Biomedical subjects

T Inagami

Publications and source records attributed to T Inagami.

At least 145 records · Page 8Linked to original sources

PubMed1994

Protein tyrosine phosphatase inhibition by angiotensin II in rat pheochromocytoma cells through type 2 receptor, AT2.

Two major isoforms of angiotensin II receptors, AT1 and AT2, have been defined on the basis of their ligand selectivity. While AT1 is known to mediate typical biological actions of angiotensin II as a cardiovascular regulator, the biological function of AT2 has not yet been established. In the present study using a rat pheochromocytoma cell line, which expresses AT2 exclusively, we found that angiotensin II inhibits phosphotyrosine phosphatase activity in vivo as measured by the inhibition of hydrolysis of [32P]-phosphate from the 32P-labeled synthetic peptide substrate, Raytide. This phosphotyrosine phosphatase inhibition was completely reversed by pertussis toxin, which indicates a G-protein coupled mechanism. In SDS-polyacrylamide gel electrophoresis we found that the phosphotyrosine group of an 85 kDa protein was a substrate mainly preserved, presumably as a consequence of the plausible intracellular phosphotyrosine phosphatase inhibition by angiotensin II.

Adrenal Gland Neoplasms↗

PubMed1994

Atrial natriuretic factor as a volume regulator.

Atrial natriuretic factor, originally isolated from the atrium of the heart, has been found to consist of three major groups: atrial natriuretic peptide (ANP), B-form natriuretic peptide (BNP), and C-form natriuretic peptide (CNP). In addition, ANP exists in its precursor form, pro-ANP, an active ANP with a longer peptide chain (urodilatin) and an antiparallel dimer of active ANP. Sites and production of these diverse forms of the peptides are also diverse, depending on pathologic states. Three major subtypes of ANP receptors exist; these include a clearance receptor and two types of a transmembrane receptor with guanylyl cyclase structures in their intracellular domain. The latter exists at least in two forms, one of which is found mainly in the brain. All the actions of ANP mediated by the transmembrane form of ANP receptors are mediated by cGMP generated by the guanylyl cyclase in the cytosolic domain of the receptor. Among the numerous effects of ANP, its major effects are stimulation of natriuresis and diuresis by the kidney through its hemodynamic and tubular effects. In addition, ANP causes vasodilatation and fluid volume reduction by direct actions on vascular smooth muscle cells, and inhibition of secretion of hormones, such as aldosterone, from adrenal cortex and norepinephrine from peripheral adrenergic neurons. Centrally mediated effects on the regulation of the fluid volume may also be important.

Atrial Natriuretic Factor↗

PubMed1994

Reperfusion enhances the local release of endothelin after regional myocardial ischemia.

The role of endothelin, a potent long-acting vasoconstrictor peptide, in the pathogenesis of the "no-reflow" phenomenon was investigated in nine closed-chest mongrel dogs undergoing 90 minutes of proximal left anterior descending artery occlusion and 3.5 hours of reperfusion. Endothelin levels were measured serially from the coronary sinus (CS) and aorta (Ao) by radioimmunoassay and correlated with regional myocardial blood perfusion. Prolonged anesthesia, surgery, and vascular instrumentation did not change endothelin levels in four sham animals. A progressive and parallel increase in CS and Ao endothelin levels occurred during coronary occlusion. A further increase in CS levels was observed during the reperfusion period, resulting in significantly higher values of the peptide at 30 and 60 minutes (30 minutes: CS 22.1 +/- 3.5 vs Ao 15.1 +/- 5.1 pg/ml; 60 minutes: CS 21.1 +/- 4.5 vs Ao 15.0 +/- 3.6 pg/ml; p < 0.05 by analysis of variance). Microvascular perfusion determined semiquantitatively with fluorescent beads was significantly reduced in the central ischemic zone (CIZ) compared with that in the nonischemic zone (NIZ) (CIZ endocardium 1.14 +/- 0.4 beads/m2, CIZ midmyocardium 1.19 +/- 0.3 beads/m2, NIZ 3.8 +/- 0.6 beads/m2; p < 0.05). A significant correlation was noted between mean reperfusion levels of endothelin in the CS and endocardial flow in the CIZ (r = -0.88; p = 0.009). This study demonstrates that reperfusion per se enhanced the spillover of endothelin from the cardiac interstitium. Local release of endothelin may contribute to the progressive decrease in microvascular flow in the reperfused bed.

Animals↗

PubMed1994

Role of carboxyl tail of the rat angiotensin II type 1A receptor in agonist-induced internalization of the receptor.

Binding of angiotensin II (Ang II) to its receptor type 1A (AT1A) is known to trigger its internalization. We studied the role of cytosolic segments of AT1A in the internalization, and obtained results indicating a functional role of the cytosolic carboxyl terminal tail of AT1A in the internalization. Deletion of 50 amino acids from the carboxyl terminus abolished the receptor internalization. Deletion mutants lacking 13 and 32 amino acid residues in the carboxyl terminal cytosolic region were internalized to the same extent as wild type AT1A; however, internalization of a mutant lacking the last 42 residues was partially suppressed. Thus, residues 310 through 327 were shown to be essential for the internalization. We propose that a short domain in the cytoplasmic tail (residues 310 to 327) may play a dominant role in the agonist-induced receptor internalization of AT1A. Our results also suggest that the molecular determinants of the AT1A receptor involved in receptor internalization are distinct from those participating in the desensitization process.

Amino Acid Sequence↗

PubMed1994

Molecular structure and function of angiotensin type 2 receptor.

More than two isoforms have been identified for angiotensin receptors based on their ligand selectivity. The objective of this study is to determine the molecular structure of angiotensin type 2 receptor (AT2), whose physiological functions are still an enigma despite extensive studies on its distribution in fetal tissues. We expression-cloned a cDNA of an affinity-purified AT2 from rat pheochromocytoma cells (PC12w). The AT2 cDNA clone comprises 2,868 nucleotides and encodes a 363 amino acid protein with seven putative transmembrane domains. The dissociation constant for its binding to 125I-CGP42112A, an AT2-specific ligand, was 0.11 +/- 0.01 nM. Its binding to 0.5 nM 125I-[Sar1,Ile8]-Ang II was not inhibited by Dup 753 but by PD123319 (IC50 = 1.7 +/- 0.2 nM). These binding features are characteristic of angiotensin type 2 receptor. The amino acid sequence analysis of the purified AT2 corroborated the amino terminus of the deduced primary structure of AT2. Angiotensin type 1 receptor (AT1) is the most closely related to AT2 but with only 32% amino acid sequence identity. Angiotensin II attenuated membrane-associated protein tyrosine phosphatase activity in the COS-7 cells stably expressing AT2 through a pertussis toxin-sensitive G protein. However, the physiological function of AT2 in the fetal kidney is still unresolved.

Amino Acid Sequence↗

PubMed1994

Angiotensin II type 1 receptor gene abnormality in a patient with Bartter's syndrome.

Administration of a selective angiotensin I type 1 receptor (AT1) antagonist in animals not only nullifies the vasopressor action of angiotensin II, but also induces chloriduria and kaliuria, juxtoglomerular apparatus (JGA) hypertrophy and hyperreninemia, features characteristic of human Bartter's syndrome. We, therefore, explored the possibility that Bartter's syndrome may involve an AT1 abnormality. Using a pair of AT1-specific oligonucleotide primers and two different DNA polymerases (Taq and Pfu), we amplified the approximately 1 kb AT1 coding region of genomic DNA isolated from leukocytes of five patients with Bartter's syndrome by PCR and analyzed the sequence of the product. While the sequence of all clones from four patients were identical to that already reported for the normal human AT1 DNA sequence, 50% of the clones from one patient with Bartter's syndrome were found to have A-->G transition at nucleotide 931 which causes an amino acid substitution (arg-->gly) on the carboxy-terminal cytosolic tail of AT1. This mutation was not found in DNA from 50 normal controls which were screened by restriction enzyme digestion pattern of the PCR products of this region. As PCR-amplified AT1 DNA clones from four other individuals with Bartter's syndrome did not display any abnormality in the coding region, the possibility exists that Bartter's syndrome consists of multiple disease entities, where an AT1 gene abnormality represents a specific subgroup of the syndrome and/or some abnormality includes mutations outside of the coding region.

Animals↗

PubMed1994

Cloning, expression and regulation of angiotensin II receptors.

Angiotensin II isoform 1 (AT1) receptor cDNAs were cloned by expression cloning from bovine adrenal and rat vascular smooth muscles. Human AT1 receptor was also cloned. Seven transmembrane structures emerged. A single type of receptor seems to interact with more than one type of G-protein. AT1 consists of subtypes AT1A and AT1B, and the regulation of the receptors occurs at many stages. The isoform AT2 was also expression cloned from rat pheochromocytoma cells. Although its ligand binding is not affected by GTP analogs, it is a seven transmembrane domain receptor. It mediates the inhibition of phosphotyrosine phosphatase by angiotensin II and AT2 specific CGP42112A; the inhibition was abolished by pertussis toxin. Thus, AT2 belongs to a new class of angiotensin receptors with unique signalling and regulatory mechanisms.

Amino Acid Sequence↗

PubMed1994

Molecular genetics of the SA gene.

1. We have recently identified a candidate gene for rat genetic hypertension, termed SA, by identifying an mRNA species that shows markedly higher expression in the kidneys of spontaneously hypertensive rats (SHR) than in those of Wistar-Kyoto rats (WKY). 2. Subsequent genetic co-segregation analyses by ourselves and others indicated that the SA gene locus did indeed influence blood pressure. Moreover, in a preliminary association study, we found an association of a polymorphism of the human SA gene with essential hypertension. 3. Further studies to identify functions of the SA gene products are required before reaching a definite conclusion.

Animals↗

PubMed1994

Angiotensin II-mediated pressor effect of rat joining peptide.

In an attempt to determine the mechanism of pressor action of centrally administered rat joining peptide (rJP), a pro-opiomelanocortin (POMC)-derived peptide, we investigated its action on the angiotensin and adrenergic system in the brain stem. In conscious spontaneously hypertensive rats with chronic cannulas in the cisterna magna and abdominal aorta, the pressor effect of synthetic rJP in the cisterna magna was markedly inhibited by pretreatment with losartan, an antagonist of angiotensin (ANG) II receptor specific for its AT1 subtype, and also by the nonspecific antagonist [Sar1,Ile8]ANG II but not by AT2-specific PD-123319. Pretreatment with captopril did not alter the pressor response. Adrenergic receptor antagonists, yohimbine and propranolol, did not change the pressor response. The intracisternal joining peptide administration (10 and 30 nmol) increased the concentration of immunoreactive ANG II in cerebrospinal fluid 2.4- and 5.7-fold, respectively. These results indicate that the pressor response to rJP is mediated by the release of central ANG II and AT1 receptor. This study details a biological response to rJP, the only POMC-derived peptide whose action has not been identified previously.

Angiotensin II↗

PubMed1994

Does plasma immunoreactive ouabain originate from the adrenal gland?

It was reported recently that the endogenous digitalis-like factor ouabain may mainly originate from the adrenal gland. To ascertain the pathophysiological significance of endogenous ouabain and to examine if it originates in the adrenal gland, we determined plasma immunoreactive ouabain levels in patients with various cardiovascular and endocrine diseases. Plasma immunoreactive ouabain levels were also determined in the adrenal venous blood by adrenal venous sampling. Plasma immunoreactive ouabain levels were significantly increased in patients with essential hypertension, primary aldosteronism, Cushing's syndrome, pheochromocytoma, acromegaly, and chronic renal failure. Plasma immunoreactive ouabain levels were decreased in patients with primary aldosteronism after unilateral adrenalectomy, acromegaly after pituitary adenomectomy, and chronic renal failure after hemodialysis. Plasma immunoreactive ouabain levels in patients after bilateral adrenalectomy were similar to those in healthy subjects. There was no significant step-up of immunoreactive ouabain levels in the adrenal vein from the peripheral vein in three patients, whereas one patient with hypertension and right adrenal tumor but without any known adrenal hormone excess showed higher plasma immunoreactive ouabain levels in the right adrenal vein than those in the peripheral vein. These results suggest an important pathophysiological significance of endogenous ouabain in various cardiovascular and endocrine diseases. It is unlikely that the adrenal gland is a major source of plasma ouabain, although a possible excess production of ouabain by the adrenal tumor remains to be elucidated.

Adrenal Gland Neoplasms↗

PubMed1994

Epidermal growth factor-enhanced human angiotensin II type 1 receptor.

The human angiotensin II type 1 (AT1) receptor gene was isolated and its promoter function analyzed by deletion mutant promoter/luciferase constructs in transfected Cos 7 cells. We found that epidermal growth factor enhanced the human AT1 promoter activity twofold to threefold. The region between -227 and -366 bp from the 5' end of the cDNA was mapped for a base sequence responsive to the epidermal growth factor stimulation. By computer analysis, PEA3 transcription factor was located in this region and was shown to bind to the promoter by gel shift assay in Cos 7 and HepG2 cells. These results indicated that the human AT1 receptor enhanced by epidermal growth factor may be due to PEA3 binding to the human AT1 promoter.

Base Sequence↗

PubMed1994

Enhanced phospholipase D activity in vascular smooth muscle cells derived from spontaneously hypertensive rats.

When cultured in the presence of fetal calf serum, aortic vascular smooth muscle cells (VSMC) derived from spontaneously hypertensive rats (SHR) grow faster than those from normotensive control Wistar-Kyoto (WKY) rats. In order to investigate the mechanism underlying this growth abnormality, we measured phospholipase D (PLD) activity in VSMC taken from both SHR and WKY rats. Upon stimulation with serum, platelet-derived growth factor (PDGF) and porbol 12-myristate 13-acetate (TPA), phosphatidylethanol (PEt) was produced in the presence of ethanol. The responses of the VSMC from SHR (SHR-cells) to all stimuli were significantly greater than those of the VSMC from WKY rats (WKY-cells), which suggests an enhanced PLD activity in the SHR-cells. Since PLD is regarded as an enzyme involved in signal transduction leading to cell proliferation, this PLD hyper-reactivity in the SHR-cells may account at least partially for the growth abnormality in the SHR-cells.

Animals↗

PubMed1994

Angiotensin II receptors: cloning and expression.

To identify the mechanisms of action of isoforms angiotensin II receptors (AT1A, AT1B, and AT2) and to overcome the difficulties encountered in attempts to purify the receptors, we have expression-cloned their cDNAs from bovine and rat sources and isolated human cDNA and rat and human genomic DNA. The AT1A and AT1B cDNAs were found to encode respective receptor proteins with 359 amino acid residues, whereas, AT2 encodes a 363 amino acid residue receptor protein. Both AT1 and AT2 were found to conform with the seven transmembrane receptor structural motif, but showed only 32% amino acid residue identity to each other. The AT1 receptor was shown to be coupled to, at least, three different G proteins activating phospholipase C, inhibiting adenylyl cyclase and opening an L-type Ca(2+)-channel, whereas, AT2 was found to inhibit a phosphotyrosine phosphatase activity without affecting guanylyl cyclase by a pertussis-toxin-sensitive, presumably G-protein-mediated mechanism.

Amino Acid Sequence↗

PubMed1994

Molecular biology of angiotensin II receptors: an overview.

ISOFORMS OF ANGIOTENSIN II RECEPTORS: So far, three isoforms of angiotensin II receptors have been identified by complementary DNA cloning, all with seven transmembrane domain structures. AT1A and AT1B are the most common isoforms. They are coupled to phospholipase C through Gq/G11 proteins and to a calcium channel, and negatively coupled to adenyl cyclase. AT2 is only remotely related to the AT1 family. KNOWN STRUCTURAL DETAILS OF ANGIOTENSIN II RECEPTORS: Ligand-binding domains are being defined in the space surrounded by transmembrane helices. Coupling to Gq seems to involve the second cytosolic loop. Receptor proteins undergo transition to a low-affinity form, which is desensitized and internalized. CHROMOSOME LOCATION: In the rat, AT1A, AT1B and AT2 are located on chromosomes 17, 2 and X, respectively. SIGNALING PATHWAY: Studies with receptors are revealing several different pathways of angiotensin signaling that modulate protein tyrosine phopsphorylation.

Amino Acid Sequence↗

PubMed1994

The renin-angiotensin system.

Unravelling of the molecular mechanisms of the action of RAS has been slow. Nature has been rather stingy in revealing bits and pieces of information. Each step of development has depended on the innovation of an appropriate methodology. The uniqueness of the RAS lies in: The function and regulation of the highly specific enzyme renin which specifically catalyses the conversion of the prohormone angiotensinogen to Ang I by an extracellular mechanism. The production of the agonist Ang II takes place in two steps. Ang II and its metabolites exert exceedingly diverse pathophysiological effects, presumably through the complex and multifunctional receptors. The exquisite mechanisms involved in the regulation of renin release and receptor regulation are fascinating. The intricate mechanisms that nature has devised for the checks and balances to maintain steady blood flow and electrolyte balance present a great challenge to biochemists in their attempts to clarify the mechanisms involved at both molecular and cellular levels. In relation to the pathophysiology of hypertension, particularly essential hypertension, there is no question that the RAS plays a pivotal role. Although numerous mechanisms could explain its hypertensinogenic effects, no single mechanism can be identified as the major determinant at the present stage of our knowledge. However, there is an important consensus that the effect of Ang II is manifested slowly at even subpressor doses of Ang II through long-term effects involving remodelling of the cardiovascular and renal system.

Amino Acid Sequence↗

PubMed1994

Increased expression of vascular angiotensin II type 1A receptor gene in glucocorticoid-induced hypertension.

OBJECTIVES: We recently demonstrated that glucocorticoid increases the number of angiotensin II type 1 (AT1) receptors and their gene expression in cultured vascular smooth muscle cells. To clarify whether this mechanism participates in glucocorticoid-induced hypertension, we investigated the effect of dexamethasone on the modulation of vascular AT1 receptor messenger RNA (mRNA) expression in rats. METHODS: The effects were studied of administering dexamethasone orally to rats at a dose of 0.1 mg/day on the modulation of the expression of vascular AT1 receptor mRNA and the blood pressure. The effects of enalapril maleate, an angiotensin converting enzyme inhibitor, were also evaluated. RESULTS: The dexamethasone-treated rats showed a rise in systolic blood pressure beginning on day 3. On day 5 the blood pressure rose significantly. Before dexamethasone administration the vascular AT1 receptor mRNA was difficult to detect by Northern blot analysis. However, the level of vascular AT1A receptor mRNA began to increase on day 2, when the blood pressure was not yet elevated, and increased further on day 5. The concurrent administration of dexamethasone and enalapril maleate attenuated the elevation of blood pressure. However, as in the dexamethasone-treated rats, the level of AT1A receptor mRNA began to increase on day 2 and increased further on day 5. In rats treated with enalapril maleate alone, the AT1 receptor mRNA was difficult to detect during the experiment. CONCLUSION: Glucocorticoid increased the amount of vascular AT1 receptor mRNA and contributed to the elevation of blood pressure.

Animals↗

PubMed1993

Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition.

There are two major isoforms of the angiotensin II receptor, type 1 (AT1) and type 2 (AT2). AT2 is distinguished from AT1 with respect to its ligand selectivity, its insensitivity to non-hydrolyzable GTP analogues, and its as yet unidentified biological functions. In the present study we have expression-cloned AT2 cDNA from a cDNA library of a rat pheochromocytoma cell line (PC12w). Rat AT2 cDNA encodes a 363-amino acid protein that has seven transmembrane domains. AT1 is the closest in homology to AT2 but with only a 32% identity of amino acid sequence. Stably expressed in COS-7 cells, the receptor showed selective binding to AT2-specific ligands PD123319 and CGP42112A but not to the AT1-specific ligand, losartan. Northern blot analysis revealed that the mRNA of rat AT2 was expressed not only in PC12w cells but also in the adrenal glands and in the inferior olive of the brain, both of which are known to contain AT2 type binding sites. The expressed AT2 receptor mediated angiotensin II-induced inhibition of protein tyrosine phosphatase, an action that was dependent on a pertussis toxin-sensitive G-protein-coupled mechanism in COS-7 cells. The AT2-specific ligand CGP42112A was an agonist rather than antagonist in the inhibition of phosphotyrosine phosphatase. AT2 did not cause a decrease in cGMP in PC12w or COS-7 cells expressing AT2 stably. These results indicate that the AT2 receptor is structurally and functionally different from AT1 and suggest novel functional roles of the renin-angiotensin system in cross-talk with phosphotyrosine signaling by modulating protein phosphotyrosine levels.

Amino Acid Sequence↗

PubMed1993

Transduction of mitogenic activity of platelet-derived growth factor (PDGF) AB by PDGF-beta receptor without participation of PDGF-alpha receptor in vascular smooth muscle cells.

In cultured rat vascular smooth muscle cells, platelet-derived growth factor (PDGF) beta receptor was expressed at a high level, whereas PDGF-alpha receptor was not detected. PDGF-BB showed a high binding activity at 4 degrees C in the cells and was not displaced by PDGF-AA or -AB. This result indicates that PDGF-AB as well as PDGF-AA does not bind to the cells lacking PDGF-alpha receptor at 4 degrees C. However, at 37 degrees C, PDGF-AB bound to the cells and induced the internalization of PDGF-beta receptor. DNA synthesis was also stimulated potentially by PDGF-AB as well as PDGF-BB in the cells, although PDGF-AA was completely inactive. At 37 degrees C, PDGF-AB caused tyrosine phosphorylation of a group of proteins including PDGF-beta receptor and phospholipase C-gamma 1, but at a slower rate than PDGF-BB. At 4 degrees C, PDGF-AB did not stimulate protein tyrosine phosphorylation, whereas PDGF-BB did. A chemical cross-linking experiment showed that PDGF-beta receptor was dimerized by PDGF-AB as well as PDGF-BB. These results indicate that PDGF-beta receptor binds PDGF-AB without participation of PDGF-alpha receptor at 37 degrees C (but not at 4 degrees C), and PDGF-AB as well as PDGF-BB acts as a potent mitogen in the vascular smooth muscle cells.

Animals↗