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Biomedical subjects

Y Kambayashi

Publications and source records attributed to Y Kambayashi.

At least 37 records · Page 2Linked to original sources

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↗

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↗

Activation of protein kinase C by oxidized diacylglycerols.

The effects of 1,2-diacylglycerol hydroperoxide (DAG-OOH), its alcohol (DAG-OH), "unoxidized" 1,2-diacylglycerol (DAG), 1,2-dioleoylglycerol (DOG), and phorbol 12-myristate acetate (PMA) on the activity of protein kinase C (PKC) isolated from rat brain were examined in the presence and absence of phosphatidylserine (PS) and calcium ion. Both DAG-OOH and DAG-OH stimulated the activity of PKC dose- and time-dependently. The ability of additives for PKC activation increased in the order of DOG, DAG << DAG-OOH, DAG-OH < PMA. DAG-OOH and DAG-OH activated PKC even in the absence of PS and calcium ion as PMA does. If DAG-OOH and DAG-OH are released from the oxidized biomembranes by the action of phospholipase C, these components may act like PMA and serve to activate the PKC-dependent signal transduction system.

Animals↗

Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells.

The biological action of adrenomedullin, a novel hypotensive peptide, on bovine aortic endothelial cells, was examined. The specific binding of adrenomedullin to these cells was observed, and adrenomedullin was found to induce intracellular cAMP accumulation in a dose-dependent manner. EC50 for the cAMP accumulation was about 100 times lower than the apparent IC50 for the binding assay. Adrenomedullin also induced increase of intracellular free Ca2+ in endothelial cells in a dose-dependent manner. The Ca2+ response to adrenomedullin was biphasic with an initial transient increase due to the release from thapsigargin-sensitive intracellular Ca2+ storage and a prolonged increase by influx through the ion channel on the plasma membrane. This intracellular free Ca2+ increase resulted from phospholipase C activation and inositol 1,4,5-trisphosphate formation, and seemed to cause nitric oxide synthase activation by monitoring intracellular cGMP accumulation. Both cAMP accumulation and Ca2+ increased responses to adrenomedullin were mediated by cholera toxin-sensitive G protein, but the two signal transduction pathways were independent. Thus, the results suggest that adrenomedullin elicits the hypotensive effect through at least two mechanisms, a direct action on vascular smooth muscle cells to increase intracellular cAMP and an action on endothelial cells to stimulate nitric oxide release, with both leading to vascular relaxation.

Adrenomedullin↗

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↗

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↗

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.

3T3 Cells↗

Conflicting view support by medical information systems.

One of the advantages of current database systems is the capability to enforce global integrity constraints on a large amount of data. Input of contradicting data will be rejected by database systems in order to maintain correctness. On the other hand, in medical information systems it may be necessary to realize two or more databases in one system, where there are some controlled contradictions among these databases. For example, if a doctor wants to hide the real disease name from the patient in critical condition, the database viewed by the patient should be different from the real database, although each of these databases should be conflict free and large amounts of data are shared by both. This kind of problem was not discussed for commercial business-oriented databases. Data sharing and data security are important functions required for medical information systems. There are, however, cases when we need to show non-real data to some users. Security mechanisms usually prevent a user from retrieving critical data. If a request for retrieval of some data is rejected by the system, a user may find there is something secret being kept from him. In order to cope with these situations, we introduce the POSTGRES database system. POSTGRES is a generalized relational database system developed at the University of California. The form of POSTGRES rule is as follows: On event (To) object WHERE POSTQUEL-qualification Then Do [instead] POSTQUEL-command(s) The POSTGRES rule shows that event is retrieve, replace, delete, append, new (i.e., replace or append) or old (i.e., delete or replace). The concept of objects is introduced in object-oriented databases. In relational database systems, an object corresponds to each data value, an attribute, or a relation. The optional keyword "instead" indicates that the action indicated by POSTQUEL-command(s) is to be performed instead of the action which caused the rule to activate. This keyword plays very important role for our purpose. By preparing a standard story which is consistent with the patient's condition, the doctor can make a smooth explanation to the patient showing the stories instead of the critical data. So, if medical personnel and system designers prepare the standard stories or data for the explanation and store such data in the system, the users of medical information systems (doctors) can take the benefit of the data replacement. For such a purpose, medical personnel and system designers classify the patients' data according to their age, sex, occupational history, personal history, and diseases. Standard story or data should be prepared for each user class in advance. Using POSTGRES rule system in medical information systems, doctors can make a smooth explanation of a patientUs condition in serious cases, when hiding real data is required. The validity of POSTGRES rule system is proved where data exchange is needed for data protection. For example, when a doctor writes a prescription of a placebo through the ordering system, POSTGRES rule system will identify its existence. We believe that POSTGRES rule system can be applied to many fields in medical data processing.

Confidentiality↗

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.

Amino Acid Sequence↗

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↗

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↗

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↗

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↗

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↗

Peptide growth factors markedly decrease the ligand binding of angiotensin II type 2 receptor in rat cultured vascular smooth muscle cells.

Of two major isoforms of angiotensin II receptors, AT1 and AT2, biological roles of AT2 remain unclear. Using vascular smooth muscle cells, we investigated the regulation of expression of AT2 by growth factors in comparison with that of AT1. The cultured rat aorta smooth muscle cells had detectable AT2 binding sites, which were reduced significantly by treatment with platelet derived growth factor-BB. On the other hand, AT1 binding sites were increased under the same conditions. Other growth factors, such as epidermal growth factor and endothelin-1, also suppressed AT2 receptors to varying extents. A negative correlation between DNA synthesis promoted by these growth factors and the binding capacity of AT2 sites was observed. This study indicated that the expression of AT2 is downregulated in cultured vascular smooth muscle cells by growth factors in contrast to that of AT1, which was slightly upregulated.

Analysis of Variance↗

Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide.

To elucidate the ligand-receptor relationship of the natriuretic peptide system, which comprises at least three endogenous ligands, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and three receptors, the ANP-A receptor or guanylate cyclase-A (GC-A), the ANP-B receptor or guanylate cyclase-B (GC-B), and the clearance receptor (C-receptor), we characterized the receptor preparations from human, bovine, and rat tissues and cultured cells with the aid of the binding assay, Northern blot technique, and the cGMP production method. Using these receptor preparations, we examined the binding affinities of ANP, BNP, and CNP for the C-receptor and their potencies for cGMP production via the ANP-A receptor (GC-A) and the ANP-B receptor (GC-B). These analyses revealed the presence of a marked species difference in the receptor selectivity of the natriuretic peptide family, especially among BNPs. Therefore, we investigated the receptor selectivity of the natriuretic peptide family using the homologous assay system with endogenous ligands and receptors of the same species. The rank order of binding affinity for the C-receptor was ANP greater than CNP greater than BNP in both humans and rats. The rank order of potency for cGMP production via the ANP-A receptor (GC-A) was ANP greater than or equal to BNP much greater than CNP, but that via the ANP-B receptor (GC-B) was CNP greater than ANP greater than or equal to BNP. These findings on the receptor selectivity of the natriuretic peptide family provide a new insight into the understanding of the physiological and clinical implications of the natriuretic peptide system.

Animals↗

Biological characterization of human brain natriuretic peptide (BNP) and rat BNP: species-specific actions of BNP.

We examined the diuretic-natriuretic activities of rat BNP and human BNP in anesthetized rats in vivo and their vasorelaxant activities for rat thoracic aorta and porcine coronary artery in vitro. Rat BNP was almost equipotent to rat ANP in diuresis and natriuresis with relative potencies of 1.6 and 2.5, respectively, while human BNP exerted no significant activity. Rat ANP, rat BNP and human BNP relaxed PGF2 alpha-contracted rat aortic strips with IC50 values of 0.62, 0.64 and 12.1 nM, respectively, while they relaxed PGF2 alpha-contracted porcine coronary arteries with IC50 values of 0.04, 1.10 and 0.02 nM, respectively. These results strongly suggest that the biological action of BNP is species-specific.

Amino Acid Sequence↗