AT1a Receptor mRNA Expression Research Articles

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<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.

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.

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.

Evidence for a causal role of the renin-angiotensin system in vascular dysfunction associated with insulin resistance.

Excess production of superoxide anion in response to angiotensin II plays a central role in the transduction of signal molecules and the regulation of vascular tone. We examined the ability of insulin resistance to stimulate superoxide anion production and investigated the identity of the oxidases responsible for its production. Rats were fed diets containing 60% fructose (fructose-fed rats) or 60% starch (control rats) for 8 weeks. In aortic homogenates from fructose-fed rats, the superoxide anion generated in response to NAD(P)H was more than 2-fold higher than that of control rats. Pretreatment of the aorta from fructose-fed rats with inhibitors of NADPH oxidase significantly reduced superoxide anion production. In the isolated aorta, contraction induced by angiotensin II was more potent in fructose-fed rats compared with control rats. Losartan normalized blood pressure, NAD(P)H oxidase activity, endothelial function, and angiotensin II-induced vasoconstriction in fructose-fed rats. To elucidate the molecular mechanisms of the enhanced constrictor response to angiotensin II, expressions of angiotensin II receptor and subunits of NADPH oxidase were examined with the use of angiotensin II type 1a receptor knockout (AT1a KO) mice. Expression of AT1a receptor mRNA was enhanced in fructose-fed mice, whereas expression of either AT1b or AT2 was unaltered. In addition, protein expression of each subunit of NADPH oxidase was increased in fructose-fed mice, whereas the expression was significantly decreased in fructose-fed AT1a KO mice. The novel observation of insulin resistance-induced upregulation of AT1 receptor expression could explain the association of insulin resistance with endothelial dysfunction and hypertension.

Attenuated renovascular constrictor responses to angiotensin II in adenosine 1 receptor knockout mice.

In the present experiments we examined the renovascular constrictor effects of ANG II in the chronic and complete absence of A1 adenosine receptors (A1AR) using mice with targeted deletion of the A1AR gene. Glomerular filtration rate (GFR) was not different between A1AR +/+ and A1AR -/- mice under control conditions (450.5 +/- 60 vs. 475.2 +/- 62.5 microl/min) but fell significantly less in A1AR -/- mice during infusion of ANG II at 1.5 ng/min (A1AR +/+: 242 +/- 32.5 microl/min, A1AR -/-: 371 +/- 42 microl/min; P = 0.03). Bolus injection of 1, 10, and 100 ng of ANG II reduced renal blood flow and increased renal vascular resistance significantly more in A1AR +/+ than in A1AR -/- mice. Perfused afferent arterioles isolated from A1AR +/+ mice constricted in response to bath ANG II with an EC50 of 1.5 +/- 0.4 x 10(-10) mol/l, whereas a right shift in the dose-response relationship with an EC50 of 7.3 +/- 1.2 x 10(-10) mol/l (P < 0.05) was obtained in arterioles from A1AR -/- mice (P < 0.05). The expression of AT1A receptor mRNA was not different in kidney RNA from A1AR +/+ or A1AR -/- mice. We conclude that chronic A1AR deficiency diminishes the effectiveness of ANG II to constrict renal resistance vessels and to reduce GFR.

A method for the identification of pseudorabies virus protein and angiotensin AT 1A receptor mRNA expression in the same CNS neurons

Neurotropic viruses have been used over the last 10 years to map the distribution of chains of synaptically connected neurons in the CNS. The peptide content of infected neurons has been determined in a number of cases immunohistochemically. However, it has been unclear whether specific mRNA can be assessed in virus-infected neurons. We have established a technique which enables the identification of viral protein and mRNA in the same neuron. In the present study pseudorabies virus retrogradely transported from the kidney was localised using immunohistochemistry and mRNA for the angiotensin II AT 1A receptor was detected by hybridisation histochemistry. Virus protein was visualised using an immunohistochemical procedure with diaminobenzidine as the chromogen and the same sections were exposed to radioactively labelled ( 35S) riboprobes, hybridising the angiotensin II AT 1A receptor. The combination of these two approaches resulted in the identification of neurons shown to project polysynaptically to the kidney and express AT 1A mRNA. These data provide neuroanatomical support for previous physiological observations that ablation of the lamina terminalis and administration of losartan, the AT 1 receptor antagonist, blocks the inhibition of renal sympathetic nerve activity following centrally injected Ang II in rats and sheep [5].

Transcriptional Suppression of Rat Angiotensin AT1a Receptor Gene Expression by Interferon-γ in Vascular Smooth Muscle Cells

Angiotensin (Ang) II stimulates proliferation of vascular smooth muscle cells (VSMC) via its specific receptor AT1 subtype, possibly leading to atherosclerosis in hypertension. On the other hand, a cytokine interferon (IFN)-γ has been shown to have an anti-atherosclerotic effect. In the present study, we examined a possible role of IFN-γ in AT1 receptor gene regulation in VSMC. A firefly luciferase expression vector driven by the rat AT1a receptor gene promoter (∼3.2 kb) was transfected into the cultured rat VSMC, and luciferase expression was determined to estimate the transcription function of the AT1a receptor gene promoter. RT-PCR was also carried out to determine mRNA expression of AT1a receptor in VSMC. IFN-γ treatment decreased AT1a receptor mRNA expression as well as luciferase expression in a dose-dependent manner. The analysis with deletion DNA fragments showed that the IFN-responsive element was located between −987 and −331 positions, where multiple GAS (gamma interferon activated site)-like elements were identified. The expression suppression was reversed by either a MAPKK inhibitor PD98059 or a Jak-2 inhibitor AG-490. These results suggest that IFN-γ can inhibit AT1 receptor expression at gene transcription level, and that the transcription suppression is dependent on MAP kinase and Jak-2. Inhibition of AT1a receptor expression may possibly be implicated in the anti-atherosclerotic action of IFN-γ in VSMC.

Angiotensin II AT1AReceptor mRNA Expression Is Induced by Estrogen–Progesterone in Dopaminergic Neurons of the Female Rat Arcuate Nucleus

Brain angiotensin II (Ang II) inhibits pituitary prolactin release by an indirect mechanism requiring stimulation of dopamine formation and release. We report that [125I]Sar1-Ang II binding to AT1 receptors and AT1A receptor mRNA expression increase selectively in the dorsomedial arcuate nucleus of 17beta-estradiol-primed ovariectomized rats after treatment with progesterone. In hormone-treated rats, arcuate nucleus AT1A receptor mRNA expression is associated with tyrosine hydroxylase-positive neurons. No AT1A receptor mRNA was detected in tyrosine hydroxylase-positive cells of the arcuate nucleus of intact male rats. Conversely, in the anterior pituitary, where local or circulating Ang II stimulates prolactin release, [125I]Sar1-Ang II binding to AT1 receptors and AT1B receptor mRNA expression are decreased in 17beta-estradiol/progesterone-treated ovariectomized rats. Thus, AT1A receptors in the dorsal arcuate nucleus and AT1B receptors in the anterior pituitary are regulated inversely by estrogen/progesterone treatment, supporting the hypothesis of a dual role for brain and pituitary Ang II on prolactin release. The colocalization of AT1A receptor mRNA and tyrosine hydroxylase in neurons of the arcuate nucleus furthermore indicates that within this area central Ang II acts directly on dopaminergic neurons. These results support the hypothesis that central Ang II inhibits pituitary prolactin release indirectly via modulation of dopaminergic activity in the arcuate nucleus.

Angiotensin II Type 2 Receptor mRNA Expression in the Developing Cardiopulmonary System of the Rat

Recent studies have shown that angiotensin II has a trophic action on the heart. The presence of two types of angiotensin II receptors, type 1 (AT1) and type 2 (AT2), has been reported in the rat heart. This in situ hybridization study describes the tissue and cell location of AT2 receptor mRNA in the developing rat cardiopulmonary system, from 15 days of gestation to adulthood. Expression of AT1A receptor mRNA was studied in parallel for direct comparison. The aortic arch and pulmonary artery expressed high levels of AT2 receptor mRNA from 15 days of gestation up until 15 days postpartum, whereas expression of this mRNA was observed only just before and after birth in the coronary arteries. AT2 receptor mRNA was not detected in any cardiac muscle of the fetus, neonate, or adult. The annulus of all four heart valves expressed AT2 mRNA from 21 days of gestation until 10 days postpartum, but no labeling was seen in the valve leaflets. The subendocardial atrial tissue showed a high level of AT2 receptor mRNA expression during the early postnatal period, but no expression was observed in the atrial myocytes from fetal stages to adulthood. The bronchi and trachea, but not the lung parenchyma, showed a high level of AT2 receptor mRNA expression starting from 17 days of gestation until 10 days postpartum. AT2 receptor mRNA expression in the cardiopulmonary system is therefore transient, developmentally regulated, and mostly located in vascular structures. By these three characteristics, its expression contrasts with that of AT1A, which is continuously expressed in the cardiac muscle to adulthood. This spatiotemporal pattern of expression of angiotensin II receptor mRNAs during development suggests a possible role for angiotensin II in organogenesis.

Comparative expression of vasopressin and angiotensin type-1 receptor mRNA in rat hypothalamic nuclei: a double in situ hybridization study.

Angiotensin II (Ang) injected intracerebroventricularly stimulates neurohypophyseal vasopressin (AVP) release into the peripheral circulation. As we have shown previously, central actions of Ang II in the rat forebrain are mediated by the AT1A receptor subtype. In the present paper, we attempted to clarify the cellular localization of the AT1A receptor mRNA in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, in order to reappraise the conflicting data on the nature of the angiotensin II receptor involved in Ang induced vasopressin release. For this purpose, double in situ hybridization was performed using a radioactive AT1A receptor riboprobe and a digoxygenin labeled AVP oligoprobe, and immunohistochemical localization of the glial marker glial fibrillary acidic protein (GFAP) on the same brain slice. The results show neuronal expression of AT1A receptor mRNA mainly in dorsal and medial parvocellular parts of the PVN, its localization in some magnocellular PVN neurons and the absence of its expression in AVP producing neurons either in the PVN or in the SON. Thus, while indirect evidence indicates the involvement of the AT1A receptor subtype in the regulation of CRH and oxytocin release, the stimulation of vasopressinergic neurons is likely due to indirect mechanisms, or to a yet unknown type of angiotensin receptor.

The angiotensin receptor subtype AT1A predominates in rat forebrain areas involved in blood pressure, body fluid homeostasis and neuroendocrine control.

Subtypes of the angiotensin II (Ang II) type-1 (AT1) receptor are probably involved in distinct actions of the peptide, since their distribution in peripheral organs and regulation of their gene expression are different. We investigated the distribution of AT1A and AT1B receptor subtype mRNAs in the rat forebrain and pituitary using sensitive cRNA probes for in situ hybridization. High level of AT1A receptor mRNA expression is observed in the subfornical organ (SFO) and in the anterior hypothalamus, particularly the periventricular tissue surrounding the anterior portion of the 3rd ventricle (AV3V), which contains the organum vasculosum of the lamina terminalis (OVLT), the median preoptic nucleus and the preoptic periventricular nucleus as well as in the hypothalamic periventricular nucleus and in the parvocellular part of the paraventricular nucleus (PVN). Moderate to strong AT1A labeling was found in the anterior olfactory nucleus, the piriform cortex and the nucleus of the lateral olfactory tract. Very low AT1B receptor mRNA expression was found in the SFO and the PVN. In contrast, strong AT1B receptor mRNA expression coincided with low AT1A receptor mRNA expression in the anterior pituitary. Labeling was cytoplasmic at the light microscopic level. We thus suggest that the AT1A receptor is responsible for the central actions of Ang II in the rat forebrain whereas direct actions of Ang II on the anterior pituitary are mediated by the AT1B receptor subtype.