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T Inagami

Publications and source records attributed to T Inagami.

At least 91 records · Page 5Linked to original sources
PubMed1997

A domain for G protein coupling in carboxyl-terminal tail of rat angiotensin II receptor type 1A.

To delineate domains essential for Gq protein coupling in the C-terminal region (C-tail) of rat angiotensin II (Ang II) receptor type 1A (AT1A), we modified the putative cytosolic regions of the receptor by truncation or alanine substitution and determined resultant changes in the guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) effect on Ang II binding and inositol trisphosphate production by the agonist. Independently, we studied the effect of synthetic C-tail peptides (P-5) and its alanine substitution analogs on [35S]GTPgammaS binding to Gq. Effects of GTPgammaS on Ang II binding (shift to a low affinity form) and inositol trisphosphate production in the deletional mutant receptor 1-317 AT1A was similar to wild type AT1A, whereas in shorter C-terminal deletion mutants 1-309, 1-311, 1-312, 1-313 AT1A, and substitutional mutants Y312A, F313A, and L314A these activities were markedly reduced. The binding of [35S]GTPgammaS to Gq was promoted by the synthetic C-terminal peptide P-5 but not when mutated at Tyr312, Phe313, or Leu314. Results indicate that Tyr312, Phe313, and Leu314 in cytosolic carboxyl-terminal region of rat AT1A are essential for coupling and activation of Gq.

Amino Acid Sequence↗
PubMed1997

Interaction of the adaptor protein Shc and the adhesion molecule cadherin.

In mitogenic signaling pathways, Shc participates in the growth factor activation of Ras by interacting with activated receptors and/or the Grb-2.Sos complex. Using several experimental approaches we demonstrate that Shc, through its SH2 domain, forms a complex with the cytoplasmic domain of cadherin, a transmembrane protein involved in the Ca2+-dependent regulation of cell-cell adhesion. This interaction is demonstrated in a yeast two-hybrid assay, by co-precipitation from mammalian cells, and by direct biochemical analysis in vitro. The Shc-cadherin association is phosphotyrosine-dependent and is abrogated by addition of epidermal growth factor to A-431 cells maintained in Ca2+-free medium, a condition that promotes changes in cell shape. Shc may therefore participate in the control of cell-cell adhesion as well as mitogenic signaling through Ras.

3T3 Cells↗
PubMed1997

Renin and angiotensin II receptor expression in the brains of DES-treated Syrian hamsters.

BACKGROUND: The renin angiotensin system (RAS) promotes vasoconstriction. Expression of RAS is induced by different factors. METHODS: In this study, forebrain sections of hamster brains were studied by immunohistochemical methods to determine the location of renin-positive and angiotensin II receptor-positive cells. The brain sections were obtained from diethylstilbesterol- (DES-) treated hamsters, adult non-DES-treated hamsters, elderly non-DES-treated hamsters, neonatal hamsters, and 15-day fetal hamsters. Circulating renin activity was determined for all but the neonatal and 15-day fetal hamsters. RESULTS: Renin-positive and angiotensin II receptor-positive vascular smooth muscle cells were observed in DES-treated hamsters. No positive cells were observed in neonatal, 15-day fetal, and adult non-DES-treated hamsters. Some expression was observed in elderly hamsters. CONCLUSIONS: Therefore, focal expression of the renin angiotensin system in brain vasculature was induced by the synthetic estrogen DES.

Age Factors↗
PubMed1997

Rat chromosome 1: regional localization of seven genes (Slc9a3, Srd5a1, Esr, Tcp1, Grik5, Tnnt3, Jak2) and anchoring of the genetic linkage map to the cytogenetic map.

Seven genes were regionally localized on rat Chromosome (Chr) 1, from 1p11 to 1q42, and two of these genes were also included in a linkage map. This mapping work integrates the genetic linkage map and the cytogenetic map, and allows us to orient the linkage map with respect to the centromere, and to deduce the approximate position of the centromere in the linkage map. These mapping data also indicate that the Slc9a3 gene, encoding the Na+/H+ exchanger 3, is an unlikely candidate for the blood pressure loci assigned to rat Chr 1. These new localizations expand comparative mapping between rat Chr 1 and mouse or human chromosomes.

Animals↗
PubMed1997

A review of mutagenesis studies of angiotensin II type 1 receptor, the three-dimensional receptor model in search of the agonist and antagonist binding site and the hypothesis of a receptor activation mechanism.

OBJECTIVE: To seek the mechanism whereby agonists, competitive antagonists and insurmountable antagonists affect the receptor function differently, by reviewing recent mutagenesis studies of angiotensin II type 1 receptor (AT1) in which the binding of the agonist and antagonists and receptor signaling were affected. AT1 RECEPTOR STRUCTURE AND LIGAND BINDING SITES: We built a model of seven transmembrane spanning domains of the AT1 receptors using bacteriorhodopsin as a template. The carboxy terminal of angiotensin II binds to Lys199 in transmembrane domain 5, whereas the guanidinium group of Arg2 binds to Asp281 in transmembrane domain 7. Results of studies using mutagenesis supporting proposed ligand-docking models are discussed. HYPOTHESIS FOR THE LIGAND-INDUCED RECEPTOR SIGNALING MECHANISM: We submit a set of hypotheses for a mechanism whereby the ligand binding induces changes in the receptor conformation by the rotation of transmembrane helices as the initial event for the subsequent activation of a G protein. In this mechanism antagonists are not capable of rotating the helices but agonists are able to do so, which results in the formation of a hydrogen bond between Asp74 in transmembrane domain 2 and Tyr292 in transmembrane domain 7. This mechanism also provides plausible explanation for the activation of monoamine receptors. COMPETITIVE AND INSURMOUNTABLE ANTAGONISTS: Competitive antagonists share the same binding sites with agonists, but insurmountable antagonists do not, and binding of the latter does not preclude agonist binding, for example, to Asp281. CONCLUSION: This hypothesis of the intrareceptor signaling mechanism and the receptor model indicate that some amino acid residues essential for the signaling play their roles in the intrareceptor activation mechanism, whereas others participate directly in ligand binding.

Animals↗
PubMed1997

Regulation of growth of the adrenal gland in DOC-salt hypertension. Role of angiotensin II receptor subtypes.

To investigate the role of the renin-angiotensin system in the regulation of adrenal growth in deoxycorticosterone (DOC)-salt hypertensive rats, and the adrenal gene expression of angiotensin AT1 and AT2 receptors, three groups of uninephrectomized rats + DOC pellet + 0.9% NaCl were given water (DOC), losartan (DOC-L), or ramipril (DOC-R) by gavage. Controls had sham surgery and water gavage. Tail-cuff systolic and mean intra-arterial blood pressures were significantly higher in the three DOC groups than in controls and not different among the groups. Adrenal weight of DOC was slightly but not significantly greater than that of controls, while those of DOC-L and DOC-R were greater than that of controls (P < .01). Northern blots showed that AT1 and AT2 gene expression was significantly reduced in DOC (by 33% and 60%), while that of AT1 (but not AT2) was significantly reduced further (versus control and DOC) in DOC-L and DOC-R. There were negative correlations between adrenal weight and AT1 (r = -.80, P < .0001) or AT2 (r = -.60, P < .005). We conclude that DOC-salt hypertension downregulates adrenal AT1 and AT2 gene expression by different mechanisms. Removal of the effects of angiotensin by losartan or ramipril downregulates AT1 further and promotes adrenal growth, indicating the presence of an AT1-mediated growth-inhibitory action of angiotensin II on the adrenal gland. These observations constitute an additional example of a growth-inhibitory role for the AT1 receptor, opposite to its more common growth-promoting actions in other organs and tissues.

Adrenal Glands↗
PubMed1997

Induction of renin in medial smooth muscle cells by balloon injury.

We studied the expression of each component of the renin-angiotensin system (renin, angiotensin I-converting enzyme, angiotensinogen, and angiotensin II type I receptor) in balloon-injured rat carotid artery. We assessed the expression levels of the respective mRNAs by competitive polymerase chain reaction. Renin mRNA concentration was markedly increased 24 hours after balloon injury and remained higher than that in the control at 7 days after balloon injury. Angiotensin-converting enzyme mRNA concentration was decreased 24 hours after balloon injury and was increased at 14 days after balloon injury. No significant change in angiotensinogen mRNA concentration was observed throughout the study period. Angiotensin type I receptor mRNA concentration was increased beginning 3 days after balloon injury and remained higher than that in the control at 14 days after balloon injury. Immunohistochemical analysis showed that renin was transiently expressed in medial smooth muscle cells after balloon injury. Administration of quinapril markedly reduced neointimal formation and was accompanied by an attenuation of the increase in the concentrations of angiotensin type I receptor and angiotensin-converting enzyme mRNAs. The upregulation of renin mRNA in balloon-injured rat carotid artery preceded and may play a role in neointimal formation.

Analysis of Variance↗
PubMed1997

Expression of the subtype 2 angiotensin (AT2) receptor protein in rat kidney.

In situ hybridization studies have suggested that the subtype 2 angiotensin (AT2) receptor gene is expressed in fetal and newborn rat kidney but is undetectable in the adult animals. In the present study, we investigated the expression of AT2 receptor protein in the fetal (days 14 and 19 of fetal life), newborn (day 1 postpartum), and adult (4-week-old and 3-month-old) rat kidney. Polyclonal anti-peptide antiserum was raised against the amino terminus of the native AT2 receptor. The selectivity of the antiserum was validated by recognition of the AT2 receptor in a stably transfected COS-7 cell line by Western blot and immunocytochemical analysis. As a positive control, the AT2 receptor signal was detected strongly in the adrenal gland. Positive immunohistochemical staining was observed in the mesenchymal cells and ureteric buds of the 14-day fetal kidney and in the glomeruli, tubules, and vessels in the 19-day fetal and newborn kidney. Glomeruli expressing the AT2 receptor were localized mainly in the outer layer of the renal cortex. In the young (4-week-old) and mature (3-month-old) adult rat on normal sodium intake, renal AT2 receptor immunoreactivity was present in glomeruli but substantially diminished compared with that of newborn rats. In both young and mature adult rats, dietary sodium depletion increased the renal AT2 receptor signal, mainly in the glomeruli and interstitial cells. Preimmune and preadsorption controls were negative. Western blot analysis detected a single 44-kD band in the fetal and newborn rat kidney and in the young and mature adult rat kidney. Dietary sodium depletion increased the density of the AT2 receptor band in mature adult rat kidneys. These data provide evidence that the AT2 receptor protein is expressed in the fetal and newborn rat kidney, diminishes in adult life, and is reexpressed in the adult in response to sodium depletion.

Adrenal Glands↗
PubMed1997

Candesartan prevents the progression of glomerulosclerosis in genetic hypertensive rats.

The renin-angiotensin system (RAS) has been implicated in the development of hypertensive glomerulosclerosis. However, there are no experimental findings clearly demonstrating activation of glomerular RAS in hypertensive nephropathy. Using the stroke-prone spontaneously hypertensive rat (SHRSP) as an animal model of hypertensive glomerulosclerosis, we examined the relationship between the sequential changes in urinary albumin excretion (UAE), renal morphology, and glomerular mRNA expression for transforming growth factor-beta (TGF-beta) and fibronectin (FN) and glomerular mRNA levels for RAS components, and determined the effects of the angiotensin II (Ang II) type 1 (AT-1) receptor antagonist (candesartan) and equihypotensive hydralazine on these parameters. In SHRSP, UAE was normal at nine weeks of age and increased by 12 weeks. Plasma renin activity, plasma Ang II concentration, and angiotensin converting enzyme (ACE) activity were not higher in 9- and 12-week-old SHRSP than in WKY. RNase protection assay revealed higher glomerular mRNA levels for angiotensinogen, ACE, and AT-1a and AT-1b receptors in 9-, 12-, and 14-week-old SHRSP than in WKY. The glomerular mRNA levels for TGF-beta and FN in SHRSP were increased from nine weeks of age. SHRSP had a greater glomerulosclerosis index (GSI) at 24 weeks of age than did WKY. Administration of candesartan for two weeks, but not of hydralazine, markedly reduced UAE and normalized mRNA levels for TGF-beta, FN, and RAS components. Candesartan administration for 12 weeks virtually prevented the progression of glomerulosclerosis in rats. We conclude that in SHRSP, RAS activation and increased sensitivity to Ang II in glomeruli play important roles in the progression of glomerulosclerosis.

Albuminuria↗
PubMed1996

Inhibition of cell proliferation and activation of protein tyrosine phosphatase mediated by angiotensin II type 2 (AT2) receptor in R3T3 cells.

Angiotensin II (Ang II) type 2 receptor (AT2) mediated inhibition of cell proliferation and activation of protein tyrosine phosphatase (PTP) were investigated in R3T3 fibroblast cells selectively expressing the AT2 subtype. Ang II did not alter the cell number of serum depleted R3T3 cells but inhibited basic fibroblast growth factor (bFGF)-stimulated cell proliferation in a dose dependent manner (IC50; 1-5 nM). This inhibitory response was abolished by an concomitant incubation with an AT2 antagonist, PD123319, and was mimicked by an AT2 agonist, CGP42112A. Stimulation of Ang II resulted in a rapid and transient increase in PTP activity as determined by using para-nitrophenyl phosphate (p-Npp) as a substrate in serum depleted R3T3 cells. This PTP activation was mimicked by CGP42112A. We conclude that the AT2 receptor is involved in the inhibition of cell proliferation and activation of protein tyrosine phosphatase.

3T3 Cells↗
PubMed1996

Chimeric mice carrying 'regional' targeted deletion of the angiotensin type 1A receptor gene. Evidence against the role for local angiotensin in the in vivo feedback regulation of renin synthesis in juxtaglomerular cells.

We have developed chimeric mice carrying 'regional' null mutation of the angiotensin type 1A (AT1A) receptor, the AT1 receptor subtype exclusively present in mouse juxtaglomerular (JG) cells. The chimeric mouse (Agtr1a -/- <--> +/+) is made up of wild-type (Agtr1a +/+) cells or cells homozygous for Agtr1a deletion (Agtr1a -/-). In the latter, the AT1A coding exon was replaced with a reporter gene, lacZ. In Agtr1a -/- <--> +/+ mice, these two clones of cells are found to be clustered and display patchy distributions in the kidney and heart. Tracking of lacZ activities in hetero- (Agtr1a +/-) and homozygous (Agtr1a -/-) deletion mutant offspring from Agtr1a -/- <--> +/+ mice revealed that the promoter activity of Agtr1a is localized in JG cells, afferent arteriolar walls, glomerular mesangial region and endothelial cells, and apical and basolateral proximal tubule membranes. The JG apparatuses of Agtr1a -/- mice are markedly enlarged with intense expression of renin mRNA and protein. In Agtr1a -/- <--> +/+ mice, these changes were proportional to the degree of chimerism. Within a given Agtr1a -/- <--> +/+ mouse, however, the degree of JG hypertrophy/hyperplasia and the expression of renin mRNA and protein were identical between Agtr1a +/+ and Agtr1a -/- cells. Thus, in the in vivo condition tested, the local interaction between angiotensin and the AT1 receptor on the JG cells has little functional contribution to the feedback regulation of JG renin synthesis.

Angiotensins↗
PubMed1996

Insulin and insulin-like growth factors induce expression of angiotensin type-2 receptor in vascular-smooth-muscle cells.

Angiotensin type-2 receptor (AT2) is abundant in fetal tissues, including aorta, and its expression level declines after birth. In the present study, the regulation of its expression was studied in cultured vascular-smooth-muscle cells (VSMC). The maximum number of binding sites of AT2 increased in VSMC after they were cultured without serum in the presence of insulin, which was essential for its expression. AT2 expression was inhibited by treatment with phorbol ester. Northern blot analyses revealed that insulin-dependent expression is due to elevation of mRNA level of AT2. Similar induction was observed when insulin-like growth factor (IGF)-I or IGF-II was used instead of insulin. The study on the dose dependencies of these factors revealed that the induction of AT2 expression was mediated through the activation of IGF-I receptors. The insulin-induced expression of AT2 was detected in the aorta of genetically obese (fa/fa) Zucker rats, which reportedly have approximately tenfold-higher plasma concentrations of insulin than their lean littermates. The insulin-dependence seems characteristic of VSMC, because it was not observed for pheochromocytoma cells or adrenal glands. These results suggest that the expression of AT2 is regulated by at least two mechanisms, that is, IGF-I receptor dependent and IGF-I receptor independent, and that the former may play an important role in the expression of AT2 in VSMC.

Animals↗
PubMed1996

Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. Possible requirement of Gq-mediated p21ras activation coupled to a Ca2+/calmodulin-sensitive tyrosine kinase.

In cultured rat vascular smooth muscle cells, angiotensin II (Ang II) induced a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor, which was insensitive to pertussis toxin but was abolished by the phospholipase C inhibitor, U73122. The Ang II-induced MAPK activation was not affected by the protein kinase C inhibitor, GF109203X, and was only partially impaired by pretreatment with a phorbol ester, whereas both treatments completely prevented MAPK activation by the phorbol ester. Intracellular Ca2+ chelation by TMB-8, but not extracellular Ca2+ chelation or inhibition of Ca2+ influx, abolished Ang II-induced MAPK activation. The calmodulin inhibitor, calmidazolium, and the tyrosine kinase inhibitor, genistein, completely blocked MAPK activation by Ang II as well as by the Ca2+ ionophore A23187. Ang II caused a rapid increase in the binding of GTP to p21(ras), and this was inhibited by genistein, TMB-8, and calmidazolium but not by pertussis toxin or GF109203X. These data suggest that Ang II-induced MAPK activation through the Ang II type 1 receptor could be mediated by p21(ras)activation through a currently unidentified tyrosine kinase that lies downstream of Gq-coupled Ca2+/calmodulin signals.

Adenosine Triphosphate↗
PubMed1996

Transcription of the rat angiotensin II type 2 receptor gene.

The promoter region of the rat angiotensin II type 2 receptor gene was cloned and the nucleotide sequences were determined. A computer homology search for a 1.2 Kb promoter region showed that there are several consensus cis DNA elements such as C/EBP, NF-IL6, GRE and AP1 in this region. Primer extension experiments showed that there is one transcription initiation site 15 bp-downstream of the TATA box. Deletion mutants of the 1.2 Kb segment were prepared and fused to a luciferase reporter gene. These type 2 receptor promoter-luciferase constructs were introduced into PC12W cells, a pheochromocytoma cell line expressing the type 2 receptor, and luciferase activity was measured. It showed that (1) a DNA segment between -1208 bp and -749 bp suppresses the promoter activity of type 2 receptor gene, (2) a positive regulatory element is present in a DNA segment between -749 bp and -216 bp; and (3) a DNA segment between -44 bp +58 bp is important for the basal promoter activity of the type 2 receptor gene.

Adrenal Gland Neoplasms↗
PubMed1996

Localization of AT2 angiotensin II receptor gene expression in rat brain by in situ hybridization histochemistry.

To localize the gene expression of AT2 angiotensin II receptors in rat brain we performed in situ hybridization histochemistry using 35S-labeled antisense riboprobes. The AT2 receptor mRNA expression pattern was compared in consecutive brain sections, from 2 week old rats, with the receptor expression by means of [125I]Sar1-ANG II binding and displacement with AT2 selective ligands followed by autoradiography. Expression of AT2 receptor mRNA was found in several thalamic nuclei (ventral posterolateral, mediodorsal, central medial, paracentral, and paraventricular), the medial geniculate nuclei, the nucleus of the optic tract, the subthalamic nucleus, the interposed nucleus of the cerebellum, and in the inferior olive. In these areas the AT2 receptor gene expression corresponds well with [125I]Sar1-ANG II binding. In addition, AT2 receptor mRNA expression was found in the red nucleus where no [125I]Sar1-ANG II binding was present. No significant hybridization of the AT2 receptor antisense probe was found in septal nuclei, the locus coeruleus, the dorsolateral geniculate nucleus, or the cerebellar cortex, areas rich in [125I]Sar1-ANG II binding. Our results indicate that some brain regions may be involved in AT2 receptor formation, transporting the receptor protein to other brain areas. However, in most structures, both the formation and expression of receptors occur, suggesting the existence of local AT2 receptor circuits, or that of AT2 autoreceptors. Other structures express only the receptor protein, indicating that these AT2 receptors are produced elsewhere. Our present data are the basis for further studies on the clarification of AT2 receptor pathways in the brain.

Angiotensin II↗
PubMed1996

Ouabain increases aldosterone release from bovine adrenal glomerulosa cells: role of renin-angiotensin system.

To evaluate the potential physiological significance of ouabain or a ouabainlike substance, we investigated the effect of nanomolar concentrations of ouabain on aldosterone release by cultured bovine adrenal glomerulosa cells. Ouabain (10 nM) increased aldosterone release from 0.35 to 0.89 ng.mg-1.4 h-1 in the serum-containing medium. Losartan prevented this increase. When angiotensinogen was added to the nonserum medium, 10 nM ouabain enhanced the aldosterone release. Losartan again blocked the increase. These findings together with a stimulation of renin release by ouabain indicate that angiotensin II generated by the adrenal cell renin-angiotensin system in the presence of exogenous serum or exogenous angiotensinogen is necessary for the ouabain-induced stimulation of aldosterone release. Ouabain (10 nM) enhanced the intracellular calcium concentration increase elicited by 0.1 nM angiotensin II severalfold. Addition of 1 nM ouabain enhanced the aldosterone secretion resulting from the addition of 1 nM angiotensin II. Nanomolar levels of ouabain, therefore, interact with both locally formed and exogenous angiotensin II to stimulate aldosterone production. A suggested mechanism is that ouabain increases calcium stores in the endoplasmic reticulum, thereby increasing the agonist response.

Aldosterone↗