Toshihiro Ichiki

Angiotensin II Type 2 Receptor Is Essential for Left Ventricular Hypertrophy and Cardiac Fibrosis in Chronic Angiotensin II–Induced Hypertension

Background—The roles of angiotensin II (Ang II) in the regulation of heart function under normal and pathological conditions have been well documented. Although 2 types of Ang II receptor (AT1 and AT2) are found in various proportions, most studies have focused on AT1-coupled events. In the present study, we examined the hypothesis that signaling by AT2 is important to the development of left ventricular hypertrophy and cardiac fibrosis by Ang II infusion in mice lacking the AT2 gene (Agtr 2−/Y). Methods and Results—Male Agtr 2−/Y and age-matched wild-type (WT) mice were treated long-term with Ang II, infused at a rate of 4.2 ng · kg−1 · min−1 for 3 weeks. Ang II elevated systolic blood pressure to comparable levels in Agtr 2−/Y and WT mice. WT mice developed prominent concentric cardiac hypertrophy, prominent fibrosis, and impaired diastolic relaxation after Ang II infusion. In contrast, there was no cardiac hypertrophy in Agtr 2−/Y mice. Agtr 2−/Y mice, however, did not show signs of heart failure or impairment of ventricular relaxation and only negligible fibrosis after Ang II infusion. The absence of fibrosis may be a clue to the absence of impairment in ventricular relaxation and account for the normal left ventricular systolic and diastolic performances in Agtr 2−/Y mice. Conclusions—Chronic loss of AT2 by gene targeting abolished left ventricular hypertrophy and cardiac fibrosis in mice with Ang II–induced hypertension.

Anxiety-like behavior in mice lacking the angiotensin II type-2 receptor.

The main biological role of angiotensin II type 2 receptor (AT2) has not been established. We made use of targeted disruption of the mouse AT2 gene to examine the role of the AT2 receptor in the central nervous system (CNS). AT2-deficient mice displayed anxiety-like behavior compared with wild-type mice. However, AT2-deficient mice showed no depressant-like activity and no change in hexobarbital-induced sleeping time as compared with findings in wild-type mice. Both noradrenergic and corticotropin-releasing factor (CRF) neuronal systems appear to be involved in this anxiety-like behavior. Diazepam, captopril (angiotensin I converting enzyme inhibitor), prazosin (alpha1 antagonist) reversed the anxiety-like behavior in these AT2-deficient mice, whereas yohimbine (alpha2 antagonist), phenylephrine (alpha1 agonist), clonidine (alpha2 agonist), isoproterenol (beta1/beta2 agonist), propranolol (beta1/beta2 antagonist) and alpha-helical CRF9-41 (CRF receptor antagonist) has no apparent effects on anxiety-like behavior in AT2-deficient mice. In addition, concentrations of plasma adrenocorticotropic hormone (ACTH) and corticosterone in AT2-deficient mice did not differ from these in wild-type mice, hence, there are probably no endocrine abnormalities involving the hypothalamic-pituitary-adrenal axis (HPA). The amygdala appears to play an important role in many of the responses to fear and anxiety. The number of [3H]prazosin but not [125I]CRF binding sites in the amygdala was significantly reduced in AT2-deficient mice. These findings indicate that the noradrenergic system is involved in mediating the anxiety-like behavior in AT2-deficient mice.

Targeted Deletion of Angiotensin II Type 2 Receptor Caused Cardiac Rupture After Acute Myocardial Infarction

Background—Accumulating evidence has suggested that the cardiac renin-angiotensin system is activated during the remodeling process after myocardial infarction (MI). Although 2 types of angiotensin II receptors (AT1 and AT2) are upregulated in the infarcted tissue, the contribution of AT2 to the subsequent fibrogenetic phase of wound healing is less certain. This study was conducted to evaluate the role of AT2 in wound healing after MI using an in vivo intervention study in mice with MI. Methods and Results—We examined myocardial hypertrophy, cardiac fibrosis, and morphological evidence of fibrillar collagen accumulation at the infarcted and noninfarcted regions in male mice lacking the AT2 receptor (Agtr 2−/Y) and age-matched wild-type (WT) animals. Of the Agtr 2−/Y mice, 63.6% died of cardiac rupture, whereas 23.5% of the WT mice died of the same cause within 1 week. The extent of fibrosis and that of collagen gene expression in Agtr 2−/Y mice were significantly reduced compared with WT mice at 1 week after coronary ligation. Furthermore, MI resulted in a marked increase in the prostaglandin E2 (PGE2) level at 4 days after surgery in Agtr 2−/Y mice. In WT mice, the PGE2 level was also elevated after MI but to a significantly lesser extent than in Agtr 2−/Y mice. Conclusions—A chronic loss of AT2 by gene targeting prevented the collagen deposition and caused cardiac rupture. The markedly elevated PGE2 may be a mechanism that inhibits collagen synthesis in the infarcted region of Agtr 2−/Y mice.

Multiple Growth Factors Modulate mRNA Expression of Angiotensin II Type-2 Receptor in R3T3 Cells

Previous studies showed that angiotensin II type-2 receptor (AT2) sites were increased when R3T3 cells were growth arrested and decreased when they were stimulated with fibroblast growth factor or serum. We examined the effects of several other growth factors on the expression of AT2 mRNA to clarify the relation between the AT2 receptor and growth factors. R3T3 cells were cultured in the medium containing 10% FCS until they were confluent and then serum was removed. AT2 mRNA was increased after serum was depleted, and the expression level reached a plateau after 2 days of serum depletion. The presence of serum (10%), fibroblast growth factor (10 ng/mL), or lysophosphatidic acid (1 mumol/L) reduced the AT2 mRNA expression. Phorbol ester (1 to 100 nmol/L) also suppressed the AT2 mRNA expression in a dose-dependent manner. Interleukin-1 beta (1 ng/mL) enhanced the AT2 mRNA expression 1.6-fold and the AT2 receptor number 1.4-fold. Insulin (100 nmol/L) enhanced AT2 mRNA expression 1.4-fold and the AT2 receptor number 1.6-fold. These results suggest that AT2 mRNA expression is modulated by multiple growth factors in both positive and negative directions. The presence of potential cis DNA elements that respond to interleukin-1 beta (CCAAT enhancer binding protein site), insulin [insulin response sequence of phospho(enol)pyruvate carboxykinase gene], and phorbol ester (AP-1 site) in the promoter region of the mouse AT2 gene suggests that the effects of these growth factors and phorbol ester may be mediated via these cis DNA elements.

ANGIOTENSIN RECEPTORS: MOLECULAR BIOLOGY AND SIGNALLING

1. The active peptide hormone angiotensin II (AngII) is formed from its prohormone angiotensinogen by way of inactive angiotensin I. The highly specific protease, renin, responsible for the initiation of this system was elusive and considered unstable. We isolated it in a pure and stable form from the kidney of the pig, human, rat, and land submandibular glands of the mouse. It was shown that there is only one type of renin with highly stringent substrate specificity, except certain strains of the mouse which have two gene products.

Cloning of the cDNA and the genomic DNA of the mouse angiotensin II type 2 receptor

Of the two major isoforms of the angiotensin II receptors, type 1 (AT1) and type 2 (AT2), little is known about the structure and features of AT2. We cloned a mouse AT2 cDNA from a mouse fetus cDNA library and an AT2 genomic DNA from a 129SV mouse genomic DNA library. The amino acid sequence of the mouse AT2 (363 residues) deduced from a mouse cDNA clone showed seven membrane-spanning domains. Amino acid identity of the mouse AT2 with mouse AT1 is 37%, and 98% with rat AT2. The genomic DNA (4.4 kb) contained three exons and two introns and the entire coding region was contained in the third exon.

Expression, Genomic Organization, and Transcription of the Mouse Angiotensin II Type 2 Receptor Gene

Although the rat angiotensin II type 2 receptor (AT2) was cloned and shown to be a member of the seven transmembrane domain-type receptor family, its signaling mechanism and biological roles have not been established. To acquire additional information on the structure and functions of AT2 genomic DNA, we cloned the mouse AT2 gene and examined its expression, transcription, and genomic organization. The amino acid sequence of the mouse AT2 cDNA showed a 98.5% sequence identity with the rat AT2. In mouse fetus, mRNA of the AT2 was highly expressed in the eviscerated carcass and brain. This expression decreased rapidly after birth. In 10-week-old mice, mRNA of the AT2 could be detected in the brain by Northern blot analysis. However, reverse transcription-polymerase chain reaction showed that mRNA of the AT2 was expressed in all organs examined, indicating that the AT2 is expressed at a low level in other organs. Southern blot analysis of the genomic DNA of the mouse liver digested with BamHI, EcoRI, and HindIII resulted in single bands, indicating that the AT2 gene probably exists at a single locus in the mouse genome. The nucleotide sequence of the AT2 gene (4.5 kb of the EcoRI fragment) revealed the presence of three exons. An entire coding sequence was included in the third exon. Primer extension experiments showed the presence of two transcription initiation sites in the mouse AT2 gene. A DNA segment of about 1.5 kb of the promoter region (-1497 to +56 bp) of the mouse AT2 gene was fused to a luciferase reporter gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Pain threshold, learning and formation of brain edema in mice lacking the angiotensin II type 2 receptor

The main biological role of angiotensin II type 2 receptor (AT2) has not been established. We made use of targeted disruption of the mouse AT2 gene to examine the functional role of the AT2 receptor in the central nervous system (CNS). We have previously shown that AT2-deficient mice displayed anxiety-like behavior in comparisons with wild-type mice. In the present study, we analyzed the pain threshold, learning behavior and brain edema formation using the tail-flick test, the tail-pinch test, the passive avoidance task and cold injury, respectively. In the passive avoidance task and cold injury, no differences were found between wild-type mice and AT2-deficient mice. In contrast, the pain threshold was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. The immunohistochemical distribution of beta-endorphin in the brain was analyzed quantitatively in AT2-deficient mice and wild-type mice, using microphotometry. The fluorescence intensity of beta-endorphin in the arcuate nucleus of the medial basal hypothalamus (ARC) was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. We found that the AT2 receptor does not influence learning behavior and brain edema formation. As AT2-deficient mice have increased sensitivity to pain and decreased levels of brain beta-endorphin, AT2 receptors may perhaps mediate regulation of the pain threshold.

Transcriptional Regulation of the Mouse Angiotensin II Type 2 Receptor Gene

The promoter region of the mouse angiotensin II type 2 receptor gene was cloned, and the nucleotide sequences were determined. A computer homology search for a 1.5-kb promoter region showed that there are several consensus cis DNA elements such as C/EBP, NF-IL6, and AP-1 in this region. Primer extension experiments showed that there are two transcription initiation sites 16 bp apart in the mouse type 2 receptor gene. Deletion mutants of this 1.5-kb segment were prepared and fused to a luciferase reporter gene. These type 2 receptor promoter-luciferase constructs were introduced into PC12W cells, which are from a pheochromocytoma cell line expressing the type 2 receptor, and luciferase activity was measured. It showed that a DNA segment between nucleotides -1497 and -874 suppresses the promoter activity of the type 2 receptor gene and that a DNA segment between nucleotides -47 and +56 is important for the basal promoter activity of the type 2 receptor gene. This proximal segment showed very weak promoter activity when introduced into vascular smooth muscle cells. Gel mobility shift assay with nuclear extracts from PC12W cells showed the presence of three DNA binding proteins that bound to a DNA probe between nucleotides -47 and +8. One DNA binding protein was only very weakly expressed in nuclear extracts from vascular smooth muscle cells, which do not express the type 2 receptor. Two other DNA binding proteins were not observed in nuclear extracts from vascular smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II receptors AT1 and AT2--new mechanisms of signaling and antagonistic effects of AT1 and AT2.

A series of pharmaceutical successes in the treatment of not only essential hypertension but also vascular hypertrophic and hyperplastic diseases, congestive heart failure, and renal degenerative diseases, with angiotensin-converting enzyme inhibitors and angiotensin (Ang) II receptor antagonists indicates that angiotensin may play a pivotal role in the genesis and maintenance of high blood pressure and resultant stroke, atherosclerosis, and heart and kidney diseases. There is more than one form of Ang II receptors. Using expression cloning, we isolated the AT1 cDNA from bovine adrenocortical cells from the kidney of spontaneously hypertensive rats and AT2 cDNA from rat PC12W cells and we showed that it was not the mas oncogene product. Further, we showed that in rodents, AT1 consists of two subtypes, AT1a and AT1b, which share a high degree of sequence homology in their coding regions, although mechanisms of their respective transcriptional control seemed to be different. By computer-assisted modeling and site-directed mutagenesis, we have delineated the docking site of Ang II. AT1a (and AT1b) serves most of the commonly recognized actions of Ang II. In addition, this G protein-coupled receptor (GPCR) also activates a tyrosine kinase mechanism that may be an underlying cause of Ang II-mediated hypertrophic and hyperplastic changes of cardiovascular tissues. In the vascular system, the phospholipase C (PLC) activated by Ang II seems to be PLC-β rather than PLC-γ1.