Transgenic mouse models of angiotensin receptor subtype function in the cardiovascular system

Angiotensin II mediates is biological actions via different subtypes of G protein-coupled receptors, termed AT(1) and AT(2) receptors. In rodents, two AT(1) receptors have been identified, AT(1A) and AT(1B), whereas in humans a single AT(1) receptor exists. Recently, a number of transgenic animal models have been generated which overexpress or lack functional angiotensin II receptor subtypes. This review focuses on the physiological significance of angiotensin II receptor subtype diversity in the cardiovascular system. In the mouse, AT(1A) receptors are the major regulators of cardiovascular homeostasis by determining vascular tone and natriuresis. In addition, AT(1A) receptors mediate growth-stimulating signals in vascular and cardiac myocytes. AT(1B) receptors participate in blood pressure regulation, and their functions become apparent when the AT(1A) receptor gene is deleted. Deletion of the mouse gene for the AT(2) receptor subtype led to hypersensitivity to pressor and antinatriuretic effects of angiotensin II in vivo, suggesting that the AT(2) receptor subtype counteracts some of the biological effects of AT(1) receptor signalling.     

Angiotensin receptors--evolutionary overview and perspectives

The structure of the angiotensin molecule has been well preserved throughout the vertebrate scale with some amino acid variations. Specific angiotensin receptors (AT receptors) that mediate important physiological functions have been noted in a variety of tissues and species. Physiological and pharmacological characterization of AT receptors and, more recently, molecular cloning studies have elucidated the presence of AT receptor subtypes. Comparative studies suggest that an AT receptor subtype homologous to the mammalian type 1 receptor subtype (AT(1)), though pharmacologically distinct, is present in amphibians and birds, whereas AT receptors cloned from teleosts show low homology to both AT(1) and AT(2) receptor subtypes. Furthermore, receptors differing from both the AT(1)-homologue receptor and AT(2) receptor exist in some non-mammalian species. This may suggest that the prototype AT receptor evolved in primitive vertebrates and diverged to more than one type of AT receptor subtype during phylogeny. Furthermore, phenotypic modulation of AT receptors appears to occur during individual development/maturation.     

Sarcosine1,glycine8 angiotensin II is an AT1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT1 and AT2, revealed anomalous characteristics of sarcosine1,glycine8 Ang II (Sar1,Gly8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT(2) receptors, while dipsogenic responses to Ang II are mediated by AT1 receptors, suggests that Sar1,Gly(8) Ang II is AT1 selective. Sar1,Gly8 Ang II competed for 125I-sarcosine1,isoleucine8 Ang II binding to AT1 receptors in pituitary, liver and adrenal (the latter with the AT2 selective antagonist PD 123,319) with Ki's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the Ki of Sar1,Gly8 Ang II for AT2 receptors in rat adrenal (with the selective AT1 antagonist losartan) was 52 nM. 125I-Sar1,Gly8 Ang II (0.5-3 nM) bound to AT1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity (Kd=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT2 receptor. 125I-Sar1,Gly8 Ang II selectively labeled AT1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar1,Gly8 Ang II to be the first angiotensin peptide analog to show AT1 receptor selectivity. 125I-Sar1,Gly8 Ang II offers a new means to selectively radiolabel AT1 receptors and may help to characterize ligand docking sites and agonist switches for AT1 versus AT2 receptors.     

Angiotensin receptor subtypes in rat, rabbit and monkey tissues: relative distribution and species dependency

The displacement of [125I]Sar1, Ile8 angiotensin II binding by the receptor subtype selective angiotensin II antagonists, DuP-753 and WL-19 (PD121981) was used to define the relative proportion of angiotensin subtype AT1 and subtype AT2 receptors, respectively in various tissues (aorta, heart, adrenal cortex, kidney cortex and brain) of the rat, rabbit and monkey. The relative abundance of these receptor subtypes varied greatly not only among different tissues of the same species but also within the same tissue of different species. The relative affinity of the DuP-753 and WL-19 for the angiotensin receptor subtypes did not vary markedly suggesting that the two angiotensin receptor subtypes in these tissues and species are similar.     

Genetic deletion of angiotensin AT2 receptor leads to increased cell numbers in different brain structures of mice

Angiotensin II (Ang II) is a potent vasoactive peptide and displays growth factor-like properties. Different high-affinity Ang II receptor subtypes (AT1A, AT1B and AT2) have been cloned. They are expressed in various brain structures. Additionally, it has been assumed that Mas could interact directly or indirectly with the renin-angiotensin system. The AT1 receptor mediates pressor and mitogenic effects of Ang II, whereas physiological function and signaling mechanisms of the AT2 receptor remain poorly understood. Recent reports have shown that Ang II could mediate apoptosis through AT2 receptors. Since the AT1A, AT2 and Mas knockout mice provide new tools for uncovering potential actions of Ang II, the cell number in different brain structures of male adult wild-type mice and mice deficient for AT1A, AT2 or Mas was evaluated to get more insight into the role of Ang II in central nervous system development. In nearly all investigated brain structures (cortex, hippocampus, amygdala, thalamus), the cell number was significantly higher in AT2-deficient mice in comparison to wild-type mice. To the contrary, in AT1A-deficient mice the cell number was significantly less than in controls in the lateral geniculate and the medial amygdaloid nucleus. However, cell numbers were not changed in Mas-knockout mice compared to their wild-types. These results show the contrary effects of both angiotensin receptors on cell growth and represent the first demonstration of their action on neuronal cell development evidenced in the adult mouse brain.     

Nitric oxide synthase mRNA levels correlate with gene expression of angiotensin II type-1 but not type-2 receptors, renin or angiotensin converting enzyme in selected brain areas.

Recent data suggest that there is interaction between peripheral angiotensin II and nitric oxide. However, sparse information is available on the mutual interaction of these two compounds in the brain. The potential intercourse of nitric oxide with brain neuropeptides needs to be substantiated by assessing its local production and gene expression of the synthesizing enzymes involved. The aim of the present study was to evaluate whether the gene expression of brain nitric oxide synthase (bNOS) is related to the sites of gene expression of different components of the rat brain renin angiotensin system (renin, angiotensin converting enzyme (ACE) or angiotensin receptors of AT1 and AT2 subtypes). The levels of corresponding mRNAs were measured and correlated in nine structures of adult rat brain (hippocampus, amygdala, septum, thalamus, hypothalamus, cortex, pons, medulla and cerebellum). As was expected, positive correlation was observed between renin and angiotensin-converting enzyme mRNAs. Moreover, a significant correlation was found between brain NO synthase and AT1 receptor mRNAs, but not with mRNA of the AT2 receptor, ACE and renin. Parallel distribution of mRNAs coding for bNOS and AT1 receptors in several rat brain structures suggests a possible interaction between brain angiotensin 11 and nitric oxide, which remains to be definitely demonstrated by other approaches.     

Role of brain renin angiotensin system in neurodegeneration: An update

Renin angiotensin system (RAS) is an endocrine system widely known for its physiological roles in electrolyte homeostasis, body fluid volume regulation and cardiovascular control in peripheral circulation. However, brain RAS is an independent form of RAS expressed locally in the brain, which is known to be involved in brain functions and disorders. There is strong evidence for a major involvement of excessive brain angiotensin converting enzyme (ACE)/Angiotensin II (Ang II)/Angiotensin type-1 receptor (AT-1R) axis in increased activation of oxidative stress, apoptosis and neuroinflammation causing neurodegeneration in several brain disorders. Numerous studies have demonstrated strong neuroprotective effects by blocking AT1R in these brain disorders. Additionally, the angiotensin converting enzyme 2 (ACE2)/Angiotensin (1–7)/Mas receptor (MASR), is another axis of brain RAS which counteracts the damaging effects of ACE/Ang II/AT1R axis on neurons in the brain. Thus, angiotensin II receptor blockers (ARBs) and activation of ACE2/Angiotensin (1–7)/MASR axis may serve as an exciting and novel method for neuroprotection in several neurodegenerative diseases. Here in this review article, we discuss the expression of RAS in the brain and highlight how altered RAS level may cause neurodegeneration. Understanding the pathophysiology of RAS and their links to neurodegeneration has enormous potential to identify potentially effective pharmacological tools to treat neurodegenerative diseases in the brain.     

Adenoviral-mediated neuron specific transduction of angiotensin II type 2 receptors

The angiotensin II (Ang II) type 2 receptor (AT2R) is localized at specific nuclei within adult rat brain. However, a lack of specific approaches for manipulating the activity of neuronal AT2R has meant that the physiological actions of these sites in the brain remain to be established. Therefore, in this study, our aim was to develop a method by which AT2R can be specifically overexpressed in neurons and in rat brain, with the ultimate goal of a producing a system where discrete increases in AT2R levels in brain nuclei could reveal (and be linked to) physiological actions. Here, we have constructed an AT2R recombinant adenoviral vector, Ad5-SYN-AT2R-IRES-EGFP, which contains the AT2R gene and an IRES-linked EGFP reporter gene, both driven by the neuron-specific synapsin I (SYN) gene promoter. This vector efficiently transduces the AT2R into neuronal cells in culture and results in the expression of high levels of AT2R. These expressed receptors are functional in terms of inhibition of Erk mitogen activated protein kinases (Erk MAPK) and stimulation of neuronal K+ current. Furthermore, microinjection of this vector into adult rat brain elicits a long lasting ( approximately 1 month) expression of AT2R within neurons. In summary, we have developed a viral vector that can be used for the efficient transduction of AT2R into neurons both in vitro and in vivo, the use of which may help to define the physiological functions of brain AT2R in adult rats.     

Differential beta-arrestin binding of AT1 and AT2 angiotensin receptors

Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, beta-arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT1 receptors, whereas AngII-bound AT2 receptors do not internalize. Although the activation of the rat AT1A receptor with AngII causes translocation of beta-arrestin2 to the receptor, no association of this molecule with the AT2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.     

Renal effects of angiotensin II receptor subtype 1 and 2-selective ligands injected into the paraventricular nucleus of conscious rats.

We determined the effects of losartan and CGP42112A (selective ligands of the AT1 and AT2 angiotensin receptors, respectively) and salarasin (a relatively nonselective angiotensin receptor antagonist) on urinary volume and urinary sodium and potassium excretion induced by administration of angiotensin II (ANG II) into the paraventricular nucleus (PVN) of conscious rats. Both the AT1 and AT2 ligands and salarasin administered in the presence of ANG II elicited a concentration-dependent inhibition of urine excretion, but losartan inhibited only 75% of this response. The IC50 for salarasin, CGP42112A, and losartan was 0.01, 0.05, and 6 nM, respectively. Previous treatment with saralasin, CGP42112A and losartan competitively antagonized the natriuretic responses to PVN administration of ANG II, and the IC50 values were 0.09, 0.48, and 10 nM, respectively. The maximum response to losartan was 65% of that obtained with saralasin. Pretreatment with saralasin, losartan, and CGP42112A injected into the PVN caused shifts to the right of the concentration-response curves, but the losartan concentrations were disproportionately greater compared with salarasin or CGP42112A. The IC50 values were 0.06, 0.5, and 7.0 for salarasin, CGP42112A, and losartan, respectively. These results suggest that both AT1 and AT2 receptor subtypes in the PVN are involved in ANG II-related urine, sodium, and potassium excretion, and that the inhibitory responses to AT2 blockade are predominant.     

The angiotensin II AT2 receptor is an AT1 receptor antagonist

The vasopressor angiotensin II activates AT(1) and AT(2) receptors. Most of the known in vivo effects of angiotensin II are mediated by AT(1) receptors while the biological functions of AT(2) receptors are less clear. We report here that the AT(2) receptor binds directly to the AT(1) receptor and thereby antagonizes the function of the AT(1) receptor. The AT(1)-specific antagonism of the AT(2) receptor was independent of AT(2) receptor activation and signaling, and it was effective on different cells and on human myometrial biopsies with AT(1)/AT(2) receptor expression. Thus, the AT(2) receptor is the first identified example of a G-protein-coupled receptor which acts as a receptor-specific antagonist.     

Characterization of a specific antibody to the rat angiotensin II AT1 receptor.

We raised a polyclonal antibody against a decapeptide corresponding to the carboxyl terminus of the rat angiotensin II AT1 receptor. This antibody was demonstrated to be specific for the rat receptor according to a number of approaches. These included (a) the ultrastructural localization of immunogold-labeled receptor on the surfaces of zona glomerulosa cells in the adrenal cortex, (b) the specific labeling of Chinese hamster ovarian (CHO) cells transfected with AT1 receptors, (c) the identification of a specific band on Western blots, (d) the immunocytochemical co-localization of angiotensin receptors on neurons in the lamina terminalis of the brain shown to be responsive to circulating angiotensin II, as shown by the expression of c-fos, and (e) the correlation between the expression of the mRNA of the AT1 receptor and AT1 receptor immunoreactivity.(J Histochem Cytochem 47:507-515, 1999)     

Brain renin angiotensin system (RAS) in stress-induced analgesia and impaired retention.

Physiological stress is known to produce analgesia and memory disruption. Brain renin angiotensin system (RAS) has been reported to participate in stress response and plays a role in the processing of sensory information. Angiotensin receptors (AT), particularly AT1 subtypes have been reported to be distributed in brain areas that are intimately associated with stress response. The purpose of present study was to examine the modulation of AT1 receptor in the immobilization stress and angiotensin II (AngII)-induced analgesia and impaired retention, and to determine whether resultant behavioral changes involve common sensory signals. Result of present experiments showed that immobilization stress in mice and rats, and intracerebroventricular (ICV) administration of AngII (10 and 20 ng) in rats produced an increase in tail-flick latency. Similarly, post training administration of AngII or immobilization stress produced impairment of retention tested on plus-maze learning and on passive avoidance step-down task. Both these responses were sensitive to reversal by prior treatment with losartan (10 and 20 mg/kg), an AT1 AngII receptor antagonist. On the other hand, naloxone, an opiate antagonist preferentially attenuated the stress and AngII-induced analgesia and retention deficit induced by immobilization stress, but failed to reverse the AngII induced retention deficit. These results suggest immobilization stress-induced analgesia and impaired retention involves the participation of brain RAS. Further, failure of naloxone to reverse AngII-induced retention impairment shows. AngII-induced behavioral changes are under control of different sensory inputs.     

Identification of two subtypes in the rat type I angiotensin II receptor.

A rat adrenal cDNA library was screened by colony hybridization using a rat cDNA fragment of type I angiotensin II receptor (AT1A) previously isolated from the kidney. Two cDNA clones were identified, designated as AT1B, to have a nucleotide sequence highly homologous to and yet distinct from AT1A. The amino acid sequence of AT1B consists of 359 amino acid residues and has 96% identity with AT1A. No conspicuous difference in the ligand binding characteristics was observed between AT1A and AT1B. The mRNA for AT1B was expressed in many tissues as is the case with AT1A, and most abundantly expressed in the adrenal glands in the Sprague-Dawley rats. The existence of two subtypes in the rat type I angiotensin II receptor might explain the diverse actions of angiotensin II in various tissues.     

A brain leptin-renin angiotensin system interaction in the regulation of sympathetic nerve activity

The sympathetic nervous system, leptin, and renin-angiotensin system (RAS) have been implicated in obesity-associated hypertension. There is increasing evidence for the presence of both leptin and angiotensin II receptors in several key brain cardiovascular and metabolic control regions. We tested the hypothesis that the brain RAS plays a facilitatory role in the sympathetic nerve responses to leptin. In rats, intracerebroventricular (ICV) administration of losartan (5 μg) selectively inhibited increases in renal and brown adipose tissue (BAT) sympathetic nerve activity (SNA) produced by leptin (10 μg ICV) but did not reduce the SNA responses to corticotrophin-releasing factor (CRF) or the melanocortin receptor agonist MTII. In mice with deletion of angiotensin II type-1a receptors (AT(1a)R(-/-)), increases in renal and BAT SNA induced by leptin (2 μg ICV) were impaired whereas SNA responses to MTII were preserved. Decreases in food intake and body weight with ICV leptin did not differ in AT(1a)R(-/-) vs. AT(1a)R(+/+) mice. ICV leptin in rats increased AT(1a)R and angiotensin-converting enzyme (ACE) mRNA in the subfornical organ and AT(1a)R mRNA in the arcuate nucleus, suggesting leptin-induced upregulation of the brain RAS in specific brain regions. To evaluate the role of de novo production of brain angiotensin II in SNA responses to leptin, we treated rats with captopril (12.5 μg ICV). Captopril attenuated leptin effects on renal and BAT SNA. In conclusion, these studies provide evidence that the brain RAS selectively facilitates renal and BAT sympathetic nerve responses to leptin while sparing effects on food intake.     

Changes in expression of angiotensin receptor subtypes in the rat aorta during development

Quantitative autoradiography was used to characterize angiotensin AT1 and AT2 receptors, in the rat aorta at three developmental ages; embryonic day 18 (E18), and postnatal weeks 2 and 8. The expression of angiotensin receptors was higher in the aorta of E18 and 2-week-old rat. A major proportion of the angiotensin receptors expressed in the aorta at these two ages was AT2 (84 and 81% respectively). Conversely, in the aorta of 8-week-old rats, AT1 was the predominant angiotensin receptor subtype (71%). In 8-week-old rats, the AT2 subtype was also present (28%). In pre- and postnatal rats, [125I]Sar1-angiotensin II binding to AT1 receptors was sensitive to GTP gamma S whereas binding to AT2 receptors was not. AT2 receptors may serve an important role during stages of rapid growth of the aorta, and also have a significant function in the adult vasculature.     

Chimeric AT1/AT2 receptors reveal functional similarities despite key amino acid dissimilarities in the domains mediating agonist-dependent activation

Chimeric AT1/AT2 angiotensin II (AngII) receptors in which the sixth and/or seventh transmembrane-spanning domains of the AT2 receptor were substituted into the AT1 receptor were used to investigate the activation mechanisms of the two receptor subtypes. Numerous reports have identified amino acid residues in the sixth and seventh transmembrane-spanning domains of the AT1 receptor involved in the intrareceptor activation mechanism following agonist binding. Many of these residues are not conserved in the AT2 receptor; the corresponding AT2 receptor residues are, in fact, disruptive of AngII-dependent activation when substituted into the AT1 receptor. Surprisingly, the chimeric AT1/AT2 receptors--which also lack these crucial AT1 residues--exhibited AngII-induced activation of phosphoinositide hydrolysis with efficacies and potencies similar to the wild-type AT1 receptor. Consistent with earlier reports, a AT1[Y292F] point mutant demonstrated greatly decreased agonist-induced activation of phosphoinositide hydrolysis. However, a AT1[Y292F/N295S] double-point mutant allowed for normal agonist-induced activation with a pharmacodynamic profile indistinguishable from the wild-type receptor. Despite amino acid dissimilarities, the same corresponding domains and even the same residue loci in both of the AngII receptor subtypes are equally able to mediate agonist-induced receptor activation. This suggests that these corresponding domains in the AT1 and the AT2 receptors are crucial to the activation mechanism, demonstrating greater structural flexibility than previously believed regarding AT1 receptor activation and supporting the possibility of a common activation mechanism for the two receptor subtypes.