AT1A Receptor mRNA Research Articles

Effects of Ginkgo biloba leaf extract on local renin-angiotensin system through TLR4/NF-κB pathway in cardiac myocyte.

The present study investigated the effects of Ginkgo biloba leaf extract (GBE50) on lipopolysaccharide (LPS) induced Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway and its effects on angiotensinogen (ATG) and AT1a receptor, so as to explore the mechanism of GBE50 in prevention and treatment of left ventricular remodeling. In vitro cultured neonatal rat ventricular myocytes (NRVMs) were divided into 4 groups including i) control group: DMEM medium; ii) LPS group: iii) LPS + GBE50 group; iv) LPS + caffeic acid phenethyl ester (CAPE, specific inhibitor of NF-κB) group. Nuclear translocation of NF-κB p65 was detected by immunocytochemical method after intervention for 24 h. Expression of TLR4, ATG, AT1a receptors and β-myosin heavy chain (β-MHC) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein content of cardiomyocytes was measured by Coomassie Brilliant Blue method. Under LPS stimulation, expression level of TLR4 mRNA in NRVMs was significantly increased (P<0.01), nuclear translocation of NF-κB p65 was increased, expression levels of ATG, AT1a receptor and β-MHC mRNA and the protein content in cells were also increased significantly (P<0.01). GBE50 and CAPE significantly inhibited nuclear translocation of NF-κB p65. GBE50 and CAPE treatments also reduced the increased mRNA levels of TLR4, ATG, AT1a receptor and β-MHC and protein content in cell caused by LPS stimulation. We concluded that, GBE50 can inhibit the activation of local renin-angiotensin system by inhibiting the activation of TLR4/NF-κB and TLR4/NF-κB, signaling pathway inhibition may be one of the mechanisms of the role of Ginkgo biloba leaf extract in preventing myocardial remodeling.

Effect of Chronic Mild Stress on AT1 Receptor Messenger RNA Expression in the Brain and Kidney of Rats.

The purpose of the study was to determine whether exposure to chronic mild stress (CMS) affects expression of angiotensin II Type 1a receptor (AT1aR) messenger RNA (mRNA) in the brain and kidney. Male Sprague-Dawley rats were divided into an unchallenged control group, which remained at rest, and an experimental group, exposed to CMS produced by a series of unexpected, disturbing stimuli applied at random over a period of 4 weeks. After sacrificing the animals, samples of the septal/accumbal and hypothalamic/thalamic diencephalon, brain medulla, cerebellum, and the renal medulla were harvested for determination of AT1aR mRNA. Expression of AT1a receptor mRNA was significantly greater in the rats in the CMS condition than in the controls (septal/accumbal diencephalon: 1.689 [0.205] versus 0.027 [0.004], hypothalamic/thalamic diencephalon: 1.239 [0.101] versus 0.003 [0.001], brain medulla: 2.694 [0.295] versus 0.028 [0.003], cerebellum: 0.013 [0.002] versus 0.005 [0.001; p < .001 for all comparisons], and renal medulla: 409.92 [46.92] versus 208.06 [30.56; p < .01]). There was a significant positive correlation between AT1a mRNA expression in the septal/accumbal diencephalon and brain medulla (p < .025). The results provide evidence that CMS significantly enhances expression of the AT1aR gene in the brain and kidney and indicate that changes in expression of AT1aR mRNA in different brain regions during CMS may be causally related. It is suggested that the up-regulation of AT1a receptors by chronic stress may potentiate negative effects of angiotensin II in pathologies associated with activation of the renin-angiotensin system.

Abstract P061: Selective Deletion of Intracellular Renin in the Brain Causes Hypertension and Elevated Sympathetic Nervous System Activity

Renin expression is regulated by two distinct promoter-1st exon combinations that target renin either for secretion (exon 1a for secreted renin) or cytoplasmic retention (exon 1b for intracellular renin, icREN). We developed icREN knockout (KO) mice by selectively deleting exon 1b. icREN KO mice are essentially brain-specific knockouts of icREN because icREN is predominantly expressed in the brain. Notably, systolic blood pressure measured by telemetry was increased in icREN KO mice (130±2 mmHg, n=8 vs 122±2 mmHg in controls, n=7, P&lt;0.01). The low- to high-frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability, a parameter of sympathetic nerve activity (SNA), was increased in icREN KO mice (KO: 1.24±0.21, n=7 vs control: 0.70±0.11, n=7, P&lt;0.05). Body weight (BW) was normal in icREN KO mice compared to controls, but the BW gain and fat accumulation induced by high fat diet (HFD) were attenuated in male icREN KO mice (BW at 16 wks of HFD- KO: 36.8±1.2 g, n=8 vs control: 41.9±1.4 g, n=9; relative fat mass at 14 wks of HFD- KO: 27.7±1.7%, n=8 vs control: 34.4±2.3%, n=9, both P&lt;0.05). The resting metabolic rate measured by respirometry was increased in icREN KO mice (0.156±0.005 kcal/h, n=46, P&lt;0.05) vs controls (0.145±0.003 kcal/h, n=53), whereas food consumption and absorbed calories were not different. We previously reported that the brain renin-angiotensin system facilitates renal SNA (RSNA) response to acute intracerebroventricular (ICV) injection of leptin. Interestingly, the RSNA response to ICV leptin was greater in icREN KO mice (KO: 214±40 % baseline, n=5 vs control: 114±18 % baseline, n=10, P&lt;0.01). AT1a receptor mRNA was upregulated in the paraventricular nucleus of icREN KO mice (P&lt;0.05). Chronic ICV injection of losartan not only abolished the elevated blood pressure in icREN KO mice, but reduced it to below baseline in controls (systolic blood pressure, 111±3 mmHg in KO, n=5; 124±4 mmHg in controls, n=6). These data suggest that icREN deletion increases the activity of brain renin-angiotensin system and elevates blood pressure and metabolic rate through sympathetic activation. We conclude that this novel icREN isoform contributes to cardiovascular and metabolic control possibly as part of an inhibitory neural circuit.

Intracellular Renin in the Brain Contributes to Cardiovascular and Metabolic Control

Renin gene expression is regulated by two distinct promoter‐first exon combinations that target renin either for secretion (exon 1a initiates secreted renin) or cytoplasmic retention (exon 1b initiates intracellular renin, icREN). Interestingly, icREN is expressed predominantly in the brain. We selectively deleted exon 1b and developed icREN KO mice to examine the role of icREN in cardiovascular and metabolic control. Systolic blood pressure in the light phase as measured by telemetry was increased in KO mice (126±2 mmHg, n=8 vs 116±2 mmHg, n=7, P&lt;0.01). The low‐ to high‐frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability was increased in KO mice (1.24±0.21, n=7 vs 0.70±0.11, n=7, P&lt;0.05). Body weight was normal in KO mice fed normal chow. However, high fat diet‐induced obesity was attenuated in male KO mice (body weight, 32.4±1.3 g, n=5 vs 36.6±1.4 g, n=9; relative fat mass, 26.1±2.3 %, n=5 vs 34.4±2.3 %, n=9, both P&lt;0.05). The resting metabolic rate measured by respirometry was increased in KO mice (0.156±0.005 kcal/h, n=46, P&lt;0.05) vs controls (0.145±0.003 kcal/h, n=53). We previously reported that the brain renin‐angiotensin system facilitates renal sympathetic nerve activity (RSNA) response to acute intracerebroventricular injection of leptin. Interestingly, the RSNA response to leptin was greater in KO mice compared to controls (214±40 % baseline, n=5 vs 114±18 % baseline, n=10, P&lt;0.01). Further, in KO mice, AT1a receptor mRNA was upregulated in the paraventricular nucleus (P&lt;0.05). These data suggest that this novel icREN isoform contributes to cardiovascular and metabolic control possibly through the modulation of AT1a receptor expression in the brain.

Abstract 002: Activation of AT1 receptors on Dendritic Cells Restrains Angiotensin II-Dependent Hypertension Through a CCR7-dependent Mechanism

Dendritic cells (DCs) trigger an adaptive immune response by presenting processed antigens to T lymphocytes. Given the emerging importance of adaptive immunity in hypertension, we posited that DCs may sample neoantigens in the kidney in order to prime pro-hypertensive T cells. Using CD11c-Cre mT/mG mice in which DCs fluoresce green, we detected robust green signals within the kidney after 2 wks of angiotensin (Ang) II-induced hypertension, confirming infiltration of DCs into the hypertensive kidney. As activation of type 1 angiotensin (AT1) receptors on other immune cell lineages has been shown to alter inflammatory responses, we explored the role of the DC AT1 receptor in blood pressure regulation by generating mice with DC-specific deletion of the AT1A receptor (CD11c Cre+ Agtr1aflox/flox = DC KO) and littermate controls (Agtr1aflox/flox = WT). Compared to WTs, the DC KOs had an ≈87% reduction in AT1A receptor mRNA expression in DCs (p&lt;0.0001) but preserved AT1A expression in B and T lymphocytes, kidney, and heart (p=NS). Next, we infused uni-nephrectomized DC KO mice and wild-type (WT) controls with Ang II (500ng/kg/min) for 4 wks. At baseline, the mean arterial blood pressure (MAP) was similar in WT and DC KO mice (123±2 vs. 126±2 mm Hg; p=NS). By contrast, after the 1st week of Ang II infusion, DC KO mice had markedly exaggerated MAP elevations compared to WTs (174±4 vs. 158±5 mm Hg; p=0.026). Expression of the chemokine receptor CCR7 on mature DCs and T cells directs both cell lineages to the lymph node where the DC can display a processed antigen and activate the T cell. We detected higher levels of CCR7 mRNA in the kidneys from the Ang II-infused DC KOs compared to WTs (2.98±0.90 vs. 1.00±0.16 au; p=0.04), and posited that enhanced DC priming of T cells in the DC KOs contributed to their exaggerated hypertension. Consistent with this possibility, uni-nephrectomized CCR7-deficient (KO) mice had a significantly blunted chronic hypertensive response to Ang II (165±4 vs. 178±2 mm Hg; p=0.03) and ameliorated albuminuria (2.1±0.2 vs. 3.7±0.8 mg/24hrs; p=0.02) versus WT controls. We conclude from these data that stimulation of AT1 receptors on DCs constrains Ang II-induced blood pressure elevation, possibly by limiting the CCR7-dependent activation of T lymphocytes by DCs.

AT1a Receptor Knockdown in the Median Preoptic Nucleus Attenuates the Sustained Component of Hypertension Resulting from Chronic Intermittent Hypoxia

Obstructive sleep apnea produces hypertension and increases sympathetic nerve activity during the hypoxic sleeping events as well as during the waking hours. To model the hypoxemia experienced during sleep apnea, rodents are exposed to chronic intermittent hypoxia (CIH). The median preoptic nucleus (MnPO) is a forebrain region that contributes to this sustained increase in mean arterial pressure (MAP) from CIH. This region integrates information from forebrain circumventricular organs and projects to the paraventricular nucleus to influence sympathetic nerve activity. Our lab has previously shown that in response to CIH there is increased ΔFosB expression in the MnPO and that after a 7 day hypoxia protocol MnPO AT1a receptor mRNA is increased 7 fold whereas AT1b shows no change. In this study, we tested the hypothesis that MnPO AT1a receptors contribute to CIH hypertension using adult male Sprague Dawley rats injected with a neuron specific adenoviral vector with either shRNA to knock down expression of AT1a receptors in the MnPO or a scrambled RNA sequence. All rats showed an increase in MAP during the hypoxic light period, but rats receiving the AT1a knock down did not exhibit the sustained increase in MAP during the normoxic dark phase (P&lt;.05). This data indicates that the AT1a receptor in the MnPO is necessary for the sustained component of hypertension resulting from CIH. P01 HL‐88052

Regional Variation in Aortic AT1b Receptor mRNA Abundance Is Associated with Contractility but Unrelated to Atherosclerosis and Aortic Aneurysms

BackgroundAngiotensin II (AngII), the main bioactive peptide of the renin angiotensin system, exerts most of its biological actions through stimulation of AngII type 1 (AT1) receptors. This receptor is expressed as 2 structurally similar subtypes in rodents, termed AT1a and AT1b. Although AT1a receptors have been studied comprehensively, roles of AT1b receptors in the aorta have not been defined.Methodology/ResultsWe initially compared the regional distribution of AT1b receptor mRNA with AT1a receptor mRNA in the aorta. mRNA abundance of both subtypes increased from the proximal to the distal aorta, with the greatest abundance in the infra-renal region. Corresponding to the high mRNA abundance for both receptors, only aortic rings from the infra-renal aorta contracted in response to AngII stimulation. Despite the presence of both receptor transcripts, deletion of AT1b receptors, but not AT1a receptors, diminished AngII-induced contractility. To determine whether absence of AT1b receptors influenced aortic pathologies, we bred AT1b receptor deficient mice into an LDL receptor deficient background. Mice were fed a diet enriched in saturated fat and infused with AngII (1,000 ng/kg/min). Parameters that could influence development of aortic pathologies, including systolic blood pressure and plasma cholesterol concentrations, were not impacted by AT1b receptor deficiency. Absence of AT1b receptors also had no effect on size of aortic atherosclerotic lesions and aortic aneurysms in both the ascending and abdominal regions.Conclusions/SignificanceRegional abundance of AT1b receptor mRNA coincided with AngII-induced regional contractility, but it was not associated with AngII-induced aortic pathologies.

Regulation of angiotensin II type 2 receptor gene expression in the adrenal medulla by acute and repeated immobilization stress

While the renin-angiotensin system is important for adrenomedullary responses to stress, the involvement of specific angiotensin II (Ang II) receptor subtypes is unclear. We examined gene expression changes of angiotensin II type 1A (AT(1A)) and type 2 (AT(2)) receptors in rat adrenal medulla in response to immobilization stress (IMO). AT(2) receptor mRNA levels decreased immediately after a single 2-h IMO. Repeated IMO also decreased AT(2) receptor mRNA levels, but the decline was more transient. AT(1A) receptor mRNA levels were unaltered with either single or repeated IMO, although binding was increased following repeated IMO. These effects of stress on Ang II receptor expression may alter catecholamine biosynthesis, as tyrosine hydroxylase and dopamine β-hydroxylase mRNA levels in PC12 cells are decreased with Ang II treatment in the presence of ZD7155 (AT(1) receptor antagonist) or with CGP42112 (AT(2) receptor agonist) treatment. Involvement of stress-triggered activation of the hypothalamic-pituitary-adrenocortical or sympathoadrenal axis in AT(2) receptor downregulation was examined. Cultured cells treated with the synthetic glucocorticoid dexamethasone displayed a transcriptionally mediated decrease in AT(2) receptor mRNA levels. However, glucocorticoids are not required for the immediate stress-triggered decrease in AT(2) receptor gene expression, as demonstrated in corticotropin-releasing hormone knockout (Crh KO) mice and hypophysectomized rats, although they can regulate basal gene expression. cAMP and pituitary adenylate cyclase-activating polypeptide also reduced AT(2) receptor gene expression and may mediate this response. Overall, the effects of stress on adrenomedullary AT(1A) and AT(2) receptor expression may contribute to allostatic changes, such as regulation of catecholamine biosynthesis.

In Vivo Expression of Angiotensin-(1-7) Lowers Blood Pressure and Improves Baroreflex Function in Transgenic (mRen2)27 Rats

Transgenic (mRen2)27 rats are hypertensive with impaired baroreflex sensitivity for control of heart rate compared with Hannover Sprague-Dawley rats. We assessed blood pressure and baroreflex function in male hemizygous (mRen2)27 rats (30-40 weeks of age) instrumented for arterial pressure recordings and receiving into the cisterna magna either an Ang-(1-7) fusion protein or a control fusion protein (CTL-FP). The maximum reduction in mean arterial pressure achieved was -38 ± 7 mm Hg on day 3, accompanied by a 55% enhancement in baroreflex sensitivity in Ang-(1-7) fusion protein-treated rats. Both the high-frequency alpha index (HF-α) and heart rate variability increased, suggesting increased parasympathetic tone for cardiac control. The mRNA levels of several components of the renin-angiotensin system in the dorsal medulla were markedly reduced including renin (-80%), neprilysin (-40%), and the AT1a receptor (-40%). However, there was a 2-fold to 3-fold increase in the mRNA levels of the phosphatases PTP-1b and dual-specificity phosphatase 1 in the medulla of Ang-(1-7) fusion protein-treated rats. Our finding that replacement of Ang-(1-7) in the brain of (mRen2)27 rats reverses in part the hypertension and baroreflex impairment is consistent with a functional deficit of Ang-(1-7) in this hypertensive strain. We conclude that the increased mRNA expression of phosphatases known to counteract the phosphoinositol 3 kinase and mitogen-activated protein kinases, and the reduction of renin and AT1a receptor mRNA levels may contribute to the reduction in arterial pressure and improvement in baroreflex sensitivity in response to Ang-(1-7).

AT1 Receptors in the Collecting Duct Directly Modulate the Concentration of Urine

Mice lacking AT(1) angiotensin receptors have an impaired capacity to concentrate the urine, but the underlying mechanism is unknown. To determine whether direct actions of AT(1) receptors in epithelial cells of the collecting duct regulate water reabsorption, we used Cre-Loxp technology to specifically eliminate AT(1A) receptors from the collecting duct in mice (CD-KOs). Although levels of AT(1A) receptor mRNA in the inner medulla of CD-KO mice were significantly reduced, their kidneys appeared structurally normal. Under basal conditions, plasma and urine osmolalities and urine volumes were similar between CD-KO mice and controls. The increase in urine osmolality in response to water deprivation or vasopressin administration, however, was consistently attenuated in CD-KO mice. Similarly, levels of aquaporin-2 protein in inner and outer medulla after water deprivation were significantly lower in CD-KO mice compared with controls, despite its normal localization to the apical membrane. In summary, these results demonstrate that AT(1A) receptors in epithelial cells of the collecting duct directly modulate aquaporin-2 levels and contribute to the concentration of urine.

The Mechanism of Distinct Diurnal Variations of Renin-Angiotensin System in Aorta and Heart of Spontaneously Hypertensive Rats

Diurnal variations in plasminogen activator inhibitor-1 mRNA expression are different between the spontaneously hypertensive rats (SHRs) and the Wistar-Kyoto (WKY) rats, and between the aorta and the heart. To elucidate the mechanisms, we examined diurnal changes in the circulating renin-angiotensin system in the SHR and WKY rats. Diurnal variations in plasma renin activity (PRA), plasma angiotensin I, and aldosterone concentrations were similar between the SHR and WKY rats. On the other hand, plasma angiotensin II (Ang II) concentration in the SHR was lower than that in the WKY rats at most time points, but increased to the level of the WKY rats in the late light phase. Treatment with AT1 receptor antagonist candesartan increased plasma Ang II concentration except at ZT 8 and lessened its diurnal variation in the SHR. At the peak in plasma Ang II in the SHR, Ang II regulated genes such as transforming growth factor-β1 and p22phox were upregulated in the aorta. On the other hand, these genes were upregulated throughout the day in the heart of SHR. Candesartan treatment increased AT1a receptor mRNA expression in the heart but not in the aorta of SHR. These findings suggest that an AT1 receptor-mediated mechanism might cause a surge in plasma Ang II concentration at the late light phase in the SHR. Homologous down-regulation of AT1a receptor by Ang II may dampen the effect of a surge in plasma Ang II concentration in the heart of SHR.

Increased Angiotensin II AT 1 receptor mRNA and binding in spleen and lung of AT 2 receptor gene disrupted mice

To clarify the relationship between Angiotensin II AT 1 and AT 2 receptors, we studied AT 1 receptor mRNA and binding expression in tissues from AT 2 receptor gene disrupted (AT 2 −/−) female mice, where AT 2 receptors are not expressed in vivo, using in situ hybridization and quantitative autoradiography. Wild type mice expressed AT 1A receptor mRNA and AT 1 receptor binding in lung parenchyma, the spleen, predominantly in the red pulp, and in liver parenchyma. In wild type mice, lung AT 2 receptors were expressed in lung bronchial epithelium and smooth muscle, and were not present in the lung parenchyma, the spleen or the liver. This indicates that AT 1 and AT 2 receptors were not expressed in the same cells. In AT 2 −/− mice, we found higher AT 1A receptor mRNA and AT 1 receptor binding in lung parenchyma and in the red pulp of the spleen, but not in the liver, when compared to littermate wild type controls. Our results suggest that impaired AT 2 receptor function upregulates AT 1 receptor transcription and expression in a tissue-specific manner and in cells not expressing AT 2 receptors. AT 1 upregulation explains the increased sensitivity to Angiotensin II characteristic of the AT 2 −/− phenotype, consistent with enhanced AT 1 receptor activation in a number of tissues.

Angiotensin II-induced hypertension regulates AT1 receptor subtypes and extracellular matrix turnover in mouse retinal pigment epithelium

Accumulation of specific deposits and extracellular molecules under the retinal pigment epithelium (RPE) has been previously observed in eyes with age-related macular degeneration (AMD) and may play a role in the pathogenesis of AMD. Even though age is the major determinant for developing AMD, clinical studies have revealed hypertension (HTN) as another systemic risk factor. Angiotensin II (Ang II) is considered the most important hormone associated with HTN. To evaluate the relationship of Ang II to AMD, we studied whether mouse RPE expresses functional Ang II receptor subtypes and whether HTN-induced Ang II regulates expression of these receptors as well as critical ECM molecules (MMP-2 and type IV collagen) involved in ECM turnover in RPE. We used 9-month-old C57BL/6 male mice infused with Ang II alone or Ang II in combination with the AT1 receptor antagonist candesartan or the AT2 receptor antagonist PD123319 for 4 weeks to determine whether HTN-associated Ang II was important for ECM regulation in RPE. We found that mouse RPE expressed both Ang II receptor subtypes at the mRNA and protein levels. Infusion with Ang II induced HTN and elevated plasma and ocular Ang II levels. Ang II also regulated AT1a and AT1b receptor mRNA expression, the intracellular concentration of calcium [Ca 2+] i, MMP-2 activity, and type IV collagen accumulation. Concurrent administration of Ang II with the AT1 receptor blocker prevented the increase in blood pressure and rise in ocular Ang II levels, as well as the calcium and MMP-2 responses. In contrast, the type IV collagen response to Ang II was prevented by blockade of AT2 receptors, but not AT1 receptors. Plasma Ang II levels were not modified by the AT1 or AT2 receptor blockade. Since the effects of Ang II on MMP-2 and type IV collagen require inhibition of both Ang II receptor subtypes, these receptors may play a role as a potential therapeutic targets to prevent ECM turnover dysregulation in the RPE basement membrane, suggesting a pathogenic mechanism to explain the link between HTN and AMD.

Angiotensin II stimulates the reactivity of the pituitary-adrenal axis in leptin-resistant Zucker rats, thereby influencing the glucose utilization

The HPA axis is hyperactive under conditions of leptin and insulin resistance as well as after ANG II administration. We hypothesized that a hyperreactivity of the HPA axis to ANG contributes to an impaired glucose utilization in obesity, since leptin resistance and an overactive renin-angiotensin-aldosterone system are features of obesity. Zucker rats were treated with ANG via subcutaneous minipumps (0, 0.9, and 9.0 mug/h; 4 wk). PA axis reactivity and glucose homeostasis were characterized after CRH treatment and during an oral glucose tolerance test (OGTT). The elevated plasma profile of corticosterone after CRH stimulation in saline-treated OZR compared with LZR confirmed that the sensitization of the PA axis depended on leptin resistance. Irrespective of the rat strain, circulating ANG levels and blood pressure were selectively increased after administration of 9 mug/h ANG (high ANG). Only high ANG induced an elevation of the corticosterone and glucose response after CRH stimulation in OZR but did not affect the ACTH secretion. During OGTT, corticosterone and consequently glucose increased in OZR after high ANG, whereas the insulin secretion was decreased. In the adrenal glands of OZR, AT(1A) receptor mRNA levels increased after high ANG. We conclude that the impairment of glucose utilization after ANG stimulation is potentiated in leptin-resistant rats as a result of a hyperreactive PA axis, thereby confirming the functional importance of a dysregulation within the HPA axis in metabolic syndrome or obesity. The ACTH-independent stimulation of corticosterone release and the selective increase of AT(1A) receptor mRNA in the adrenals of OZR indicated a sensitization of adrenals toward ANG, causing a stimulation of the PA axis.

Challenged sodium balance and expression of angiotensin type 1A receptor mRNA in the hypothalamus of Wistar and Dahl rat strains

The present study investigates the influence of a chronic high Na + diet (8% Na +) on the expression of the angiotensin type 1A (AT 1A) receptor gene in the lamina terminalis and paraventricular nucleus of the hypothalamus (PVH) in normotensive Wistar (W) rats, as well as in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats. Three weeks of 8% Na + diet led to a higher blood pressure in DS rats compared to DR and W rats. Moreover, the high Na + diet was correlated with a decreased expression of AT 1A receptor mRNA in the median preoptic nucleus (MnPO) and in the PVH of DS rats, compared to DR and W rats. Contrastingly, the AT 1A receptor mRNA expression was not altered by the high Na + diet in the forebrain circumventricular organs of all the rat strains. Interestingly, a furosemide-induced Na + depletion was correlated with an increased expression of AT 1A receptor mRNA in the PVH, MnPO and SFO of both the DS and DR rats. It is concluded that chronic high Na + diet did differently regulate the expression of AT 1A receptor mRNA in two hypothalamic integrative centers for hydromineral and cardiovascular balance (the PVH and MnPO) in DS rats, compared to DR and W rats. However, the AT 1A receptor mRNA expression was similarly regulated in DS and DR rats in response to an acute Na + depletion, suggesting a distinct high Na +-induced regulation of the AT 1A receptor gene in the PVH and MnPO of DS rats.

Effects of dietary protein restriction on nephron number in the mouse

In rats, maternal protein restriction reduces nephron endowment and often leads to adult hypertension. Sex differences in these responses have been identified. The molecular and genetic bases of these phenomena can best be identified in a mouse model, but effects of maternal protein restriction on kidney development have not been examined in mice. Therefore, we determined how combined prenatal and postnatal protein restriction in mice affects organ weight, glomerular number and dimensions, and renal expression of angiotensin receptor mRNA, in both male and female offspring. C57/BL6/129sv mice received either a normal (20% wt/wt; NP) or low (9% wt/wt; LP) protein diet during gestation and postnatal life. Offspring were examined at postnatal day 30. Protein restriction retarded growth of the kidney, liver, spleen, heart, and brain. All organs except the brain weighed less in female than male offspring. Protein restriction increased normalized (to body weight) brain weight, with females having relatively heavier brains than males. The effects of protein restriction were not sex dependent, except that normalized liver weight was reduced in males but increased in females. Glomerular volume, but not number, was greater in female than in male mice. Maternal protein restriction reduced nephron endowment similarly in male and female mice. Renal expression of AT(1A) receptor mRNA was approximately sixfold greater in female than male NP mice, but similar in male LP and female LP mice. We conclude that maternal protein restriction reduces nephron endowment in mice. This effect provides a basis for future studies of developmental programming in the mouse.

T. violacea lowers blood pressure and down regulates AT1a gene expression in a hypertensive rat model

The in vivo effects of Tulbagia violacea, also known as wild garlic, in a salt-sensitive rat model and its effects on AT1a receptor mRNA expression in kidney tissues was investigated. Three month old Dahl salt-sensitive received, daily, for 14 days T. violacea 50mg/kg (TvL); captopril 10 mg/kg b.w. (Cap) or DMSO (Con). Blood pressures were monitored prior to the commencement of the study and biweekly and urine volume and sodium concentration was measured on days 0, 7 and 14. On day 15, animals were anaesthetized (sodium thiopentane, 50 mg/kg), blood samples for aldosterone levels were taken and kidneys removed for AT1a mRNA expression. Tvl and Cap groups showed reduced AT1a mRNA expressions respectively when compared to the control group. Decreases in systolic blood pressures mirrored decreases in AT1a mRNA expressions in all groups. Tvl and Cap showed reductions in SBP of 10.8% and 11% respectively from day 0 to day 14. Urine output in the Cap and Tvl groups increased and sodium excretion increased from Day 0 to 14. The actions of captopril and TVL on blood pressure, urinary volume and sodium excretion may be attributed to an interruption of the renin-angiotensin pathway.

Nocturnal hypertension in mice consuming a high fructose diet

Objective To investigate the effect of fructose consumption on the light/dark pattern of blood pressure, heart rate and autonomic neural function in mice. Background Insulin resistant diabetes is associated with hypertension and autonomic dysfunction. There is evidence that the increasing incidence of diabetes may be related to dietary changes, including consumption of high levels of fructose. Design/methods C57/BL mice, instrumented with radiotelemetric arterial catheters, were fed a control or high fructose diet (60%). Cardiovascular parameters measured were light/dark pattern of mean arterial pressure (MAP), heart rate (HR) and variability (time and frequency domain). We also measured plasma insulin, glucose, lipids and angiotensin II (Ang II) as well as glucose tolerance. In situ hybridization was used to measure brainstem expression of tyrosine hydroxylase (TH) and Ang AT1a mRNA. Results Fructose diet (8 weeks) produced an increase in MAP, variance and low frequency domain (14 ± 3 vs. 33 ± 4 mm Hg 2, variance and 10 ± 2 vs. 26 ± 4 mm Hg 2, LF, control vs. fructose, P < 0.01). The changes occurred only at night, a period of activity for mice. Glucose tolerance was attenuated in the fructose group. Fructose also increased plasma cholesterol (80 ± 1 vs. 126 ± 2 mg/dl, control vs. fructose, P < 0.05) and plasma Ang II (18 ± 5 vs.65 ± 12 pg/ml, control vs. fructose, P < 0.05). Depressor responses to α 1-adrenergic blockade with prasozin were augmented in fructose-fed mice. Using quantitative in situ hybridization, we found that Ang AT1a receptor and TH mRNA expression were significantly increased in the brainstem locus coeruleus. Conclusion A high fructose diet in mice produced nocturnal hypertension and autonomic imbalance which may be related to activation of sympathetic and angiotensin systems.

High Fructose Diet in Mice Activates Brainstem Angiotensin AT1a and Catecholaminergic Systems

A high fructose diet produces glucose resistance in animals and humans. Previous studies from our laboratory showed that fructose feeding in mice produced an increase in plasma angiotensin (Ang) II associated with a mild hypertension and sympathetic activation which was observed only during the dark phase. The aim of this study was to evaluate the effect of consumption of a high fructose diet on brainstem Ang and catecholaminergic systems. Using in situ hybridization methods we identified and quantified Ang AT1a and tyrosine hydroxylase (TH) mRNA in brainstem regions involved in cardiovascular regulation, locus coeruleus (LC) and dorsal vagal complex (DVC). Male C57BL mice were fed a normal chow (Control, n=6) or a fructose-rich diet (60%, n=7) for 8 weeks. Dietary fructose produced no change in body weight, glucose or insulin. Fructose produced impairment in glucose tolerance and increased plasma cholesterol. There was a significant increase in expression of Ang AT1a and TH mRNA in the LC of fructose-fed mice (60–70% increase) as measured using phosphor imaging methods. Fructose did not change mRNA expression in the DVC. Emulsion photomicrographs showed that there is a clear co-distribution of Ang AT1a and TH mRNA in the LC and DVC. Results show that a high fructose diet activates Ang AT1a receptor and TH mRNA in a cardiovascular responsive brainstem region. These central changes may mediate the dietary induced cardiovascular effects

Effects of cytokine treatment on angiotensin II type 1A receptor transcription and splicing in rat cardiac fibroblasts

Angiotensin II (ANG II) plays important roles in cardiac extracellular matrix remodeling via its type 1A (AT(1A)) receptor. The cytokines tumor necrosis factor-alpha and interleukin-1beta (IL-1beta) were shown previously to upregulate AT(1A) receptor mRNA and protein, thereby increasing the profibrotic response to ANG II in cardiac fibroblasts. The present experiments implicate increased nuclear factor-kappaB (NF-kappaB)-dependent transcription and also, to a lesser extent, altered mRNA splicing in the mechanism of receptor upregulation. Cytokine stimulation was found to increase AT(1A) heterogeneous nuclear RNA levels, which strongly suggests that mRNA upregulation occurs transcriptionally. The transcription factor NF-kappaB was previously deemed necessary for cytokine-induced AT(1A) receptor mRNA upregulation. Computer analysis of upstream DNA sequences revealed putative NF-kappaB elements at -365 and -2540 bp. Both isolated elements were shown to bind NF-kappaB (using gel-shift assays) and to transactivate a minimal promoter (using reporter assays), although the element at -365 bp appeared stronger. Three splice variants of AT(1A) receptor mRNA that have different 5' untranslated regions were detected in rat tissues, namely, exons 1-2-3 (predominant), 1-2-3+6, and 1-3. Cytokine treatment of fibroblasts upregulated all splice variants, but exon 1-3 increased more than the others. This differential upregulation, albeit of modest magnitude, was statistically significant with IL-1beta treatment. Exon 2 contains an inhibitory minicistron and a predicted inhibitory hairpin structure. Luciferase reporter assays indicated that each splice variant translates at a different efficiency, with exon 1-2-3+6 (both minicistron and hairpin) < exon 1-2-3 (minicistron only) < exon 1-3 (neither minicistron or hairpin). These results provide evidence that cytokines increase AT(1) protein levels by altering both transcription and splicing.