliação cardiopulmonar anatômica, ecocardiograma e histologia cardíaca. RESULTADOS: Os SHRs apresentaram menor PC, adiposidade, glicose, colesterol, triacilglicerol, leptina e insulina, quando comparados aos WKYs. Nos SHR, a ingestão calórica aumentou com a DH. Já nos WKYs, a DH elevou a eficiência energética, a adiposidade e a leptina e reduziu a glicemia. Na avaliação cardiovascular, os SHR apresentaram maior PAS, umidade pulmonar, hipertrofia e fibrose intersticial miocárdica em relação aos WKYs (p<0,01); mas a função cardíaca foi similar entre as cepas. A DH reduziu o diâmetro sistólico ventricular nos WKY e acentuou a relação E/A mitral, as espessuras diastólicas do septo interventricular e da parede posterior bem como a fibrose intersticial do ventrículo esquerdo. CONCLUSÃO: Embora não tenha afetado significativamente o perfil nutricional dos SHRs, o tratamento acentuou a remodelação cardíaca e precipitou o aparecimento de disfunção diastólica ventricular. Nos WKY, a dieta aumentou a adiposidade e a leptinemia, e promoveu modificações cardiovasculares não significantes.

BACKGROUND: Although a high fat diet (HFD) promotes nutritional and heart disorders, few studies have assessed its influence in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). OBJECTIVE: To evaluate and compare the nutritional and cardiovascular profiles of WKY and SHR on a high fat diet. METHODS: 20 WKY and 20 SHR were divided into four groups: Control-WKY (C-WKY), HFD-WKY, Control-SHR (C-SHR) and HFD-SHR. The C and HFD groups received, respectively, a normocaloric diet and a HFD for 20 weeks. The following features were evaluated: body weight (BW), adiposity, blood glucose, serum lipids, with measurements of total cholesterol and triacylglycerol levels, insulin and leptin. The cardiovascular study included the systolic blood pressure (SBP), a cardiopulmonary anatomical evaluation, an echocardiography and heart histology. RESULTS: The SHR had BW, adiposity, glucose, cholesterol, triacylglycerol, leptin and insulin levels lower than the WKY. In SHR, the caloric intake increased with HFD. In WKY, the HFD increased energy efficiency, adiposity and blood leptin, and reduced glucose. In the cardiovascular assessment, the SHR had SBP, pulmonary moisture, myocardial hypertrophy and interstitial fibrosis higher than the WKY (p <0.01); the cardiac function was similar in both strains. The HFD reduced the ventricular systolic diameter in the WKY and increased the mitral E/A ratio, the diastolic thickness of the interventricular septum and the posterior wall, as well as the interstitial fibrosis of the left ventricle. (Arq Bras Cardiol 2009; 93(5) : 487-494) CONCLUSION: Although it had not significantly affected the nutritional profile of the SHR, the treatment increased cardiac remodeling and precipitated the emergence of ventricular diastolic dysfunction. In WKY, the diet increased adiposity and leptinemia, and promoted non-significant cardiovascular changes.

FUNDAMENTO: Embora dietas hiperlipídicas (DH) promovam distúrbios nutricionais e cardíacos, poucos estudos avaliaram sua influência em ratos normotensos Wistar-Kyoto (WKY) e espontaneamente hipertensos (SHR). OBJETIVO: Evaluar y comparar el perfil nutricional y cardiovascular de WKY y SHR tratadas con DH. MÉTODOS: Un total de 20 WKY y 20 SHR se distribuyó en cuatro grupos: WKY-control (WKY-C), WKY-DH, SHR-control (SHR-C) y SHR-DH. Los grupos C y DH recibieron, respectivamente, dieta normocalórica y DH durante 20 semanas. Se evaluaron: peso corporal (PC), adiposidad, glucemia, lípidos séricos, con dosificaciones de colesterol total y triacilglicerol, insulina y leptina. El estudio cardiovascular contempló la presión arterial sistólica (PAS), evaluación cardiopulmonar anatómica, ecocardiograma e histología cardiaca. RESULTADOS: Las SHRs presentaron menor PC, adiposidad, glucosa, colesterol, triacilglicerol, leptina e insulina, cuando comparadas a las WKYs. En las SHR, la ingestión calórica aumentó con la DH. Sin embargo en las WKYs, la DH elevó la eficiencia energética, la adiposidad y la leptina y reduzco la glucemia. En la evaluación cardiovascular, las SHR presentaron mayor PAS, humedad pulmonar, hipertrofia y fibrosis intersticial miocárdica en cuanto a las WKYs (p<0,01); sin embargo la función cardiaca se halló similar entre las cepas. La DH reduzco el diámetro sistólico ventricular en los WKY y acentuó la relación E/A mitral, los espesores diastólicos del septo interventricular y de la pared posterior así como la fibrosis intersticial del ventrículo izquierdo. CONCLUSIÓN: Aunque no afectó significativamente el perfil nutricional de las SHRs, el tratamiento acentuó la remodelación cardiaca y precipitó el aparecimiento de disfunción diastólica ventricular. En los WKY, la dieta aumentó la adiposidad y la leptinemia, y promovió modificaciones cardiovasculares no significantes.

Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis.

AIM: Overexpression of human tissue kallikrein (HK), mediated by recombinant adeno-associated virus (rAAV), decreased blood pressure in spontaneous hypertensive rats (SHRs) and reduced injury to the heart, aorta and kidney. In this study, we used both an in vivo animal model and in vitro cell culture system to investigate whether rAAV-mediated HK gene therapy protects against organ damage by inhibiting cell apoptosis. METHODS: rAAV encoding HK (rAAV-HK) or LacZ (rAAV-lacZ) were delivered as a control to spontaneously hypertensive rats (SHRs) and cultured human embryonic kidney (HEK) 293 cells. RESULTS: Treatment with rAAV-HK decreased cell apoptosis in the target organs of SHRs and also inhibited lipopolysaccharide (LPS)-induced HEK 293 apoptosis. The rAAV-HK delivery system also increased the levels of apoptosis-inhibiting proteins bcl-2 and bcl-x(L), and decreased the level of Bax and the activity of caspase 3, two promoters of apoptosis. In addition to its role in the inhibition of apoptosis, rAAV-HK also activated the cell survival and proliferation signaling pathways ERK1/2 and PI3K/AKT. CONCLUSION: rAAV-mediated HK gene delivery has multiple therapeutic possibilities for treating hypertension, not only by decreasing blood pressure, but also by directly inhibiting end-organ damage.

Adenosine receptor antagonists improve short-term object-recognition ability of spontaneously hypertensive rats: a rodent model of attention-deficit hyperactivity disorder.

The strain of spontaneously hypertensive rats (SHR) is considered a genetic model for the study of attention-deficit hyperactivity disorder (ADHD), as it displays hyperactivity, impulsivity and poorly sustained attention. Recently, we have shown the involvement of adenosinergic neuromodulation in the SHR's short-term and long-term memory impairments. In this study, we investigated the performance of male and female SHR in a modified version of the object-recognition task (using objects with different structural complexity) and compared them with Wistar rats, a widely used outbred rat strain for the investigation of learning processes. The suitability of the SHR strain to represent an animal model of ADHD, as far as mnemonic deficits are concerned, was pharmacologically validated by the administration of methylphenidate, the first-choice drug for the treatment of ADHD patients. The role of adenosine A1 and A2A receptors in object discrimination was investigated by the administration of caffeine (nonselective antagonist) or selective adenosine receptor antagonists. Wistar rats discriminated all the objects used (cube vs. pyramid; cube vs. T-shaped object), whereas SHR only discriminated the most structurally distinct pairs of objects (cube vs. pyramid). Pretraining administration of methylphenidate [2 mg/kg, intraperitoneal (i.p.)], caffeine (1-10 mg/kg, i.p.), the selective adenosine receptor antagonists DPCPX (8-cyclopenthyl-1,3-dipropylxanthine; A1 antagonist, 5 mg/kg, i.p.) and ZM241385 (A2A antagonist, 1.0 mg/kg, i.p.), or the association of ineffective doses of DPCPX (3 mg/kg) and ZM241385 (0.5 mg/kg), improved the performance of SHR in the object-recognition task. These findings show that the discriminative learning impairments of SHR can be attenuated by the blockade of either A1 or A2A adenosine receptors, suggesting that adenosinergic antagonists might represent potentially interesting drugs for the treatment of ADHD.

Mechanisms of insulin resistance in hypertensive rats.

Insulin resistance and hyperinsulinemia are common findings in patients with essential hypertension. Recent evidence indicates that these impairments in glucose metabolism may play a role not only in the development of type 2 diabetes, but also in the onset and persistence of hypertension, dyslipidemia, and abdominal obesity. The accumulation of these risk factors constitutes a high-risk group of cardiovascular diseases, the so-called metabolic syndrome. Insulin resistance has also been reported in several animal models for hypertension, including the spontaneously hypertensive rat (SHR) and the fructose-fed rat (FFR). SHRs and FFRs have been employed in many studies to investigate the mechanisms and pathophysiology of insulin resistance and hypertension, but the precise mechanism of insulin resistance remains to be clarified. In this review, the possible mechanisms of insulin resistance in SHRs and FFRs are summarized.

Angiotensin II type 1 receptor blocker irbesartan ameliorates vascular function in spontaneously hypertensive rats regardless of oestrogen status.

BACKGROUND: Angiotensin II type 1 (AT1) receptor overexpression may play a decisive role in endothelial dysfunction during oestrogen deficiency in spontaneously hypertensive rats (SHRs). Similarly, exaggerated production of angiotensin II and enhanced expression of AT1 receptor have been reported in vessels of SHRs compared with normotensive rats. OBJECTIVE: To test the hypothesis that antihypertensive treatment with the AT1 receptor antagonist, irbesartan, could not only decrease blood pressure but also ameliorate endothelial dysfunction associated with both hypertension and oestrogen deficiency. METHODS: Ovariectomized and sham-ovariectomized SHRs were treated with 50 mg/kg irbesartan per day, administered with chow for 30 weeks. Sham-ovariectomized and ovariectomized rats receiving no treatment were used as control groups. At the end of the treatment period, the vascular reactivity of aortic rings was studied. RESULTS: In the irbesartan-treated groups, vasoconstriction induced by Nomega-nitro-l-arginine methyl ester (l-NAME) was increased and the response to phenylephrine exhibited greater potentiation in the presence of l-NAME, demonstrating a greater availability of basal nitric oxide in these groups. In addition, chronic treatment with irbesartan similarly enhanced the responsiveness of aortic rings from ovariectomized or sham-ovariectomized rats to acetylcholine and sodium nitroprusside. Incubation with indomethacin did not significantly alter acetylcholine- and sodium nitroprusside-induced relaxations in the irbesartan-treated rats. However, relaxations induced by acetylcholine and sodium nitroprusside in aortic rings from non-treated rats were significantly greater in the presence of indomethacin. CONCLUSION: Our data suggest that irbesartan enhances basal nitric oxide availability and ameliorates vascular relaxations in SHRs, by decreasing the production of cyclooxygenase-dependent contracting factors in smooth muscle cells, regardless of oestrogen status.

Contribution of sarcoplasmic reticulum Ca2+ to the activation of Ca2+ -activated K+ channels in the resting state of arteries from spontaneously hypertensive rats.

OBJECTIVE: Localized release of Ca2+ from the sarcoplasmic reticulum (SR) toward the plasmalemma, sometimes visualized as Ca2+ sparks, can activate Ca2+-activated K+ (KCa) channels. We have already reported that the addition of charybdotoxin (ChTX), a blocker of KCa channels, to the resting state of arteries from spontaneously hypertensive rats (SHR) caused a powerful contraction, suggesting that KCa channels were active in the resting state. This study aimed to determine whether the Ca2+ responsible for activity of KCa channels was derived from SR. METHODS: Possible mechanisms underlying the ChTX-induced contractions were examined in endothelium-denuded strips of femoral, mesenteric, small mesenteric and carotid arteries from 13-week-old SHR and normotensive Wistar-Kyoto (WKY) rats by using selective inhibitors of the Ca2+ spark process. RESULTS: ChTX (100 nmol/l) induced a contraction in the SHR arteries. The ChTX-induced contractions were increased by a moderate membrane depolarization by 15.9 mmol/l K+ and were abolished by nifedipine (100 nmol/l). When SR Ca2+ was depleted by treatment of the strips with ryanodine (10 mumol/l) plus caffeine (20 mmol/l) or with thapsigargin (100 nmol/l), the ChTX-induced contraction was decreased in femoral, mesenteric and small mesenteric arteries and was almost abolished in the carotid artery. A similar phenomenon can be observed in arteries from WKY rats after a moderate membrane depolarization. In both SHR and WKY rats, SR Ca2+-dependent ChTX-induced contraction always represents 20-30% of the maximal K+-induced contraction. CONCLUSIONS: We conclude that activation of KCa channels depended upon influx of Ca2+ through L-type Ca2+ channels and release of Ca2+ from the SR, suggesting that recycling of entering Ca2+ from the superficial SR toward the plasmalemma sufficiently elevated Ca2+ near these channels to activate them.

Type 1 parathyroid hormone receptor expression level modulates renal tone and plasma renin activity in spontaneously hypertensive rat.

These studies examine whether PTHrP(1-36), a vasodilator, modulates BP and renal vascular resistance (RVR) in spontaneously hypertensive rat (SHR). Within the kidney of normotensive rats, PTHrP(1-36) was enriched in vessels. In vessels of SHR, PTHrP was upregulated by 40% and type 1 PTH receptor (PTH1R) was downregulated by 65% compared with normotensive rats. To investigate the role of endogenous PTHrP in the regulation of BP and RVR, SHR were subjected to somatic human (h)PTH1R gene delivery. Three weeks after a single intravenous injection of pcDNA1.1 plasmid containing the hPTH1R gene under the control of the cytomegalovirus promoter, hPTH1R mRNA was detected in all of the main organs. Within the kidney, the transgene was enriched in vessels. In the isolated perfused kidney, RVR was reduced by 23% and PTHrP(1-36)-induced vasodilation, which is depressed in SHR, was restored and a vasoconstrictory response to PTH(3-34), a PTH1R antagonist, was revealed. These effects were not observed in control SHR treated with empty plasmid. BP remained unchanged, and plasma renin activity increased by 60%. Thus, in SHR renal vessels, a reduced number of PTH1R contributes to the high RVR, despite the higher expression of vasodilatory PTHrP. Moreover, these studies provide evidence for a direct link between the density of PTH1R and plasma renin activity, which might be responsible for the absence of effect of PTH1R gene delivery on BP in SHR. Overall, PTHrP significantly contributes to the homeostasis of renal and systemic hemodynamics in SHR.

Tubular cell apoptosis and proliferation in the early phase of renal damage in uninephrectomized SHR.

In the present study, we measured tubular cell apoptosis and proliferation and Bcl-2 expression during the early phase (3 months) of the process of renal fibrosis in the experimental model of uninephrectomized spontaneously hypertensive rats (SHR). Tubulointerstitial fibrosis was evaluated by automated quantitative morphometry using selective staining of the extracellular matrix with sirius red. Apoptosis was quantified by both in situ dUTP biotin nick end-labeling method (TUNEL) and by propidium iodide staining. Proliferation rate was measured by counting cells expressing the proliferating cell nuclear antigen. Bcl-2 expression was assessed by immunohistochemistry. Tubulointerstitial fibrosis increased progressively during the 3 months of follow-up. Proliferation and apoptosis rates in tubular cells increased from the first to the second month after UNX. In the third month after UNX, the proliferating tubular cell number continued to increase, whereas the apoptotic cell number was maintained, coinciding with an increase in the expression of Bcl-2. Our observations demonstrate a different profile of tubular cell proliferation and apoptosis during the genesis of early tubulointerstitial damage in UNX-SHR.

L-type Ca(2+) channel regulation by pituitary adenylate cyclase-activating polypeptide in vascular myocytes from spontaneously hypertensive rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a vasoactive peptide, modulates the L-type Ca(2+) channel current (L channel current) in vascular smooth muscle cells (VSMC) through activation and integration of two intracellular pathways, protein kinase A and protein kinase C (PKC). In the present study we compared the effects of PACAP on the L channel current in VSMC from the spontaneously hypertensive rats (SHR) and normotensive controls, Wistar Kyoto rats (WKY). We found that compared with WKY, VSMC from SHR had a higher L channel current density. Stimulation by PACAP (10 nM) caused an increase in the amplitude of the whole cell current and prolonged open time in VSMC from SHR and WKY, with the increase greater in SHR. These effects of PACAP on the L channel current was mimicked by an activator of PKC. In contrast, PACAP caused a smaller increase in cAMP accumulation in VSMC from SHR than WKY, and there was no difference in the inhibitory effect of 8-bromo-cAMP on the L channel current from both type of cells. The greater increase in amplitude of the L channel current by PACAP in VSMC from SHR persisted in the presence of adenosine cyclic 3',5'-monophosphothioate, Rp-isomer, a cAMP antagonist, but not calphostin C, a PKC inhibitor. Taken together, our results show an increase in L channel current density and an enhanced PACAP effect on the L channel current in VSMC from SHR compared with WKY. This difference in PACAP response appears to be predominately secondary to an increased PKC sensitivity.

Cysteinyl leukotrienes mediate enhanced vasoconstriction to angiotensin II but not endothelin-1 in SHR.

We assessed whether cysteinyl leukotrienes mediate the vasoconstrictor responses to angiotensin II and endothelin-1 in the mesenteric vascular bed of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) perfused ex vivo at a constant flow rate of 5 ml/min with Krebs buffer. Maximal perfusion pressure response (E(max)) but not EC(50) values to angiotensin II (P < 0.001) and endothelin-1 (P < 0.01) were significantly higher in the SHR, whereas the responses to potassium chloride remained unchanged. Inclusion of the selective 5-lipoxygenase inhibitor AA-861 or the cysteinyl leukotriene receptor antagonist MK-571 significantly reduced the vasoconstrictor responses to angiotensin II but not to endothelin-1 and potassium chloride. The reduction in E(max) to angiotensin II was more pronounced in SHR (P < 0.001) than in WKY (P < 0.05) rats. Cysteinyl leukotrienes LTC(4)-, LTD(4)-, and LTE(4) (1 microM)-evoked vasoconstrictor responses were significantly higher in SHR (P < 0.05), whereas LTB(4) failed to evoke any response in either strain. These data suggest that 5-lipoxygenase metabolites, particularly cysteinyl leukotrienes, contribute to the exaggerated vasoconstrictor responses to angiotensin II but not to endothelin-1.

Differential salt-sensitivity in the pathogenesis of renal damage in SHR and stroke prone SHR.

The spontaneously hypertensive rat (SHR) and the stroke prone SHR (SHRsp) display contrasting susceptibilities to the development of the severe hypertensive lesions of malignant nephrosclerosis, both with aging and after the provision of a high salt intake on the background of a Japanese style "stroke prone" rodent diet. The SHR is relatively resistant, whereas the SHRsp is markedly susceptible. The responsible mechanisms remain controversial. Blood pressure (BP) radiotelemetry was used to investigate the interrelationship between salt intake, systolic BP, and renal damage in 8- to 12-week-old male SHR and SHRsp given a standard North American style diet for 6 weeks, a standard diet plus 1% NaCl as drinking water for 6 weeks, or an 8% NaCl diet plus tap water for 4 weeks. After 4 weeks, BP was significantly greater in the SHRsp compared to the SHR and was significantly more sensitive to supplemental salt in the SHRsp than in SHR. Average systolic pressures during week 5 (after 4 weeks on standard diet plus tap water, standard diet plus 1% NaCl, and 8% NaCl diet plus tap water) were 188.0 +/- 3.0 mm Hg, 207.3 +/- 5.6 mm Hg, and 226 +/- 9.4 mm Hg in SHRsp compared with 171.4 +/- 3.8 mm Hg, 180.6 +/- 3.8 mm Hg, and 190.3 +/- 5.0 mm Hg in SHR. In the absence of supplemental NaCl, both strains exhibited minimal evidence of hypertensive renal damage until about 16 weeks of age. A high salt intake resulted in the development of lesions of malignant nephrosclerosis (fibrinoid necrosis and thrombosis of small vessels and glomeruli) in the SHRsp but not in the SHR; semiquantitative histologic renal damage scores in SHRsp versus SHR being 10.4 +/- 2.0 versus 0.7 +/- 0.2 after 6 weeks of standard diet plus 1% NaCl, and 32.1 +/- 2.5 versus 0.7 +/- 0.4 after 4 weeks of 8% NaCl diet plus tap water; P < .001 for both comparisons. The development of more severe hypertension in salt-supplemented SHRsp could only partly account for the severity of renal damage in SHRsp, the increase in which was disproportionate to the increase in absolute BP. However, the rate of increase of BP was greater in the SHRsp and this might have contributed to the greater renal damage observed in the SHRsp. These data indicate that the contrasting genetic susceptibility to renal damage between SHR and SHRsp is mediated, at least in part, by a differential BP salt sensitivity.

Abnormal Ca2+ signalling in vascular endothelial cells from spontaneously hypertensive rats: role of free radicals.

OBJECTIVE: To test the hypothesis that the Ca2+ signal transduction process in endothelial cells from genetically hypertensive rats (SHR) is affected by an overproduction of free radicals. METHODS: The Ca2+ response to the inositol 1,4,5-triphosphate (IP3) mobilizing agonist, ATP, was measured using the fluorescent probe, fura-2, in endothelial cells from Sprague-Dawley rats, and in young and age-matched genetically hypertensive rats (SHR). The effect of free radicals and reducing agents on the intracellular release of Ca2+ and IP3productionwas determined in resting and ATP-stimulated cells. Experiments were also performed to compare the level of expression and enzymatic activity of catalase and superoxide dismutase (SOD) in endothelial cells from SHR and Sprague-Dawley rats. RESULTS: The exposure of aortic endothelial cells from Sprague-Dawley rats to the free-radical generating system, hypoxanthine + xanthine oxidase (HX/XO), caused a time- and concentration-dependent inhibition of the ATP-induced Ca2+ response. A similar HX/XO-dependent inhibition was also observed in Sprague-Dawley cells stimulated with the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin. Incubation with the antioxidative enzymes, catalase and SOD, had no effect on the ATP-induced Ca2+ release in Sprague-Dawley cells, but led to a strong increase in the internal release of Ca2+ in cells from adult (12 weeks old) or young (3 weeks old) SHR. The effect of antioxidants was not related either to an enhancement of the ATP-induced production of IP3, or to a lower expression and activity of SOD and catalase. CONCLUSION: The present work provides evidence that the Ca2+ signalling process in SHR endothelial cells is affected by an overproduction of free radicals, resulting in a depletion of releasable Ca2+ from IP3-sensitive and insensitive Ca2+ pools. These results point towards a beneficial action of antioxidants on Ca2+ signalling in endothelial cells from models of hypertension.

Genistein, daidzein and glycitein inhibit growth and DNA synthesis of aortic smooth muscle cells from stroke-prone spontaneously hypertensive rats.

Recent studies have reported that estrogen replacement therapy (ERT) reduces the risk of cardiovascular diseases in postmenopausal women. However, mechanisms responsible for this effect are not yet completely understood, and ERT is associated with carcinogenic side effects in women and feminizing effects in men. Because soybean isoflavones, a group of natural phytoestrogens, have only weak estrogenic activity and are not known to have side effects such as carcinogenesis and feminization, we evaluated the effects of genistein, daidzein and glycitein on the growth and DNA synthesis of aortic smooth muscle cells (SMC) from stroke-prone spontaneously hypertensive rats (SHRSP). SMC were cultured in dishes and proliferated on 10% dextran-coated charcoal/fetal bovine serum, and then treated with 0.1-30 micromol/L of genistein, daidzein or glycitein to investigate cell proliferation (cell number) and DNA synthesis (cell proliferation ELISA system), respectively. We also studied their effects on platelet-derived growth factor (PDGF)-BB (20 microg/L)-induced SMC proliferation. Soybean isoflavones inhibited proliferation and DNA synthesis of SMC from SHRSP in a concentration-dependent manner. Inhibition was significant at 3 micromol/L of genistein and 10 micromol/L of both daidzein and glycitein. For significant inhibition of PDGF-BB-induced SMC proliferation, concentrations as low as 0.1 micromol/L of each isoflavone were effective. These isoflavones, with their inhibitory effects on natural and PDGF-BB-induced SMC proliferation, may be useful in attenuatating such proliferation, a basic mechanism involved in atherosclerotic vascular change, thereby preventing atherosclerotic cardiovascular diseases.

Antihypertensive and antihypertrophic effects of omapatrilat in SHR.

Vasopeptidase inhibitors, such as omapatrilat are single molecules that simultaneously inhibit neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE). In normotensive rats, a single dose of oral omapatrilat (10 mg/kg) and 1 mg/kg inhibited plasma ACE (P < .01) for 24 h and increased plasma renin activity for 8 h (P < .01). In vitro autoradiography using the specific NEP inhibitor radioligand 125I-RB104 and the specific ACE inhibitor radioligand 125I-MK351A showed omapatrilat (10 mg/kg) caused rapid and potent inhibition of renal NEP and ACE, respectively, for 24 h (P < .01). In spontaneously hypertensive rats, 10 days of oral omapatrilat (40 mg/kg/day) reduced blood pressure (vehicle 237 +/- 4 mm Hg; omapatrilat, 10 mg/kg, 212 +/- 4 mm Hg; omapatrilat 40 mg/kg, 197 +/- 4 mm Hg, P < .01) in a dose-dependent manner (10 v 40 mg/kg, P < .01). Left ventricular hypertrophy was significantly reduced by high-dose omapatrilat (vehicle 2.76 +/- 0.03 mg/g body weight; omapatrilat, 10 mg/kg, 2.71 +/- 0.02 mg/g; omapatrilat 40 mg/kg, 2.55 +/- 0.02 mg/g, P < .01) and omapatrilat also increased kidney weight compared to vehicle (both doses, P < .01). Omapatrilat caused significant inhibition of plasma ACE and increased plasma renin activity (both doses, P < .01), and in vitro autoradiographic studies indicated sustained inhibition of renal ACE and NEP (both doses, P < .01). Omapatrilat is a potent vasopeptidase inhibitor, and its antihypertensive effects are associated with inhibition of NEP and ACE at the tissue level and beneficial effects on cardiovascular structure. Relating the degree of tissue inhibition to physiologic responses may allow further definition of the role of local renin angiotensin and natriuretic peptide systems in the beneficial effects of vasopeptidase inhibitors.

Angiotensin II regulates the cell cycle of vascular smooth muscle cells from SHR.

We have demonstrated that spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) show the exaggerated growth and produce angiotensin II (Ang II). In the current study, we investigated the role of endogenous Ang II in the regulation of the cell cycle in VSMC from SHR. Levels of Ang II in conditioned medium from SHR-derived VSMC cultured without serum were significantly higher than levels in conditioned medium from Wistar-Kyoto (WKY) rat-derived VSMC. Basal DNA synthesis was higher in quiescent VSMC from SHR than that in cells from WKY rats. An Ang II type 1 receptor antagonist, CV11974, significantly inhibited the elevation in DNA synthesis in quiescent VSMC from SHR but did not affect it in cells from WKY rats. Cellular DNA content analysis by flow cytometry revealed that the proportion of cells in S phase was higher, whereas the proportion of cells in G1+G0 phase was lower in VSMC from SHR than those in cells from WKY rats. CV11974 significantly decreased the proportion of cells in S phase and correspondingly increased the proportion of cells in G1+G0 phase in VSMC from SHR, but it did not affect the proportion in cells from WKY rats. Cyclin-dependent kinase 2 (CDK2) activity, which is known to induce the progression from G1 to S phase, was higher in VSMC from SHR than in cells from WKY rats. Expression of CDK2 inhibitor p27(kip1) mRNA was markedly higher in VSMC from SHR than in cells from WKY rats. CV11974 decreased expression of p27(kip1) mRNA in VSMC from SHR, whereas CV11974 increased it in cells from WKY rats. These findings indicate that enhanced production of endogenous Ang II regulates the cell cycle especially in the progression from G1 to S phase, and increases CDK2 activity, which is independent of p27(kip1) in VSMC from SHR.