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T Inagami

Publications and source records attributed to T Inagami.

At least 109 records · Page 6Linked to original sources
PubMed1996

Translational regulation of angiotensin II type 1A receptor. Role of upstream AUG triplets.

The cDNA sequence of rat angiotensin II type 1A receptor (AT1AR) shows that AT1AR transcripts have AUG triplets in the 5'-leader region that may begin a short open reading frame encoding an 11-amino acid peptide. In this study, the mutational inactivation of the start codon of the short open reading frame in AT1AR-chloramphenicol acetyltransferase (CAT) reporter gene constructs resulted in a 2.6-fold increase in CAT activity, whereas CAT transcript levels were not affected. Furthermore, experiments with rat AT1AR cDNA-transfected Cos-7 cells revealed that mutagenesis of the upstream AUG increased the AT1AR protein up to 2.5-fold, although AT1AR transcript levels showed no changes. The synthetic peptide corresponding to the sequence of the short open reading frame significantly suppressed the amount of AT1AR product in the in vitro translation system. The inhibiting effect of the short open reading frame appears to operate at least in part at the level of translation initiation, because polysome analysis with transfected Cos-7 cells showed that mutagenesis of the upstream AUG resulted in a shift of AT1AR mRNA distribution from a smaller to larger fraction of polysomes. Taken together, these results show that the upstream AUG inhibits translational regulation, suggesting that the short open reading frame in the 5'-leader region of AT1AR transcripts has a certain role in the translation of AT1AR protein.

Animals↗
PubMed1996

Angiotensin II type 2 receptor inhibits cell proliferation and activates tyrosine phosphatase.

The angiotensin II type 2 (AT2) receptor inhibits basic fibroblast growth factor-induced proliferation of R3T3 fibroblast cells and transiently stimulates a vanadate-sensitive phosphotyrosine phosphatase, strongly suggesting that AT2 is a mitogen inhibitor. We generated AT2 gene-null mice that showed increased blood pressure, indicating the hypotensive action of AT2. However, inhibition of renomedullary AT2 by selective antagonists, as reported by Sassard and associates, show that AT2 suppresses pressure natriuresis. Thus, both AT1 and AT2 work in the direction of sodium retention, suggesting a unique role for angiotensin II in the kidney in terms of blood pressure regulation and sodium metabolism.

Angiotensin II↗
PubMed1996

Differential inducibility of angiotensin II AT2 receptor between SHR and WKY vascular smooth muscle cells.

Although the fetal aorta expresses a substantial amount of angiotensin II type 2 receptors, the expression level of angiotensin II type 2 receptors in the adult aorta and cultured vascular smooth muscle cells is very low or even absent. Prolonged serum depletion (6 to 8 days) with a supplement of insulin, transferrin and sodium selenite induced angiotensin II type 2 receptors and mRNA in cultured vascular smooth muscle cells from Wistar Kyoto rats. Insulin was found to be essential for the induction of the receptor. However, these receptors could not be induced in cultured vascular smooth muscle cells from spontaneously hypertensive rats. These results suggest that: (1) insulin plays an important role for the expression of the angiotensin II type 2 receptor gene; and (2) the type 2 receptor gene expression is differentially regulated between cultured vascular smooth muscle cells of Wistar Kyoto rats and spontaneously hypertensive rats.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1995

AT1A, AT1B, and AT2 angiotensin II receptor subtype gene expression in rat brain.

We localized the gene expression of angiotensin II receptor subtypes (AT1A, AT1B, and AT2) in 2-week-old rat brain by in situ hybridization using subtype specific riboprobes. AT1A receptor mRNA but not AT1B or AT2 receptor mRNA was expressed in the subfornical organ and paraventricular nucleus of the hypothalamus. AT1B as well as AT1A receptor mRNA were found in the cerebral cortex and hippocampus. Conversely, AT2 receptor mRNA, but not AT1A or AT1B, was expressed in the medial geniculate nucleus and inferior olive. Our results indicate that AT1A receptors are involved in the well known central functions of angiotensin II. These results also support the hypothesis of the involvement of AT2 receptors in sensory and motor function.

Angiotensin II↗
PubMed1995

Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor.

There are two major angiotensin II receptor isoforms, AT1 and AT2. AT1 mediates the well-known pressor and mitogenic effects of angiotensin II, but the signalling mechanism and physiological role of AT2 has not been established. Its abundant expression in fetal tissues and certain brain nuclei suggest possible roles in growth, development and neuronal functions. Here we report the unexpected finding that the targeted disruption of the mouse AT2 gene resulted in a significant increase in blood pressure and increased sensitivity to the pressor action of angiotensin II. Thus AT2 mediates a depressor effect and antagonizes the AT1-mediated pressor action of angiotensin II. In addition, disruption of the AT2 gene attenuated exploratory behaviour and lowered body temperature. Our results show that angiotensin II activates AT1 and AT2, which have mutually counteracting haemodynamic effects, and that AT2 regulates central nervous system functions, including behaviour.

Angiotensin II↗
PubMed1995

Disulfide bridges in extracellular domains of angiotensin II receptor type IA.

Angiotensin II receptor type IA (AT1A) has a cysteine (Cys) residue in each of four extracellular domains, and these Cys residues are believed to form two disulfide bridges. However, the question as to which pairs of Cys residues form disulfide bridges have not been experimentally determined. We constructed four mutants of rat AT1A, in which extracellular Cys residues were individually replaced by glycine (mutant C-1, C-2, C-3 and C-4). Further, we constructed two double mutants, in which two extracellular Cys residues were simultaneously substituted for by glycine. The binding affinity for angiotensin II in a double mutant C-1 + 4 (Cys18,274Gly) was similar to that in individually substituted mutants (C-1, C-2, C-3 and C-4) whereas the ligand binding of a double mutant C-2 + 4 (Cys101,274Gly) was completely abolished. The bindings of the non-peptide AT1A antagonist [125I]EXP-985 to mutants C-1, C-4 and C-1 + 4 were only slightly reduced whereas in mutant C-2, C-3 and C-2 + 4 the specific binding for [125I]EXP-985 was completely abolished. These results suggest that disulfide bridges in AT1A are formed between Cys18 and Cys274, and between Cys101 and Cys180, and the latter disulfide bond is essential for the binding of the non-peptidic antagonists [125I]EXP-985 or losartan.

Angiotensin II↗
PubMed1995

Cloning and expression of protein tyrosine phosphatase-like protein derived from a rat pheochromocytoma cell line.

A novel protein [designated protein tyrosine phosphatase-like protein (PTPLP)] which is distantly related to receptor-type protein tyrosine phosphatases (PTPases) was cloned from a rat pheochromocytoma cell line. The PTPLP was detected exclusively in the brain. Overexpression of the PTPLP decreased the basal PTPase activity of COS-7 cells for Raytide. These results suggest that PTPLP may function as a negative regulator of PTPases in neuronal tissues.

Adrenal Gland Neoplasms↗
PubMed1995

Cloning, expression and regulation of angiotensin II receptors.

Complementary DNAs for angiotensin II type 1 receptor isoforms AT1A and AT1B were cloned by expression cloning from bovine adrenal and rat vascular smooth muscles. Human AT1 receptor was also cloned. Seven transmembrane structures emerged. The AT1 type receptor interacted with more than one type of G-proteins. The ligand binding site of AT1 involving Arg167, Lys199, and Asp263 has been identified by site directed mutagenesis. The regulation of the receptors occur at many stages. The isoform, AT2, was also expression cloned from rat pheochromocytoma cells. Although its ligand binding is not affected by stable GTP analogs, it is a seven transmembrane domain receptor. It mediates the modulations of phosphotyrosine phosphatase by angiotensin II and AT2 specific CGP42112A. The modulation was abolished by pertussis toxin. Thus, AT2 belongs to a new class of angiotensin receptors with unique signalling and regulatory mechanisms. AT1 mediates cellular growth. Interestingly, AT2 expression is inversely related to the mitogenic activity of cells.

Amino Acid Sequence↗
PubMed1995

Steroid hormones upregulate rat angiotensin II type 1A receptor gene: role of glucocorticoid responsive elements in rat angiotensin II type 1A promoter.

The transcription of the rat angiotensin II type 1A receptor gene is stimulated by glucocorticoids. To clarify the molecular mechanism for glucocorticoid action in rat vascular smooth muscle cells, we investigated the effects of dexamethasone on the promoter activity of the angiotensin II type 1A receptor by using promoter/luciferase reporter gene constructs and heterologous context constructs (containing the thymidine kinase promoter) in transfected vascular smooth muscle cells. There are three putative glucocorticoid responsive elements in the promoter. However, only one glucocorticoid responsive element was found to respond to dexamethasone (1 microM). The region was located at positions, -756 to -770 bp upstream of the transcription initiation site. A glucocorticoid antagonist, RU38486, completely blocked the induction by dexamethasone, suggesting that the glucocorticoid responsive element was functional through a specific glucocorticoid receptor. Compared with the angiotensin II type 1A receptor promoter, no effect by dexamethasone was observed in vascular smooth muscle cells transfected with the angiotensin II type 1B receptor promoter/luciferase reporter gene constructs. We concluded that the dexamethasone-induced increase in the transcription of the angiotensin II type 1A receptor gene occurred through the binding to GRE up the glucocorticoid-specific receptor.

Aldosterone↗
PubMed1995

Developmental expression of renal angiotensin II receptor genes in the mouse.

The cellular distribution of angiotensin II type 1 (AT1) and type 2 (AT2) receptor mRNA was examined in mouse kidneys at several embryonic stages (12 to 18 days; 19 days = full term) and up to three weeks after birth by in situ hybridization. The expression of both AT1 and AT2 mRNAs appeared simultaneously at 14 days of gestation. However, their distributions were contrasting: AT1 mRNA was expressed in mature glomeruli and maturing S-shaped bodies throughout the stages examined. AT1 expression was also detected at 16 days of gestation in the proximal and distal tubules and peaked at the end of gestation. Both the temporal and spatial expression of AT1 coincide with the differentiation and proliferation of glomerular mesangial and tubular cells during nephrogenesis. In contrast, AT2 mRNA was present only in the mesenchymal cells adjacent to the stalk of the ureter bud at early developmental stages, and, later, extended to the mesenchymal cells located near, but outside, the nephrogenic area of superficial cortex and also the cells between collecting ducts. AT2 expression in these regions decreased markedly within three weeks after birth. Temporally and spatially, AT2 mRNA expression coincides with the epithelial-mesenchymal interactions that take place during early phases of nephrogenesis. The site of AT2 expression also overlaps closely with that of a specific group of cells which undergo apoptosis following nephrogenesis. Thus, contrary to current belief, the activation of AT1 and AT2 genes takes place in different cell types of the kidney during embryonic development, and thereby conceivably contributes to the ontogeny of those specific renal cells.

Angiotensin II↗
PubMed1995

Diversity and variability of smooth muscle phenotypes of renal arterioles as revealed by myosin isoform expression.

The contractility and distensibility of renal arterioles are important in the regulation of glomerular filtration. However, little is known regarding the characteristics of contractile proteins in these arterioles. Recently it was demonstrated that vascular smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2, which are unique molecular markers of smooth muscle cell phenotypes. SM1 is constitutively expressed in all types of smooth muscles, whereas SM2 exists only in mature smooth muscles. We characterized the expression of MHC isoforms as well as the ultrastructural myofilament assembly of renal arteriolar smooth muscles in human, rat and rabbit by immunohistochemical techniques. SM1 and alpha-smooth muscle actin were localized in both the preglomerular vessels (including the afferent arterioles) and efferent arterioles, whereas SM2 was present only in the preglomerular vessels. Renin-producing cells in the afferent arterioles (juxtaglomerular granular cells, JG cells) were positive for alpha-smooth muscle actin but negative for SM2. When renin synthesis was stimulated, the more proximal afferent arteriolar smooth muscles turned renin-positive and SM2 disappeared. Glomerular mesangial cells did not show immunoreactivities for SM1, SM2 or alpha-smooth muscle actin. The difference in MHC isoform expression in these arterioles was also reflected by ultrastructures; the afferent arteriolar smooth muscles contained abundant myofilaments including thick filaments, whereas the efferent arteriolar smooth muscles had a few myofilaments composed only of thin microfilaments. The JG cells displayed a myofilament assembly similar to that in the efferent arteriolar smooth muscles. We conclude from these observations that smooth muscles in pre-and postglomerular arterioles, the glomerular mesangial cells and JG cells differ in phenotypes, suggesting that they may have different contractile properties which may be critically involved in the regulation of glomerular filtration.

Actin Cytoskeleton↗
PubMed1995

Recent progress in molecular and cell biological studies of angiotensin receptors.

Recent developments in angiotensin II receptor research are discussed in the context of our knowledge in preceding years. Cloning of non-mammalian angiotensin II receptors without high affinity for non-peptide antagonists has permitted a new approach to the delineation of ligand-binding domains. Cloning of the second major isoform of angiotensin II receptor, AT2, and identification as a seven transmembrane domain receptor with only 32% sequence homology with the first isoform, AT1, provide the first concrete step toward our understanding of the roles of AT2. The discovery of phospholipase C-mediated pathway for AT1 in vascular smooth muscle cell signaling introduces an entirely unexpected angle to future research. New aspects of AT1 gene regulation and receptor desensitization and internalization are evolving. Molecular mechanisms and physiological implications of the differential expression of AT1A and AT1B are being clarified. The recent discovery of human AT1B may make studies on animal models interesting and more meaningful. The first paper on the genetic role of the AT1 gene in human hypertension has just been published. A promising future is expected in the further development of angiotensin-receptor research in relation to cardiac, renal, and vascular function by employing techniques of molecular biology.

Amino Acid Sequence↗
PubMed1995

Localization of the genes encoding the three rat angiotensin II receptors, Agtr1a, Agtr1b, Agtr2, and the human AGTR2 receptor respectively to rat chromosomes 17q12, 2q24 and Xq34, and the human Xq22.

Using fluorescence in situ hybridization, we determined the regional localization of the 3 rat genes encoding angiotensin II receptors at 17q12 (Agtr1a), 2q24 (Agtr1b) and Xq34 (Agtr2). In parallel, we showed that the type 2 human gene, AGTR2, also maps on the X chromosome, at band Xq22.

Animals↗
PubMed1995

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)

Animals↗
PubMed1995

Mapping of G protein coupling sites of the angiotensin II type 1 receptor.

Angiotensin II type 1 (AT1) receptors have been identified in a wide variety of tissues, including the kidney, liver, adrenal gland, cardiovascular system, and brain. AT1 receptors also mediate complex signaling mechanisms that elicit a diversity of specific physiological effects. The rat AT1A receptor has seven transmembrane domains and couples with three distinct G proteins: Gq, Gi, and Go. But it is unknown which domains of AT1A couple with and activate each type of G protein. To identify the domains responsible for the activation of various types of G protein, we studied the effect of five different synthetic peptides representing different domains of cytosolic segments of the rat AT1A receptor on the binding of the 35S-labeled stable analogue of GTP, GTP gamma S. Peptides P-3, which is located in the N-terminal region of the putative third intracellular loop of AT1A (residues 216 through 230), and P-5 (residues 306 through 320), corresponding to the N-terminal region of the C-terminal tail, were found to activate purified Gi1, Gi2, and Go proteins. These results indicate that not only the third cytosolic loop but also the C-terminal cytosolic domain of AT1A is important for Gi1, Gi2, and Go protein coupling and activation.

Amino Acid Sequence↗
PubMed1995

Differential regulation of angiotensin II receptor subtypes in rat kidney by low dietary sodium.

This study was designed to determine whether expression of renal messenger RNA (mRNA) encoding the two known angiotensin II type 1 (AT1) receptor subtypes (AT1A and AT1B) can be regulated by dietary sodium. Seven-week-old male Wistar rats were fed a low-sodium diet (0.07%, n = 9) or a normal-sodium diet (0.5%, n = 9 [control]) for 14 days. A rat AT1 complementary DNA (cDNA) probe, which hybridizes to mRNA encoding both the AT1A and AT1B receptor subtypes, and cDNA probes, which are selective for AT1A or AT1B mRNA, were used in Northern blot or in situ hybridization analysis. By use of Northern blot analysis, renal mRNA levels for the AT1 and AT1A receptors in rats fed a low-sodium diet were found to be increased twofold (P < .05) compared with control. Because renal AT1B mRNA content was not detected by Northern blot analysis, quantitative image analysis of in situ hybridization with a digoxigenin-labeled cRNA probe made from AT1B cDNA was used. In situ hybridization analysis indicated that AT1B mRNA was expressed in the proximal and collecting tubules of the kidney in rats fed a normal-sodium diet. The low-sodium diet significantly decreased the percent positive staining area of AT1B mRNA in the renal cortex (5.51 +/- 0.77% versus 2.73 +/- 0.35%, P < .05) and medulla (4.76 +/- 0.70% versus 2.01 +/- 0.43%, P < .05) compared with the control diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗