AT1 mRNA Research Articles

Construction of shRNA targeted to the rat angiotensin II type 1 receptors and its RNAi in cytoplasma

The expression vector of shRNA targeted to the rat angiotensin II receptor gene was constructed and the efficacy of siRNAs to modulate the expression of target gene in the in vitro cultured mammalian cells was investigated for antihypertensive therapy in spontaneous hypertensive rat (SHR) at post-transcriptional level. The sense and antisense RNA oligonucleotides strands targeting angiotensin II receptor mRNA were synthesized individually according to the sequence of the rat angiotensin II receptor. For preparation of duplexes, sense- and antisense-stranded oligonucleotides were mixed and annealed, and the annealed duplexes were cloned into the pGenesil-1 vector. . The rat glioma cells were transfected with constructed pGenesil-1-shRNA plasmid and scrambled plasmid. The cultured cells were collected at different phases. RT-PCR and Western blot were performed. The AT1 mRNA and protein levels behaved ultimately same. Compared to control after 48 h, AT1 mRNA levels were decreased to 35.5% +/- 3.0%, and the levels reached their lowest point after 72 h (20.7% +/- 4% of control). At 24 and 48 h, AT1 protein was reduced to 46.9% +/- 4. 2% and 36.98% +/- 3.7% respectively compared to control and a maximum reduction was observed after 72 h of incubation (28.1% +/- 4% compared to controls). Plasmid-based shRNA expression systems targeted against the rat angiotensin II receptor gene were generated successfully. The shRNAs with a 22-nt stem and a short loop were cleaved into small interfering dsRNA (siRNA) by the Dicer. The in vitro transcribed siRNA enables the effective silencing of gene expression to the target mRNA and leads to effective inhibition of translation of proteins and will be lay the foundation of application of gene silencing technology to hypertensive rats.

Carotid arteriotomy induces different temporal gene expression profiles in normotensive and hypertensive rat strains

Analysis of gene expression profiles in patients or in animal models affected by cardiovascular diseases may provide insight into therapeutic strategies. In this study, 3 rat strains, Wistar Kyoto (WKY), spontaneously hypertensive rats (SHR) and the Milan hypertensive rat strain (MHS), have been investigated to assess the influence of genetic background and/or of hypertension on gene expression in arteriotomy-injured carotid arteries (CAs). Expression profiles of genes, c-myc, AT1, AT2, ETA, ETB, Bcl-2, Bax and Bcl-X, were determined by reverse transcription-polymerase chain reaction (RT-PCR) in the acute phase, from 1 to 48 h, following CA arteriotomy. WKY, SHR and MHS show significant differences in gene expression profiles after CA arteriotomy. c-Myc mRNA is activated earlier and/or to a greater extent in hypertensive strains than in WKY (p<0.05). AT1 mRNA increases in WKY after injury, while it decreases in both SHR and MHS (p<0.05). AT2 shows the opposite behaviour, decreasing in WKY and increasing in hypertensive strains (p<0.05). ETA mRNA decreases in all strains although with different timing and levels, associated with a replacement by ETB mRNA (p<0.05). Bcl-2/Bax ratio gradually decreases in WKY, while it decreases only transiently in SHR and MHS 4 h after injury (p<0.05). Overall data indicate that therapeutic strategies for stenosis prevention should carefully consider the gene expression profile after injury, the genetic background, the kind of vascular trauma and the diseases affecting the animal model or the patient.

Intronic ANG II type 2 receptor gene polymorphism 1675 G/A modulates receptor protein expression but not mRNA splicing

The X-linked ANG II type 2 receptor (AT2) is supposed to be involved in cardiovascular disorders. Two studies associated the A allele of the AT2 gene polymorphism (PM) 1675 G/A with left ventricular hypertrophy in men and coronary ischemia in women. Because the PM is located in the short intron 1 of the AT2 gene within a sequence motif similar to the splice branch site consensus, we tested whether it might affect pre-mRNA splicing and/or modulate AT2 receptor expression. We first analyzed the AT2 mRNA splice pattern by RT-PCR in myocardial samples from 12 explanted human hearts and compared it with the respective genotypes. All 12 patients, 10 hemizygous males (7 A, 3 G allele carriers) and 2 homozygous females (2 G/G allele carriers), exhibited the same myocardial AT2 splice pattern with a relative abundance of transcript exon 1/2/3 compared with exon 1/3. Next, AT2 minigene constructs were cloned from both alleles, comprising the core promoter and the complete transcribed region up to the translation start codon, upstream of a luciferase reporter gene. These constructs were transfected into human (HT1080) and rat (PC12W) cell lines and rat vascular smooth muscle cells, and luciferase activities were assessed, as well as the splice patterns of the chimeric AT2/luciferase mRNAs. In all transfected cell types, the mRNA expressed from the AT2 constructs was spliced like the endogenous myocardial AT2 mRNA. However, luciferase activities driven by the G allele construct were significantly higher than those expressed from the A allele. Taken together, these data indicate that individuals carrying the G allele may express higher levels of AT2 receptor protein, which may be protective during the development of ventricular hypertrophy and coronary ischemia.

Expression of angiotensin II type 1 receptor in human cirrhotic livers: Its relation to fibrosis and portal hypertension

Angiotensin II (ANG-II) and its receptor (AT1) have been potential targets of therapy for liver cirrhosis. However, AT1 expression in human cirrhotic livers has not been clarified. We studied AT1 and ANG-II generating enzymes in human autopsy (20 cirrhotics and 20 normal controls) and biopsy (10 cirrhotics) livers. AT1 immunoreactivity in tissue sections was quantified by computer-aided morphometry. AT1 protein and mRNA levels were assessed by Western blotting and real-time polymerase chain reaction. Concerning ANG-II generating system, angiotensin-converting enzyme (ACE) and mast cell chymase were examined. AT1 expression was seen not only in vascular smooth muscle cells, but also in activated stellate cells/myofibroblasts and liver parenchymal cells. AT1-positive vessels and myofibroblasts were significantly increased in fibrous septa of cirrhosis, although overall hepatic AT1 expression was reduced in the cirrhotic livers compared with the controls. Augmentation of AT1-positive vessels was related to severity of portal hypertension. Expressions of ACE and chymase were enhanced in the cirrhotic livers. These results suggest that hepatic AT1 expression is shifted to and concentrated in vessels and myofibroblasts in cirrhotic settings, and increased ANG-II generation by ACE and chymase contributes to portal hypertension and liver fibrosis via binding to AT1 expressed on vessels and myofibroblasts.

The effect of hypothalamo-pituitary disconnection on the renin-angiotensin system in the late-gestation fetal sheep

The activity of the renin-angiotensin system (RAS) increases significantly in the late-gestation fetal sheep. Fetal cortisol is also increased during this time, and it is thought that the increase in cortisol may modulate the RAS changes. Previous studies have examined the effects of cortisol infusion on RAS activity, but the effects of blocking the peripartum increase in cortisol concentrations on the developmental changes in the RAS are not known. Therefore, we utilized the technique of hypothalamic-pituitary disconnection (HPD), which prevents the cortisol surge from occurring, to investigate the importance of the late-gestation increase in cortisol on the ontogenic changes in RAS activity. HPD of fetal sheep was performed at 120 days of gestational age (dGA), and fetuses were delivered between 135 and 139 dGA. Control fetuses were sham operated. HPD blocked the late-gestation cortisol increase but did not alter renal renin mRNA, renal renin or prorenin protein content, nor plasma renin levels compared with sham operated. However, HPD fetuses had increased ANG II receptor subtype 1 (AT1) mRNA and protein expression in the kidney and lungs. ANG II receptor subtype 2 (AT2) expression was not altered in these tissues at either mRNA or protein level. HPD did not change AT1 or AT2 mRNA in the left ventricle but did result in decreased protein levels for both receptors. These studies demonstrate that blockade of the naturally occurring increase in fetal cortisol concentration in late gestation is associated with tissue-specific alterations in expression of AT1 and AT2 receptors. These changes may impact on fetal tissue maturation and hence have consequences in postnatal life.

Altered expression of angiotensin II receptor subtypes and transforming growth factor-? in the heart of nitrofen-induced diaphragmatic hernia in rats

The renin-angitensin system (RAS) plays an important role as a growth factor in cardiac development. Angiotensin converting enzyme is involved in converting angiotensin I to angiotensin II (Ag-II). The effects of Ag-II are mediated by two primary receptors, type 1 (AT1) and type 2 (AT2). Ag-II stimulates transforming growth factor-beta1(TGF-beta1) and acts as a potent stimulant of myocyte growth and fetal contractile protein gene transcription. The aim of this study was to determine the expression of Ag-II receptor subtypes and TGF-beta1 in the hypoplastic heart of nitrofen-induced congenital diaphragmatic hernia (CDH). CDH was induced in pregnant rats following administration of 100 mg nitrofen on day 9.5. The fetuses were divided into three groups: normal controls (n=16), nitrofen-treated without CDH (n=16), and nitrofen-induced CDH (n=16). Reverse transcriptase-polymerase chain reaction was performed to evaluate mRNA expression of AT1, AT2, and TGF-beta1. Levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin. AT1 and AT2 mRNA expressions were significantly decreased in CDH heart compared with controls (0.43+/-0.33 vs. 1.0+/-0.48 and 0.62+/-0.23 vs. 1.4+/-0.43, respectively). TGF-beta1 mRNA expressions were also significantly decreased in CDH heart compared with controls (0.38+/-0.17 vs. 0.72+/-0.26). No significant difference was found between the hearts of controls and nitrofen-treated rats without CDH. The decreased expression of AT1, AT2, and TGF-beta1 mRNA in the hypoplastic heart suggests that the downregulation of RAS may be involved in the pathogenesis of cardiac hypoplasia in nitrofen-induced CDH.

Persistent Proteinuria Up-Regulates Angiotensin II Type 2 Receptor and Induces Apoptosis in Proximal Tubular Cells

Apoptosis is implicated in the progressive cell loss and fibrosis both at glomerular and tubulointerstitial level. In this study, we examined the potential mechanisms by which persistent proteinuria (protein-overload model) could induce apoptosis. After uninephrectomy (UNX), Wistar rats received daily injections of 0.5 g of bovine serum albumin (BSA)/100 g body weight or saline. Both at day 8 and day 28, rats receiving BSA had proteinuria and renal lesions characterized by tubular atrophy and/or dilation and mononuclear cell infiltration. In relation to control-UNX rats, renal cortex of nephritic rats showed an increment in AT2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) expression. In both groups, AT2 receptor immunostaining was mainly localized in proximal tubular cells. Rats with persistent proteinuria showed a significantly increased number of terminal dUTP nick-end labeling positive apoptotic cells compared with UNX-controls, both in glomeruli and tubulointerstitium. Double staining for apoptosis and AT2 receptor showed that most terminal dUTP nick-end labeling positive cells were found in tubules expressing AT2 receptor. Using an antibody that recognizes the active form caspase-3, we observed an increment in caspase-3 activation in rats receiving BSA with respect to those receiving saline. Rats with persistent proteinuria showed a diminution in the phosphorylation of Bcl-2 with respect to UNX-controls both at day 8 and day 28. By contrast, no changes were observed either in the Bax or in the Bcl-2 protein levels. The administration of BSA to UNX rats induced a diminution in the phosphorylation of ERK with respect to UNX-control at all times studied. The changes observed in ERK activities took place without alterations of ERK1/2 protein levels. In summary, our data suggest that persistent proteinuria causes apoptosis in tubular cells through the activation of AT2 receptor, which can, in turn, inhibit MAP kinase (ERK1/2) activation and Bcl-2 phosphorylation.

Angiotensin II type 1 receptor mRNA and its protein expression in human pancreatic cancer cell lines

The incidence of pancreatic cancer is increasing in China, and in many patients the surrounding lymphatics have already been invaded and there is blood-borne metastasis at the time of diagnosis. Additionally, pancreatic cancer is largely refractory to conventional therapies. Therefore, to improve its prognosis, it is important to resolve the problem of its growth. Angiotensin II type 1 receptor (AT1) stimulates the growth and angiogenesis of pancreatic cancer and a selective AT1 antagonist could inhibit these effects. The present study aimed to investigate the expression of AT1 in pancreatic cancer cell lines to provide the theoretical basis for its treatment. The pancreatic cancer cell lines were SW1990, PaTu8988s and PANC-1. RT-PCR was used to detect the AT1 mRNA expression, and ABC immunocytochemical staining and SDS-PAGE were used to detect the expression of AT1 protein. Both AT1 mRNA and protein were expressed in all three cell lines. The AT1 protein was found on the cell membrane and in the cytoplasm of these cells. The AT1 protein (44 x 10(3)) was also demonstrated by SDS-PAGE. The results suggest that AT1 plays an important role in the growth of pancreatic cancer and its inhibition may be a therapeutic strategy.

AT1b receptor predominantly mediates contractions in major mouse blood vessels.

In rodents, angiotensin (Ang) II type-1 (AT1) receptors exist as two pharmacologically identical subtypes: AT1a and AT1b. Recent studies have utilized mouse models with specific subtype receptor deletions to differentiate the functional difference between AT1 subtypes. However, little information is available on AT1 subtype expression in mouse vasculature. Therefore, in this study, AT1a-/- mice and wild-type littermates (AT1a+/+) were used to examine AT1 subtype expression and its functional relevance in mouse arterial vessels. Using RT-PCR and restriction enzyme digestion, we showed that AT1b accounts for most of the total AT1 mRNA in mouse abdominal aorta and femoral artery. In contrast, AT1a is the predominant subtype in kidney. To study the functional role of AT1 subtypes, we measured the in vitro contractility in vessels from AT1a-/- and AT1a+/+ mice. The Ang II concentration response curves in abdominal aorta and femoral artery were comparable between the two mouse strains. Furthermore, the Ang II response in AT1a-/- mouse vessels was completely antagonized by losartan, an AT1 antagonist. These results demonstrate that AT1b receptor is a major mediator for Ang II contractile response in mouse vessels, such as abdominal aorta and femoral artery.

Differential vasoconstrictions induced by angiotensin II: role of AT1 and AT2 receptors in isolated C57BL/6J mouse blood vessels.

Genetically altered mice are increasingly used as experimental models. However, ANG II responses in mouse blood vessels have not been well defined. Therefore, the aim of this study was to determine the role of ANG II in regulating major blood vessels in C57/BL6J mice with isometric force measurements. Our results showed that in mouse abdominal aorta ANG II induced a concentration-dependent contraction (EC50 4.6 nM) with a maximum contraction of 75.1 +/- 4.9% at 100 nM compared with that of 60 mM K+. Similarly, femoral artery also exhibited a contractile response of 76.0 +/- 3.4% to the maximum concentration of ANG II (100 nM). In contrast, ANG II (100 nM)-induced contraction was significantly less in carotid artery (24.5 +/- 6.6%) and only minimal (3.5 +/- 0.31%) in thoracic aorta. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester and the AT2 antagonist PD-123319 failed to enhance ANG II-induced contractions. However, an AT1 antagonist, losartan (10 microM), completely inhibited ANG II (100 nM) response in abdominal aorta and carotid artery. An AT1 agonist, [Sar1]-ANG II (100 nM), behaved similarly to ANG II (100 nM) in abdominal aorta and carotid artery. RT-PCR analyses showed that mouse thoracic aorta has a significantly lower AT1 mRNA level than abdominal aorta. These results demonstrate that major mouse vessels exhibit differential contractions to ANG II, possibly because of varied AT1 receptor levels.

Real-Time PCR Quantification of AT1 and AT2 Angiotensin Receptor mRNA Expression in the Developing Rat Kidney

Background/Aims: Angiotensin II (Ang II) is an important growth factor in the fetal kidney. Molecular cloning and pharmacological studies have defined two major classes of Ang II receptors designated AT1 and AT2. Two AT1 isoforms, AT1A and AT1B, exist in rodents. AT1 promotes growth and proliferation and mediates many of the known physiological actions of Ang II. AT2 appears to antagonize AT1. Our goal was to measure their relative contributions to Ang II signaling in the developing kidney. Methods: We used real-time RT-PCR to quantify AT1A, AT1B, AT2 and the housekeeping gene EF1α mRNA in kidneys from embryonic (E) day 14–20 and postnatal (P) day 1–14 rats. Results: AT2 mRNA declined from 1.4 × 10⁴ copies/10⁶ copies EF1α on E14 to 4.2 × 10³ copies/10⁶ copies EF1α on P14. In contrast, total AT1 mRNA increased gradually from 2.0 × 10³ copies/10⁶ copies EF1α on E14 to 2.0 × 10⁴ copies/10⁶ copies EF1α on P14, with AT1A accounting for about 90% of total AT1 mRNA throughout nephrogenesis. Moreover, the ratio of AT2/(AT1A + AT1B) decreased in a log-linear fashion during maturation, from 6.7 on E14 to 0.2 on P14. Conclusion: The ratio of AT2 to AT1 gene expression modulates Ang II action in the developing kidney.

Differential response of cardiac fibroblasts from young adult and senescent rats to ANG II.

The intracardiac ANG II-forming pathway is activated in the senescent myocardium, raising the possibility of enhanced ANG II effects on cardiac fibroblasts. This study established an in vitro model of cultured cardiac fibroblasts from aged rats to examine if the response of these cells to ANG II is modified in the aged heart. Levels of mRNA encoding renin, angiotensinogen, and the AT(1) receptor subtype in cardiac fibroblasts from young adult and senescent rats were quantified by RT-PCR, net collagen production by a hydroxyproline-based assay, and transforming growth factor (TGF)-beta levels using a commercial kit. In cardiac fibroblasts from young adult rats, ANG II significantly enhanced AT(1) mRNA levels, net collagen production, and TGF-beta production. In fibroblasts from the aged myocardium, ANG II downregulated AT(1) mRNA expression, had a less pronounced effect on net collagen production, and had no effect on TGF-beta production. Such age-related modification of the response of cardiac fibroblasts to ANG II may counteract the effects of augmented intracardiac ANG II production in the senescent heart, limiting fibrogenesis.

The Role of the Renin-Angiotensin System in Cholesterol and Puromycin Mediated Renal Injury

Puromycin aminonucleoside (PAN) nephropathy is a widely studied model of glomerular sclerosis (GS) in the rat, and cholesterol feeding exacerbates the injury induced by PAN. The importance of the interaction of angiotensin II (Ang II) with the AT2 receptor is unclear. We investigated the role of the renin-angiotensin system, particularly with regard to AT1 and AT2 receptor dynamics, in PAN and cholesterol-mediated GS. Sprague-Dawley rats were given a 4% cholesterol diet (group II), subcutaneous PAN (group III), or a 4% cholesterol diet and PAN (group IV) and compared with a control group given PAN vehicle (group I). After 16 weeks, kidneys were harvested and tissue Ang II concentration, angiotensin-converting enzyme (ACE) activity, and ACE, AT1, and AT2 mRNA levels were determined. Compared with control rats, proteinuria was significantly higher in groups II to IV. Kidney ACE activity and ACE mRNA levels in groups III and IV were 2- and 3-fold higher than in groups I and II, respectively. Kidney Ang II concentration also was increased in the experimental groups. Whereas kidney AT1 mRNA was significantly lower in groups III and IV, kidney AT2 mRNA was significantly increased in groups II to IV. In these experimental models of GS, there is significant activation of the tissue-based renin-angiotensin system. Puromycin with and without cholesterol decreased the AT1 receptor mRNA and increased the AT2 receptor mRNA. Up-regulation of AT2 receptors may be important in ameliorating the proliferative effects of Ang II, which presumably occur through the AT1 receptor.

Evidence of Apoptosis: Bax and Fas Expression in Acute Phase of Myocardial Infarction in Rats

Myocardial infarction (MI) remains the leading cause of death from cardiovascular diseases over the past decades. Apoptosis is ‘programmed’ cell death which leads to clearance of ‘unwanted’ cells without disruption of tissue structure or function. Recently, we reported that angiotensin receptor subtype AT1 and AT2 mRNA levels were time dependently regulated after MI. AT1 and AT2 receptor mRNA levels markedly increased at 30 min and peaked 24 h post-MI. The time-dependent increase of AT1 and AT2 receptor mRNA is associated with the early remodeling process of the non-infarcted myocardium post MI. There is a further question raised whether the up-regulation of AT2 receptor is linked to apoptosis. Therefore, we investigated the apoptotic development and apoptotic related gene expression after MI at different time points in the current study. MI was induced in Wistar rats by ligation of the left anterior descending coronary artery. Bax gene expression was found to be increased at 12 h after MI and peaked at 24 h (4.3-fold). It declined at 72 h after MI. Fas gene started to over-express at 12 h after MI as well but it reached maximum at 72 h (4.7-fold). Protein levels of Bax and Fas expression were detected in the necrotic area and area at risk (surrounding area) at different time points after MI. Apoptosis was detected in the infarcted areas. No apoptosis was detected in the sham operated rats. In the MI groups, strongest staining of apoptosis was detected in rats 3 days post operation. Weak staining was found 1 day, 7 day post MI. Very fewer apoptotic cells were detected in the rats 2 weeks after MI. Our results demonstrate that apoptotic development after MI is time dependent in the ischemic area and there could be some linkage with the over expression of AT2 receptor post MI.

Angiotensin II and cAMP regulate AT 1-mRNA expression in rat cardiomyocytes by transcriptional mechanism

Mechanisms of angiotensin II and cAMP regulating the expression of angiotensin II type 1 (AT 1) receptor mRNA were studied in neonatal rat cardiomyocytes. Angiotensin II induced a transient decrease of AT 1-mRNA expression in time- and dose-dependent manner. Maximal decrease (49.2±9.5% of control) occurred at 6 h of angiotensin II (10 nmol/l) treatment. AT 1 receptor antagonists 4-ethyl-2- n-propyl-1-[2′-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-5-carboxylic acid (DMP811) and losartan as well as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) reversed the down-regulation of AT 1-mRNA expression. 6 h of phorbol 12-myristate 13-acetate (PMA) stimulation caused a decrease of AT 1-mRNA level. Treatment by angiotensin II plus actinomycin D for 6 h produced the same effect as actinomycin D alone. These results suggest that angiotensin II down-regulates AT 1-mRNA level of rat cardiomyocytes by inhibiting the transcription of AT 1 gene, which is mediated by AT 1 receptor and related to the activation of protein kinase C. Stimulation by forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) decreased the expression of AT 1-mRNA to 68.1±21.5% of control at 6 h treatment; while increased to 207.9±27.1% of control at 48 h treatment. A series of 5′-upstream deletion mutants of AT 1A promoter were produced and then were recombined with pGL 3 basic vector utilizing luciferase as reporter gene. Among all the constructors, p(−201/+74)Luc was of the highest luciferase activity (5.9 times higher than control) after stimulation by forskolin for 48 h. Further deletion from −201 to −61 resulted in a large decrease of activity. These results indicate that cAMP induces a time-dependent bi-directional regulation of AT 1-mRNA expression. The cAMP responsible element (CRE) cis-element located in the region −201/−61 of rat AT 1A promoter is forskolin inducible, which may mediate the up-regulation of AT 1-mRNA expression induced by cAMP long-lasting stimulation.

Programming effects of short prenatal exposure to cortisol.

Recent studies have linked fetal exposure to a suboptimal intrauterine environment with adult hypertension. The aims of this study were twofold: 1) to see whether cortisol treatment administered to the ewe for 2 days at 27 days of gestation (term approximately 150 days) resulted in high blood pressure in offspring; 2) to study the effect of the same treatment on gene expression in the brain at 130 days of gestation and in lambs at 2 months of age. Mean arterial pressure was significantly higher in the adult female and male offspring of sheep treated with cortisol than in the control group (females: 89+/-2 mmHg vs. 81+/-2; P<0.05 and males: 102+/-4 mmHg vs. 91+/-3; P<0.05). Prenatal cortisol treatment led to up-regulation of angiotensinogen, AT1, MR, and GR mRNA in the hippocampus in fetuses at 130 days of gestation but not in the animals at 2 months of age. This is the first evidence that short prenatal exposure to cortisol programmed high blood pressure in the adult female and male offspring of sheep. Altered gene expression in the hippocampus could have a significant effect on the development of the hippocampus, and on postnatal behavior.

Inhibition of vascular smooth muscle cell growth by angiotensin type 2 receptor stimulation for in vitro organ culture model.

Recent data suggest that AT2 receptors in smooth muscle cells (VSMCs) of adult vessels may counterbalance AT1 receptor activity by inhibiting cell growth. The role of AT2 receptors in VSMC proliferation was investigated by using an in vitro organ culture model in which the sprouting of tubular structures was assessed. In preparations with endothelium, tubular growth was unaffected by angiotensin II (Ang II) but was inhibited by 50 +/- 9% by losartan and was increased by 110 +/- 15% by the AT2 antagonist PD123177. In endothelium-deprived preparations, growth inhibition (-49.1 +/- 0.5%) was observed when angiotensin II was added together with losartan 1 microM, whereas stimulation (59.8 +/- 14%) was induced by angiotensin II with 1 microM PD123177. In cultured VSMCs angiotensin II slightly promoted growth that was inhibited by losartan but was unaffected by PD123177. AT1a, AT1b, and AT2 mRNA expression was demonstrated in cells isolated from tubular structures grown from intact and endothelium-deprived rings, but only AT1a and AT1b mRNA was detected in cultured VSMCs. In conclusion, this paper proposes an in vitro organ culture model in which the expression of AT2 receptors in VSMCs is preserved and demonstrates AT2 receptor-mediated inhibition of VSMC proliferation in adult vessels.

Transcription Factor NF-κB Is Necessary for Up-regulation of Type 1 Angiotensin II Receptor mRNA in Rat Cardiac Fibroblasts Treated with Tumor Necrosis Factor-α or Interleukin-1β

Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta up-regulate type 1 angiotensin II receptor (AT(1)) mRNA and protein in cultured neonatal rat cardiac fibroblasts. The use of pharmacologic inhibitors and a degradation-resistant mutant I kappa B-alpha demonstrated that the transcription factor nuclear factor-kappa B (NF-kappa B) is necessary for cytokine-induced AT(1) up-regulation. The increase in AT(1) mRNA with TNF-alpha treatment is slow, reaching significance by 6-12 h and peaking by 24-48 h. Electrophoretic mobility shift assays revealed that NF-kappa B nuclear translocation was maintained for > or = 24 h with a single dose of TNF-alpha. Since prolonged NF-kappa B activation appeared necessary to maximize AT(1) up-regulation, the mechanism of persistent NF-kappa B activation was studied further. Stimulation with TNF-alpha induced a >10x increase in I kappa B kinase (IKK) activity that quickly diminished by 20 min. I kappa B-alpha and I kappa B-beta proteins were degraded during this time, and I kappa B-alpha was resynthesized subsequently by NF-kappa B-dependent transcription. However, I kappa B isoforms and IKK activity did not return completely to unstimulated values during a 12-h time course. These results suggest that low but persistent IKK activity and I kappa B degradation lead to prolonged NF-kappa B nuclear translocation and maximal AT(1) up-regulation in the continued presence of TNF-alpha.

Angiotensin and calcium signaling in the pituitary and hypothalamus.

1) In the rat pituitary, angiotensin type 1B receptors (AT1B) are located in lactotrophs and corticotrophs. 2) Activation of AT1B receptors are coupled to Gq/11 (Guanine protein coupled receptor, or GPCR); they increase phospholipase beta C (PLC) activity resulting in inositol 1,4,5 triphosphate (InsP3) and diacylglycerol (DAG) formation. A biphasic increase in [Ca2+]i triggered by InsP3 and DAG ensues. 3) As many GPCRs, AT1B pituitary receptors rapidly desensitize. 4) This was observed in the generation of InsP3, the mobilization of intracellular Ca(2+), and in prolactin release. Both homologous and heterologous desensitization was evidenced. 5) Desensitization of the angiotensin II type 1 (AT1) receptor in the pituitary shares similarities and differences with endogenously expressed or transfected AT1 receptors in different cell types. 6) In the pituitary hyperplasia generated by chronic estrogen treatment there was desensitization or alteration in angiotensin II (Ang II) evoked intracellular Ca2+ increase, InsP3 generation, and prolactin release. This correlates with a downregulation of AT1 receptors. 7) In particular, in hyperplastic cells Ang II failed to evoke a transient acute peak in [Ca2+]i, which was replaced by a persistent plateau phase of [Ca2+]i increase. 8) Different calcium channels participate in Ang II induced [Ca2+]i increase in control and hyperplastic cells. While spike phase in control cells is dependent on intracellular stores sensitive to thapsigargin, in hyperplastic cells plateau increase is dependent on extracellular calcium influx. 9) Signal transduction of the AT1 pituitary receptor is greatly modified by hyperplasia, and it may be an important mechanism in the control of the hyperplastic process. 10) In the hypothalamus and brain stem there is a predominant expression of AT1A and AT2 mRNA. 11) Ang II acts at specific receptors located on neurons in the hypothalamus and brain stem to elicit alterations in blood pressure, fluid intake, and hormone secretion. 12) Calcium channels play important roles in the Ang II induced behavioral and endocrine responses. 13) Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space to increase [Ca2+]i in polygonal and stellate astroglia of the hypothalamus and brain stem. 14) In primary cell culture of neurons from newborn rat hypothalamus and brain stem, it has also been determined that Ang II elicits an AT1 receptor mediated inhibition of delayed rectifier K(+) current and a stimulation of Ca2+ current. 15) In primary cell cultures derived from the subfornical organ or the organum vasculosum laminae terminalis of newborn rat pups, Ang II produced a pronounced desensitization of the [Ca2+]i response. 16) Hypothalamic and pituitary Ang II systems are involved in different functions, some of which are related. At both levels Ang II signals through [Ca2+]i in a characteristic way.

Organs/systems potentially involved in one model of programmed hypertension in sheep.

1. When pregnant ewes and their fetuses are exposed to the synthetic glucocorticoid dexamethasone for 2 days early in pregnancy (days 26-28; term 145-150 days), female offspring have increased blood pressure relative to a control group. In one series, this was shown to be due to increased cardiac output, concomitant with a reset mean arterial pressure/heart rate reflex. The first group of such animals had, by the age of 7 years, left ventricular hypertrophy and reduced cardiac functional capacity. 2. The elevation in blood pressure is not maintained by any change in the peripheral renin-angiotensin system (RAS). 3. There is, however, preliminary evidence that some aspects of local RAS (particularly in the kidney and brain) could have participated in the 'programming' event. The levels of mRNA for angiotensin II receptors (AT1, AT2) and angiotensinogen are increased in the kidney of such dexamethasone-treated fetuses in late gestation (130 days), some 100 days after steroid treatment. Similar increases in AT1 mRNA in the medulla oblongata of the fetal brain and large increases of mRNA for angiotensinogen occur in the hypothalamus. 4. These findings, together with evidence from the literature, suggest that both the kidney and parts of the brain are affected by events that also 'program' high blood pressure in the offspring of animals in which the intra-uterine environment has been perturbed at some stage.