AT1B Receptor Research Articles

Genetic deletion of AT1a receptors attenuates intracellular accumulation of ANG II in the kidney of AT1a receptor-deficient mice

We and others have previously shown that high levels of ANG II are accumulated in the rat kidney via a type 1 (AT(1)) receptor-mediated mechanism, but it is not known which AT(1) receptor is involved in this process in rodents. We tested the hypothesis that AT(1a) receptor-deficient mice (Agtr1a-/-) are unable to accumulate ANG II intracellularly in the kidney because of the absence of AT(1a) receptor-mediated endocytosis. Adult male wild-type (Agtr1a+/+), heterozygous (Agtr1a+/-), and Agtr1a-/- were treated with vehicle, ANG II (40 ng/min ip via osmotic minipump), or ANG II plus the AT(1) antagonist losartan (10 mg.kg(-1).day(-1) po) for 2 wk. In wild-type mice, ANG II induced hypertension (168 +/- 4 vs. 113 +/- 3 mmHg, P < 0.001), increased kidney-to-body weight ratio (P < 0.01), caused pressure natriuresis (P < 0.05), and elevated plasma and whole kidney ANG II levels (P < 0.001). Concurrent administration of ANG II with losartan attenuated these responses to ANG II. In contrast, Agtr1a-/- mice had lower basal systolic pressures (P < 0.001), smaller kidneys with much fewer AT(1b) receptors (P < 0.001), higher basal 24-h urinary sodium excretion (P < 0.01), as well as basal plasma and whole kidney ANG II levels (P < 0.01). However, intracellular ANG II levels in the kidney were lower in Agtr1a-/- mice. In Agtr1a-/- mice, ANG II slightly increased systolic pressure (P < 0.05) but had no effect on the kidney weight, urinary sodium excretion, and whole kidney ANG II levels. Losartan restored systolic pressure to basal levels and decreased whole kidney ANG II levels by approximately 20% (P < 0.05). These results demonstrate a predominant role of AT(1a) receptors in blood pressure regulation and in the renal responses to long-term ANG II administration, that AT(1b) receptors may play a limited role in blood pressure control and mediating intrarenal ANG II accumulation in the absence of AT(1a) receptors.

Developmental Programming Through Epigenetic Changes

See related article, pages 520–526 The last decade has seen an exponential number of scientific articles addressing the concept of fetal programming of adult onset diseases. Though it has long been known (as it is probably part of common knowledge in human history) that the environment and life events during the fetal period impact on the child’s development, the notion that intrauterine life can also impact on adult health and on the incidence of late onset diseases such as hypertension and type II diabetes is relatively more recent. In 1964, Rose reported the association of ischemic heart disease and infant mortality within the same families (reviewed by1). Then Forsdahl in 1977 showed that incidence of arteriosclerotic heart disease in a certain age group could be correlated with the infant mortality rate of that same population.1 Interest in these types of association rose dramatically following Professor Barker and collaborators’ extensive observations of a well documented population in England and Wales. The meticulous work of home health visitors in the early 20th century, which noted every child weight and length at birth and throughout the first year of life as well as other observations of the mother health and living milieu, generated data which clearly demonstrated an inverse relationship between birthweight and adult incidence of cardiovascular related mortality and morbidities as well as type II diabetes.1 We will focus here on hypertension and related complications. The association between birthweight and adult blood pressure was demonstrated in many parts of the world, in industrialized as well as in more developing countries, in both men and women.1 The association is first noted in early childhood (but not at birth) and seems to increase with age. Despite the significant number of studies published, which have examined to date tens …

Knockout Mice Reveal That the Angiotensin II type 1B Receptor Links to Smooth Muscle Contraction

Rodents express two isoforms of the angiotensin II type 1 (AT(1)) receptor: AT(1A) and AT(1B). It is unclear which receptor subtype mediates contraction in response to angiotensin II in various arteries. We tested the hypothesis that the AT(1B) receptor is the predominant receptor that mediates contraction in the abdominal aorta in response to angiotensin II. Isometric tension responses to angiotensin II were determined in abdominal aortic rings obtained from male wild-type and AT(1B) receptor knockout mice. The rings were suspended in an organ bath of a wire myograph and contractions to angiotensin II and other vasoconstrictors were determined. Angiotensin II contracted aortic segments from wild-type mice; however, this response was virtually absent in rings obtained from AT(1B) receptor knockout mice. Contractions in response to K(+) and U46619 (thromboxane A(2) mimetic) were not different between rings obtained from wild-type and AT(1B) receptor knockout mice. Reduced angiotensin II contraction is not related to a generalized decrease in smooth muscle function, rather it is specifically due to genetic ablation of the AT(1B) receptor. Our data support the concept that AT(1B) receptors couple to contraction in the mouse abdominal aorta, a function that parallels the single known AT(1) receptor in human vascular smooth muscle.

Angiotensin II Upregulates the Expression of L‐type Calcium Channels, Inositol Trisphosphate Receptors and Ryanodine Receptors in Rat Mesenteric Vascular Bed

In vitro effects of angiotensin II (Ang II) on the expression of CaV1.2 L-type voltage dependent calcium channel (L-VDCC), type 1 inositol 1, 4, 5 trisphosphate receptor (IP3R1) and ryanodine receptor (RyR) in mesenteric vascular beds of Sprague-Dawley rats were studied using Western blots. Ang II dose-dependently upregulated the protein expression of L-VDCC, IP3R1 and RyR in cultured mesenteric arterial beds within 24 h. These actions were not blocked by losartan or candesartan (AT1 receptor antagonists); instead, the upregulations of L-VDCC and RyR, but not IP3R1, by Ang II were blocked by PD 123,319 (an AT2 or AT1B receptor antagonist). The mRNA levels of these channels were not found to be increased by Ang II. The endothelium nitric oxide synthase (eNOS) inhibitor, L-NAME, exerted a similar upregulation of these channels, while the nitric oxide donor, sodium nitroprusside (SNP), downregulated these channels. After endothelium damage by 0.3% CHAPS, Ang II had no effects on any of these channels. The upregulations of these channels by Ang II were also abolished by the PKC inhibitor chelerythrine and by the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI). These results suggest that: Ang II upregulates L-VDCC, IP3R1 and RyR calcium release channels in the rat mesenteric bed, with effects on the L-VDCC and RyR mediated by PD 123,319-sensitive receptors (AT2 or AT1B); These actions were via posttranscriptional modulation of the channels; PKC, NAD(P)H oxidase-mediated oxidative stress and NO signaling pathways and the endothelium were involved in these actions. Supported by HL 63903.

Angiotensin AT1a shRNA Demonstrates Interactions between Brainstem Angiotensin AT1a Receptors and Angiotensin Converting Enzyme 2

Angiotensin (Ang) AT1a receptors and Ang converting enzyme 2(ACE2) are expressed in the brainstem dorsal vagal complex (DVC). Studies were conducted using adenovirus mediated inference small hairpin RNA for AT1a (Ad-AT1a-shRNA) to evaluate the interactions between AT1a and ACE2. We used a system in which Ad-AT1a-shRNA or its Ad-LacZ control was injected bilaterally into the DVC. mRNA levels were measured using in situ hybridization (ISH). Using a double cannulae inserted in the DVC, Ad-AT1a-shRNA or Ad-LacZ (both at 1x 109 cpu/ml, 200nl) was injected. This injection method allows for a side by side comparison of mRNA expression. Brainstems were collected at day 10 after treatment, a time at which receptors are known to be decreased. ISH was performed for Ang AT1a, Ang AT1b, ACE1, and ACE2, monitored using phosphor imaging and autoradiography. Results showed: Ad-AT1a-shRNA decreased Ang AT1a mRNA by 61.2 ± 6.8% compared with the control side (p<0.001). Ad-AT1a-shRNA had no effect on AT1b receptor mRNA expression. ACE2 mRNA expression on the Ad-AT1a-shRNA treated side was reduced by 29.0±14.5 % (P<0.01). Ad-AT1a-shRNA had no effect on ACE1. These results demonstrate that Ad-AT1a-shRNA can site- and subtype- specifically silence AT1a receptor mRNA. AT1a may also modulate brainstem ACE2 expression, perhaps reflecting important interactions between the systems in control of blood pressure and autonomic function.

Fat intake modifies vascular responsiveness and receptor expression of vasoconstrictors: Implications for diet-induced obesity

Angiotensin II (Ang II), endothelin-1 (ET-1) and reactive oxygen species (ROS) have been implicated in the development of pathologic changes associated with obesity including hypertension and atherosclerosis. The aim of this study was to investigate the effects of dietary fat content on vasoreactivity and receptor expression at the level of gene and protein expression. C57BL/6 mice were fed diets of normal (Control, 12.3% kcal from fat), high (HF, 41% kcal from fat) and very high (VHF, 58% kcal from fat) fat content for 15 weeks. Glucose tolerance tests were performed, and aortic rings were exposed to ET-1 (0.01-300 nM) and Ang II (100 nM) in the presence of L-nitro-arginine-methyl ester (L-NAME; 300 microM). Gene and protein expressions of angiotensin and endothelin receptors were examined by real-time PCR and immunoblotting, respectively. The effects of diet on responses to acetylcholine (ACh 0.1-300 microM), in the absence or presence of L-NAME, and to exogenous ROS/.OH were also investigated. Both high fat diets similarly impaired glucose tolerance (P<0.05). Increasing dietary fat augmented contractions to Ang II in a step-wise manner (P<0.05). Conversely, increasing dietary fat had no effect on contractions to ET-1. Exposure to ROS/.OH resulted in a rapid vasodilation that was markedly augmented in a step-wise manner with increasing dietary fat (P<0.05). Endothelium-dependent relaxation to ACh was unaffected whereas vasoconstriction to high concentrations of ACh was enhanced in VHF animals (P<0.05 vs. control). Gene expression of the AT(1B) receptor was increased in the aorta of VHF mice, and aortic ET(A) receptor protein expression was increased after both high fat diets. These findings demonstrate that changes in dietary fat intake modulate vascular reactivity in response to Ang II and ROS, as well as expression of vascular angiotensin and endothelin receptors. Dietary fat intake may thereby directly affect cardiovascular risk.

Angiotensin II Inhibition Reduces Stress Sensitivity of Hypothalamo-Pituitary-Adrenal Axis in Spontaneously Hypertensive Rats

Angiotensin II type 1 (AT(1)) receptors are expressed within organs of the hypothalamo-pituitary-adrenal (HPA) axis and seem to be important for its stress responsiveness. Secretion of CRH, ACTH, and corticosterone (CORT) is increased by stimulation of AT(1) receptors. In the present study, we tested whether a blockade of the angiotensin II system attenuates the HPA axis reactivity in spontaneously hypertensive rats. Spontaneously hypertensive rats were treated with candesartan (2 mg/kg), ramipril (1 mg/kg), or mibefradil (12 mg/kg) for 5 wk. In addition to baseline levels, CORT and ACTH responses to injection of CRH (100 microg/kg) were monitored over 4 h. mRNA of CRH, proopiomelanocortin, AT(1A), AT(1B), and AT(2) receptors was quantified by real-time PCR. All treatments induced equivalent reductions of blood pressure and had no effect on baseline levels of CORT and ACTH. However, both candesartan and ramipril significantly reduced CRH-stimulated plasma levels of ACTH (-26 and -15%) and CORT (-36 and -18%) and lowered hypothalamic CRH mRNA (-25 and -29%). Mibefradil did not affect any of these parameters. Gene expression of AT(1A), AT(1B), and AT(2) receptors within the HPA axis was not altered by any drug. We show for the first time that antihypertensive treatment by inhibition of AT(1) receptors or angiotensin-converting enzyme attenuates HPA axis reactivity independently of blood pressure reduction. This action is solely evident after CRH stimulation but not under baseline conditions. Both a reduced pituitary sensitivity to CRH and a down-regulation of hypothalamic CRH expression have the potential to reduce HPA axis activity during chronic AT(1) blockade or angiotensin-converting enzyme inhibition.

Renal microvascular vasoconstrictor and vasodilator responses are reduced in angiotensin type 1 receptor double null mice (DKO)

AngII induced afferent arteriolar (AA) diameter responses are reduced, while efferent arteriolar (EA) diameter responses are absent in kidneys of mice lacking the AT1A receptor compared to wild-type (WT) (AJP 284:F538-545, 2003), while responses to norepinephrine (NE) are not altered. We determined the renal microvascular reactivity of WT and DKO mice using the in vitro blood perfused juxtamedullary nephron technique. Kidneys were harvested from WT and DKO mice and bathed with NE (100–1000nM), AngII (100nM), or acetylcholine (ACh;10μM). In WT mice, AA (12.6±0.9μm; n=8) diameter was significantly reduced by 29±4 and 51±6%, and EA (11.7±0.4μm; n=8) diameter was reduced by 17±2 and 38±3% in response to 300 and 1000nM NE, respectively. Baseline AA diameters of DKO kidneys were significantly larger than WT (19.1±1.2μm; n=12), while EA diameters were not different (15.8±2.4μm; n=4). Significant constriction to 1μM NE was observed in AA and EA (18±5% AA; 23±6% EA) of kidneys from DKO mice, but were less than observed in WT mice. AngII produced a 23±7% and 28±7% reduction in AA (n=5) and EA (n=7) diameters of WT mice, respectively. Responses to AngII were absent in AA (−2±2%; n=12) and EA (−0.1±2%; n=4) of DKO mice as predicted. AA diameter increased significantly from 15.0±0.6 to 17.0±1.2μm in response to ACh in WT mice (n=6). AA diameter of DKO mice (n=6) was not significantly altered by ACh averaging 19.8±1.1 and 17.8±1.6μm (p=0.06), respectively. In summary, vasoconstrictor and vasodilator responses are reduced in renal arterioles of mice lacking both AT1A and AT1B receptor subtypes. We conclude that AngII plays a pivotal role in renal arteriolar vascular smooth muscle cell development and/or function.

Targeting Brain AT 1 Receptors By RNA Interference

The dipsogenic and pressor actions of angiotensin II (Ang II) in the central nervous system (CNS) have been well documented in many species and are now accepted as dogma. The major central cardiovascular effects of Ang II are elicited by a complex receptor-dependent signaling cascade initiated by the detection of circulating Ang II in regions of the brain lacking a blood brain barrier (BBB) through local production of Ang II in regions outside and inside the BBB or through the neurotransmitter actions of Ang II. It is generally accepted that most of the central neurocardiovascular actions of Ang II occur through the activation of intracellular signaling cascades that originate at the Ang II type 1 (AT1) receptor. A second Ang II receptor, termed AT2, has been implicated in some cardiovascular and behavioral responses. However, the AT2 receptor is distantly related to AT1 and can be effectively distinguished from AT1 pharmacologically. In humans, a single gene encodes the AT1 Ang II receptor, whereas in rodents a duplication resulted in 2 AT1 receptor genes, located on different chromosomes, each known to encode 1 of 2 distinct subtypes of the receptor, AT1a and AT1b. Both receptors are highly homologous, use the same signaling mechanisms, and cannot be distinguished using currently available pharmacological reagents or antisera. Despite their high similarity at the protein level, sufficient divergence is present in the sequence of the mRNA that allows them to be distinguished by RT-PCR and in situ hybridization. Through

Efferent arterioles exclusively express the subtype 1A angiotensin receptor: functional insights from genetic mouse models

Angiotensin (ANG) type 1A (AT(1A)) receptor-null (AT(1A)(-/-)) mice exhibit reduced afferent arteriolar (AA) constrictor responses to ANG II compared with wild-type (WT) mice, whereas efferent arteriolar (EA) responses are absent (Harrison-Bernard LM, Cook AK, Oliverio MI, and Coffman TM. Am J Physiol Renal Physiol 284: F538-F545, 2003). In the present study, the renal arteriolar constrictor responses to norepinephrine (NE) and/or ANG II were determined in blood-perfused juxtamedullary nephrons from kidneys of AT(1A)(-/-), AT(1B) receptor-null (AT(1B)(-/-)), and WT mice. Baseline AA diameter in AT(1A)(-/-) mice was not different from that in WT mice (13.1 +/- 0.9 and 12.6 +/- 0.9 microm, n = 7 and 8, respectively); however, EA diameters were significantly larger (17.3 +/- 1.4 vs. 11.7 +/- 0.4 microm, n = 10 and 8) in AT(1A)(-/-) than in WT mice. Constriction of AA (-40 +/- 8 and -51 +/- 6% at 1 microM NE) and EA (-29 +/- 6 and -38 +/- 3% at 1 microM NE) in response to 0.1-1 microM NE was similar in AT(1A)(-/-) and WT mice. Baseline diameters of AA (13.5 +/- 0.7 and 14.2 +/- 0.9 microm, n = 9 and 10) and EA (15.4 +/- 1.0 and 15.0 +/- 0.7 microm, n = 11 and 9) and ANG II (0.1-10 nM) constrictor responses of AA (-25 +/- 4 and -31 +/- 5% at 10 nM) and EA (-32 +/- 6 and -35 +/- 7% at 10 nM) were similar in AT(1B)(-/-) and WT mice, respectively. ANG II-induced constrictions were eliminated by AT(1) receptor blockade with 4 microM candesartan. Taken together, our data demonstrate that AA and EA responses to NE are unaltered in the absence of AT(1A) receptors, and ANG II responses remain intact in the absence of AT(1B) receptors. Therefore, we conclude that AT(1A) and AT(1B) receptors are functionally expressed on the AA, whereas the EA exclusively expresses the AT(1A) receptor.

Characterization of two polyclonal peptide antibodies that recognize the carboxy terminus of angiotensin II AT1A and AT1B receptors

1. The differential identification of the angiotensin AT1A and AT1B receptor subtypes is impaired by the existing>96% homology of both receptors. In the present study, we characterized two polyclonal rabbit peptide antibodies, namely alpha-AT1A and alpha-AT1B, that recognize the C-terminal region of mouse AT1A and AT1B receptors, respectively. 2. In immunoblotting, both antibodies detected two major AT1 receptor-specific bands at sizes of 72.5 and 87.6 kDa in mouse tissues and in Neuro-2a cell lysates. In immunohistochemistry, antibodies demonstrated AT1 receptor-specific staining in renal proximal and distal tubules, as well as in kidney glomeruli. In addition, both antibodies stained AT1 receptors in Neuro-2a cells with G-protein receptor typical distribution. Dot-blot and ELISA analysis of the alpha-AT1A antibody showed 2.5- to fourfold higher selectivity for its AT1A receptor target peptide (1A-PEP) compared with the non-specific AT1B receptor peptide (1B-PEP). In contrast, the alpha-AT1B antibody showed high binding affinity towards its target peptide 1B-PEP, but also demonstrated high cross-reactivity for the non-specific peptide 1A-PEP (1.4- to twofold in ELISA and dot-blot analysis). In contrast with the lack of recognition by the alpha-AT1B antibody, the alpha-AT1A antibody selectively recognized the AT1A receptor fused to red fluorescence protein in transiently transfected Chinese hamster ovary cells. 3. In summary, we have generated two new peptide antibodies to the mouse AT1A and AT1B receptors (alpha-AT1A and alpha-AT1B), of which the alpha-AT1A antibody has the capability to distinguish AT1A receptor types in immunological approaches.

Enhanced osmotic responsiveness in angiotensin AT1a receptor deficient mice: evidence for a role for AT1b receptors

Experiments were performed to study the role of angiotensin (Ang) AT1a and AT1b receptor subtypes in osmotic regulation of blood pressure using gene deletion and pharmacological methods. The cardiovascular effects of hypertonic saline (HS) or vasopressin (VP) delivered via vascular catheters were measured in Ang AT1a gene deletion (AT1a-/-) and control (AT1a+/+) mice. Blood pressure (BP) and heart rate (HR) were recorded in conscious mice using direct carotid catheters. Plasma osmolality and VP concentration were also measured. The major finding was that deletion of AT1a receptors resulted in enhanced BP response to osmotic stimulation. This was seen after acute HS injection (20 microl, 20% NaCl). The peak percentage change in mean arterial pressure (MAP) was 15.4+/-1.9% versus 28.1+/-2.4% (AT1a+/+versus AT1a-/-, respectively). Losartan (AT1 antagonist), but not PD123319 (AT2 antagonist), inhibited the HS-induced MAP response, specifically in AT1a-/- mice. Plasma osmolality and VP concentration were elevated after HS injection with no differences noted between groups. Vascular injection of VP (5 ng g-1) increased BP and HR, with similar MAP response between groups. Evidence shows that removal of Ang AT1a receptors results in a significant enhancement in the pressor response to acute osmotic stimulation. Studies of AT1 receptor blockade indicate that complementary Ang AT1b receptors, but not AT2 receptors, may be involved in the osmotic response.

Functional evidence that in the cardiovascular system AT angiotensin II receptors are AT prejunctionally and AT postjunctionally

Numerous studies have shown that angiotensin II causes vasoconstriction both by a direct action on smooth muscle cells (postjunctional effect) and indirectly through the facilitation of noradrenaline release from postganglionic sympathetic neurons (prejunctional effect). The receptors through which angiotensin II exerts its actions were first divided into AT1 and AT2 subtypes. A further subdivision of AT1 receptors into AT1A and AT1B subtypes was based on cloning and receptor binding studies: AT1A showed a high affinity for losartan, but low affinity for PD123319, while AT1B receptors showed nearly 100-fold lower affinity for losartan and 10,000-fold higher affinity for PD123319 relative to AT1 sites. The present review deals with functional evidence supporting the existence of AT1A and AT1B subtypes in the cardiovascular system. Taken together, all the functional results obtained in vivo and in vitro-in a wide variety of vascular tissues from different species-allow one to conclude that angiotensin II AT1 receptors are different pre- and postjunctionally and also that prejunctional and postjunctional angiotensin II receptors most probably belong to AT1B and AT1A subtypes, respectively.

Physiological regulation of brain angiotensin receptor mRNA in AT1a deficient mice

Experiments were performed to study the physiological regulation of angiotensin (Ang) AT1b receptors using Ang AT1a knockout mice (AT1aKO). Ang AT1b mRNA was analyzed in forebrain, hypothalamus, and brainstem using in situ hybridization (ISH) under baseline and water-restricted conditions. Plasma was analyzed for osmolality, vasopressin, and corticosterone. Dehydration (24 h) increased osmolality and corticosterone and decreased body weight with no difference between groups. Plasma vasopressin was not different between the groups and was not stimulated by dehydration. Under water ad libitum conditions, there were no differences in AT1b mRNA expression in medial periventricular, anterior third ventricle (AV3V), and subfornical organ (SFO) between controls and AT1aKO. In contrast, there was higher expression in the dorsal motor nucleus of the vagus (DMV) of AT1aKO vs. Controls (0.6 ± 0.1 vs. 0.9 ± 0.1 μCi/g, Control vs. AT1aKO in water ad libitum group). Dehydration increased AT1b expression in SFO in AT1aKO, but not in controls (0.6 ± 0.07 vs. 0.9 ± 0.06 μCi/g; water ad libitum vs. dehydrated). Emulsion autoradiography documents the detailed pattern of AT1b expression in brainstem of controls and AT1aKO. There was labeling in DMV, locus coeruleus, inferior olive, lateral reticular nucleus, and caudalis spinal trigemius. In conclusion, deletion of AT1a receptors produces a compensatory increase in AT1b receptor mRNA expression in brainstem, but not in hypothalamus or rostral forebrain. In addition, AT1aKO mice showed an enhanced response to dehydration in terms of AT1b mRNA expression in SFO.

Severe hypertension caused by alleles from normotensive Lewis for a quantitative trait locus on chromosome 2

Pursuing fully a suggestion from linkage analysis that there might be a quantitative trait locus (QTL) for blood pressure (BP) in a chromosome (Chr) 2 region of the Dahl salt-sensitive rat (DSS), four congenic strains were made by replacing various fragments of DSS Chr 2 with those of Lewis (LEW). Consequently, a BP QTL was localized to a segment of around 3 cM or near 3 Mb on Chr 2 by comparative congenics. The BP-augmenting alleles of this QTL originated from the LEW rat, a normotensive strain compared with DSS. The dissection of a QTL with such a paradoxical effect illustrated the power of congenics in unearthing a gene hidden in the context of the whole animal system, presumably by interactions with other genes. The locus for the angiotensin II receptor AT-1B (Agtr1b) is not supported as a candidate gene for the QTL because a congenic strain harboring it did not have an effect on BP. There are approximately 19 known and unknown genes present in the QTL interval. Among them, no standout candidate genes are reputed to affect BP. Thus the QTL will likely represent a novel gene for BP regulation.

A major role for AT 1b receptor in mouse mesenteric resistance vessels and its distribution in heart and neuroendocrine tissues

In mice, angiotensin (Ang) II type-1 (AT 1) receptors exist as AT1a, and AT 1b subtypes. In an effort to understand the role of AT 1b in regulating vascular function, AT 1 subtype mRNA and its functional relevance in the mesenteric resistance vessels were determined using wildtype (WT) and AT 1a knockout (AT 1a –/– mice) mice. With RT-PCR followed by restriction-enzyme digestion, we found that AT 1b accounted for almost all (98%) of AT 1 receptors in the mesenteric resistance vessels of WT mice. Also, the Ang II response in the vessels of AT 1a –/– mice was comparable to that of WT mice, suggesting an important role for AT 1b in regulating vasoreactivity. To further characterize AT 1b receptor distribution, several other tissues were examined. Among them, AT 1b is only predominantly expressed in hypothalamus, whereas AT 1a exists exclusively or as a major subtype in heart, pituitary, adrenal glands and brainstem. These results further underscore a tissue-specific role for AT 1b receptor in mice.

A quantitative trait locus for aortic smooth muscle cell number acting independently of blood pressure: implicating the angiotensin receptor AT1B gene as a candidate

Vascular hyperplasia may be involved in the remodeling of vasculature. It was unknown whether there were genetic determinants for aortic smooth muscle cell number (SMCN) and, if so, whether they acted independently of those for blood pressure (BP). To unravel this issue, we utilized congenic strains previously constructed for BP studies. These strains were made by replacing various chromosome 2 segments of the Dahl salt-sensitive (S) rat with those of the Milan normotensive rat (MNS). We measured and compared SMCN in aortic cross-sectional areas and BPs of these strains. Consequently, a quantitative trait locus (QTL) for SMCN was localized to a chromosome region not containing a BP QTL, but harboring the locus for the angiotensin II receptor AT1B (Agtr1b). Agtr1b became a candidate for the SMCN QTL because 1) two significant mutations were found in the coding region between S and all congenic strains possessing the MNS alleles, and 2) contractile responses to angiotensin II were significantly and selectively reduced in congenic rats harboring the MNS alleles of the SMCN QTL compared with S rats. The current investigation presents the first line of evidence that a QTL for aortic SMCN exists, and it acts independently of QTLs for BP. The relevant congenic strains developed therein potentially provide novel mammalian models for the studies of vascular remodeling disorders.

Angiotensin type 1a receptor signaling-dependent induction of vascular endothelial growth factor in stroma is relevant to tumor-associated angiogenesis and tumor growth

Angiotensin II is a multi-functional bioactive peptide and recent reports have suggested that angiotensin II is a proangiogenic growth factor. A retrospective cohort study revealed that angiotensin converting enzyme inhibitors decreased cancer risk, however, the precise mechanism is unknown. We hypothesized that endogenous angiotensin II plays a crucial role in tumor-associated angiogenesis. Tumors implanted in the subcutaneous tissue of wild-type mice developed intensive angiogenesis with vascular endothelial growth factor (VEGF) induction in tumor stroma. AT1a receptor (AT1a-R), but not AT1b receptor or AT2 receptor was expressed in tumor stroma and systemic administration of an AT1-R antagonist reduced tumor-associated angiogenesis and VEGF expression in tumor stroma. Angiotensin II up-regulates VEGF expression through the pathway including protein kinase C, AP-1 and NF-kappaB in fibroblasts, the major cellular component of tumor stroma. VEGF is a major determinant of tumor-associated angiogenesis in the present model, since angiogenesis was markedly reduced by either a VEGF neutralizing antibody or a VEGF receptor kinase inhibitor. Compared with the wild-type, tumor-associated angiogenesis was reduced in AT1a-R null mice, with reduced expression of VEGF in the stroma, and this reduction in AT1a-R null mice was not inhibited by an AT1-R antagonist. These suggest that host stromal VEGF induction by AT1a-R signaling is a key regulator of tumor-associated angiogenesis and tumor growth. AT1a-R signaling blockade may be a novel and effective therapeutic strategy against cancers.

Dietary sodium regulates angiotensin AT1a and AT1b mRNA expression in mouse brain

Previous results showed that angiotensin (Ang) AT1a and AT1b receptor mRNA are expressed in mouse hypothalamus (HYP), brainstem (BS) and anterior pituitary (PIT). To extend these findings, we developed a real-time polymerase chain reaction (PCR) method to differentiate and quantify Ang AT1a and AT1b mRNA in mouse brain. An experiment was conducted in male C57Bl/6J mice to determine the effects of low and high dietary salt (0.04 or 8% NaCl for 2 weeks) on mRNA expression. Physiological measurements showed that high salt increased water intake (15.1 ± 0.6 ml/day), whereas low salt decreased water intake (3.2 ± 0.1 ml/day). There were no significant changes in body weight, hematocrit or plasma osmolality. Real-time PCR was effective in distinguishing AT1a and AT1b receptor mRNA. The PCR efficiencies for AT1a, AT1b and 18S ribosome were tested to be identical, making it possible to quantify mRNA levels. There were differences in angiotensin receptor expression, related to diet and brain region. In hypothalamus, both the high salt and low salt diet decreased AT1a expression (to 63 ± 4% and 62 ± 1%), although there were no changes in AT1b. In brainstem, there was a marked increase in AT1a (to 365 ± 60%) and AT1b (to 372 ± 23%) after high salt, although there was only a marked decrease for AT1b (to 23 ± 5%) after low salt. In anterior pituitary, both high salt and low salt diet increased AT1a expression (to 152 ± 8% and 123 ± 9%), although there were no changes in AT1b. Results document that both AT1 receptor subtypes are present in mouse hypothalamus, brainstem and anterior pituitary, and that there is differential regulation of expression in response to changes in dietary salt.

Different AT1 receptor subtypes at pre- and postjunctional sites: AT1A versus AT1B receptors.

Angiotensin (AT) II is known to enhance responses to electrical field stimulation (EFS) via AT1 receptors located on sympathetic nerve terminals. Differences in potency exist between AT1 receptor antagonists regarding the inhibition of the prejunctional and postjunctional AT1 receptors. It is hypothesized that prejunctional AT1 receptors might belong to the AT1B receptor subtype. Accordingly, the authors investigated whether AT1B receptor inhibition by high concentrations of PD123319 could suppress ATII-augmented noradrenergic transmission (prejunctional) in the rabbit thoracic aorta by means of a noradrenaline spillover model. Additionally, the influence of PD123319 on ATII-enhanced constrictor responses to electrical field stimulation was investigated in the isolated rabbit mesenteric artery. Furthermore, the authors investigated whether PD123319 could influence the constrictor responses (postjunctional) to ATII in both preparations. In the thoracic aorta, ATII (10 nM) caused a significant enhancement of EFS-evoked [3H]-noradrenaline release by a factor of 2.0 +/- 0.1. This reinforcement could be inhibited by PD123319 (0.1, 1, and 10 microM). The constrictor response to ATII was unaffected by PD123319. In the mesenteric artery, ATII (0.5 nM) caused a significant enhancement of constrictor responses to EFS by factors of 2.9 +/- 0.3, 2.3 +/- 0.3, and 1.6 +/- 0.1 at 1, 2, and 4 Hz, respectively. This enhancement could be attenuated by PD123319 (1 and 10 microM). The constrictor response to ATII was unaffected by PD123319. It is concluded that the prejunctional AT1 receptors belong to the AT1B subtype whereas postjunctional AT1 receptors do not.