Proposed update of angiotensin receptor nomenclature.
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Animals↗
Biomedical subjects
T Inagami
Publications and source records attributed to T Inagami.
Tachyphylaxis of human forearm vascular responses does not occur rapidly after exposure to isoproterenol.
In vitro and limited in vivo data suggest that rapid desensitization of beta-adrenoceptor responses occurs after exposure to agonist. Tachyphylaxis to a beta-adrenoceptor agonist would represent a potentially important mechanism for the short-term regulation of vascular tone. The effects of a 4-hour infusion of 400 ng/min intra-arterial isoproterenol on forearm blood flow and presynaptic beta-adrenoceptor-mediated norepinephrine release were determined in eight healthy volunteers. Intra-arterial isoproterenol at 400 ng/min resulted in a significant increase in forearm blood flow in all eight subjects at all time points, with no evidence of tachyphylaxis. In fact, forearm blood flow after 4 hours of the isoproterenol infusion (22.8 +/- 3.3 mL/100 mL per minute) was significantly greater than after 7 minutes (14.6 +/- 2.8 mL/100 mL per minute), 15 minutes (15.4 +/- 2.4 mL/100 mL per minute), and 30 minutes (17.4 +/- 3.0 mL/100 mL per minute) of the infusion (P < .05). Similarly, presynaptic beta-adrenoceptor responses showed no evidence of tachyphylaxis, so forearm norepinephrine spillover values after 7 minutes (6.6 +/- 0.94 ng/min), 15 minutes (7.6 +/- 1.5 ng/min), and 4 hours (8.8 +/- 1.1 ng/min) of isoproterenol infusion were increased and similar. Minimal systemic effects were observed, and there was no evidence of tolerance, there being no difference in heart rate after 7 minutes (70.7 +/- 2.7 beats per minute) and 4 hours (72.2 +/- 3.6 beats per minute) of isoproterenol infusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Cardiovascular and respiratory effects of endothelin in the ventrolateral medulla of the normotensive rat.
We studied the relevance of the ventrolateral medulla for the cardiovascular and respiratory effects of endothelin-1 in urethane-anesthetized rats. Microinjection of endothelin-1 into the rostral ventrolateral medulla (RVLM) evoked pressor and bradycardic effects followed by sustained decreases in blood pressure, bradycardia, and respiratory depression. These effects were inhibited by endothelin-A receptor antagonists (BQ-123 and BQ-610) but not by endothelin-B antagonists. In the caudal ventrolateral medulla (CVLM) endothelin-1 decreased blood pressure, renal sympathetic nerve activity, respiratory frequency, and phrenic nerve activity, whereas heart rate increased. Pretreatment with BQ-123 in the CVLM increased respiratory frequency by 15 +/- 6 breaths per minute and prevented the effects of intra-CVLM administration of endothelin-1. In separate experiments, the intracisternal administration of endothelin-1 (20 pmol) to rats pretreated with saline in both RVLM and CVLM resulted in a hypotensive and bradycardic phase that was followed by hypertension (50 +/- 15 mm Hg), bradycardia, and 100% mortality. In a separate group, pretreatment with BQ-123 in the RVLM and CVLM completely inhibited the hypotensive phase and reduced by 83% the subsequent rise in blood pressure evoked by endothelin-1. Cardiorespiratory arrest was prevented in all the rats in this group. Selective endothelin receptor blockade in the RVLM attenuated the hypertensive period of intracisternal administration of endothelin-1 and prevented mortality by 33%, whereas in the CVLM the endothelin receptor antagonist inhibited the initial hypotension and reduced mortality by 25%. Our results support the concept that in the ventral medulla, endothelin-1 can modulate cardiovascular and respiratory function.
Animals↗
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)
Animals↗
Inhibition of protein kinase C prevents rapid desensitization of type 1B angiotensin II receptor.
The type 1B angiotensin II (AT1B) receptor cloned from rat kidney was stably expressed in Chinese hamster ovary cells. The stably expressed receptor was characterized by radioligand binding studies and functional coupling to inositol 1,4,5-triphosphate (IP3) formation. Exposure of cells expressing the AT1B receptor to angiotensin II (Ang II) resulted in a rapid and dose-dependent homologous desensitization of receptor-mediated production of IP3, with an essentially complete desensitization at an agonist concentration > 10 nmol/L. Binding studies revealed no significant change in the number of AT1B receptors in transfected cells exposed to 1 nmol/L Ang II, whereas exposure to 100 nmol/L Ang II caused a rapid decrease of cell surface receptors, with a 75% loss of receptor number seen at 1 hour. Rapid desensitization occurred in the absence of receptor internalization. Blockade of receptor internalization with concanavalin A had at most only a slight effect on the agonist-induced desensitization. This indicates that factors other than internalization are chiefly responsible for the rapid agonist-induced desensitization. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, caused rapid desensitization of the receptor-mediated IP3 response. Neither tyrosine kinase inhibitors nor a protein kinase A activator affected the receptor-mediated IP3 response. The specific PKC inhibitor GF109203X or PKC depletion by prolonged treatment with 1 mumol/L PMA completely blocked the PMA-dependent desensitization. Desensitization evoked by a low Ang II agonist concentration (1 nmol/L) was reversed by the PKC-specific inhibitor GF109203X or PKC depletion, whereas the desensitizing effect at a high agonist concentration (100 nmol/L) is only partially prevented by PKC inhibitory treatment. These results demonstrate that PKC plays a crucial role in the desensitization of the AT1B receptor. They also suggest that receptor internalization and an additional PKC-independent pathway also contribute to desensitization of the AT1B receptor in transfected cells.
Analysis of Variance↗
Identification of a cis-acting glucocorticoid responsive element in the rat angiotensin II type 1A promoter.
Enhanced vascular responsiveness to angiotensin II at the AT1 receptor has been considered one of the major contributing factors to vascular hypertrophy and high blood pressure. 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 (< 12 passages). There are three putative glucocorticoid responsive elements (GREs) in the promoter. However, only one GRE was found to respond to dexamethasone (1 mumol/L) and was located at positions -756 to -770 bp upstream from the transcription initiation site. When compared with the consensus sequence of GRE, 9 of 12 bases were identical. RU38486, a glucocorticoid antagonist, completely blocked the induction by dexamethasone, suggesting that the GRE was functional through a specific glucocorticoid receptor. The response to dexamethasone was lost in vascular smooth muscle cells at higher passage numbers (> 8 passages) but was restored when the cells were transfected with a glucocorticoid-receptor expression construct. This finding provided additional support that the response to dexamethasone was mediated by the glucocorticoid receptor. The gel mobility supershift assay showed that the GRE binds in vitro-translated rat glucocorticoid receptors in a specific manner. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Angiotensin II↗
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↗
Molecular structure and transcriptional regulation of the gene for the platelet-derived growth factor alpha receptor in cultured vascular smooth muscle cells.
PDGF has been shown to contribute to hypertrophy in vascular smooth muscle cells (VSMC). PDGF-AA differentially promotes protein synthesis in VSMC from spontaneously hypertensive rats (SHR) but not in those from Wistar-Kyoto rats (WKY). This observation has led us to postulate a role for PDGF alpha receptor (PDGFR-alpha) in the hypertensive hypertrophy of blood vessels. Western and Northern blot analyses demonstrated a high and specific expression of the PDGFR-alpha protein and mRNA in SHR cells but not in WKY cells. To clarify the mechanism of the differential expression of the PDGFR-alpha gene, we isolated the promoter region of the gene. Studies on the promoter functions indicated that this promoter is active in SHR cells but not in WKY cells. The regulatory domain responsible for this difference was narrowed to the sequence between -246 and -139, which enhanced the promoter activity of SHR fivefold over the basal activity. DNase I footprinting and gel-shift assay indicated that this sequence specifically interact with nuclear proteins from VSMC through the binding site for CCAAT/enhancer-binding proteins, and members of the C/enhancer-binding protein family play a significant role in the strain-specific transcription of the PDGFR-alpha gene.
Animals↗
Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation.
Elevated levels of endogenous angiotensin can cause hypertensive nephrosclerosis as a result of the potent vasopressor action of the peptide. We have produced by gene targeting mice homozygous for a null mutation in the angiotensinogen gene (Atg-1-). Postnatally, Atg-1- animals show a modest delay in glomerular maturation. Although Atg-1- animals are hypotensive by 7 wk of age, they develop, by 3 wk of age, pronounced lesions in the renal cortex, similar to those of hypertensive nephrosclerosis. In addition, the papillae of homozygous mutant kidneys are reduced in size. These lesions are accompanied by local up-regulation of PDGF-B and TGF-beta1 mRNA in the cortex and down-regulation of PDGF-A mRNA in the papilla. The study demonstrates an important requirement for angiotensin in achieving and maintaining the normal morphology of the kidney. The mechanism through which angiotensin maintains the volume homeostasis in mammals includes promotion of the maturational growth of the papilla.
Aging↗
Characterization of a synthetic peptide antibody recognizing rat kidney renin and prorenin.
A fourteen amino acid peptide from the cDNA predicted rat renin amino acid sequence was synthesized and used to produce a polyclonal antibody. The ability of this antibody to recognize rat renin was characterized and compared to that of a previously tested polyclonal anti-rat renin antibody raised against purified rat renin to verify its usefulness as a reagent in renin processing. The anti-renin peptide antibody and anti-rat renin antibody were evaluated for specificity of recognition of rat renin by immunohistochemical staining and immunoblotting. The ability of the anti-renin peptide antibody to inhibit renin enzymatic activity and to immunoprecipitate rat recombinant protein was also tested. Anti-renin peptide antibody and anti-rat renin antibody had identical recognition of rat renin and prorenin in immunohistochemical stains of rat kidney section and immunoblots. However the anti-renin peptide antibody does not inhibit renin enzymatic activity nor does it immunoprecipitate recombinant rat prorenin. We have produced and characterized an antirenin peptide antibody which recognizes denaturated rat renin and prorenin in a way that is identical to that of an antibody raised against native rat renin. The anti-renin peptide antibody does not recognize nondenatured renin because it did not inhibit renin enzymatic activity nor was it able to immunoprecipitate renin or prorenin. The technique of using synthetic peptides to produce antibody recognizing at renin is useful because low yields of native renin purified from rat kidney are generally insufficient for antibody production. This limits the widespread availability of antibodies that recognize rat renin.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Participation of diacylglycerol kinase in mitogenic signal transduction induced by platelet-derived growth factor in vascular smooth muscle cells.
In rat vascular smooth muscle cells, platelet-derived growth factor (PDGF), stimulated phosphatidic acid synthesis by activating both of the two alternative pathways, diacyglycerol kinase (DGK) and phospholipase D (PLD). Genistein, a tyrosine kinase inhibitor, inhibited PLD activation but not DGK activation, the latter was inhibited selectively by R 59022. PDGF-induced DNA synthesis was partially inhibited by genistein or R 59022, but these inhibitors had no effect on phorbol ester-induced DNA synthesis. Further, the specific effects of these inhibitors on PDGF-induced DNA synthesis were additive.
Alkaloids↗
Differences in signal transduction between platelet-derived growth factor (PDGF) alpha and beta receptors in vascular smooth muscle cells. PDGF-BB is a potent mitogen, but PDGF-AA promotes only protein synthesis without activation of DNA synthesis.
Cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats express both alpha and beta isoforms of the platelet-derived growth factor (PDGF) receptors at high levels (100,000 and 240,000 sites/cell, respectively). In this cell type, PDGF-BB elicited a mitogenic response; however, PDGF-AA increased only protein synthesis without activating DNA synthesis. Protein kinase C (PKC) was activated by PDGF-AA as well as PDGF-BB with concomitant translocation from cytosol to membrane fractions. However, the hypertrophic effect of PDGF-AA was not affected by depletion of cellular PKC, whereas the mitogenic action of PDGF-BB was partially attenuated by the depletion. Following incubation with PDGF-AA or -BB, phospholipase C-gamma 1 (PLC-gamma 1) and phosphatidylinositol 3-kinase were tyrosine phosphorylated; however, the phosphorylation of Ras-GTPase-activating protein was induced only by PDGF-BB. Both PDGF isoforms resulted in a prompt and transient increase in the level of 1,2-diacylglycerol (DAG), presumably through the action of PLC-gamma 1. After returning to basal levels, the rate of DAG synthesis steadily increased for at least 15 min due to activation of phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC). Incubation with PDGF-BB-activated phospholipase D (PLD) in a PKC-dependent manner resulting in the formation of phosphatidic acid (PA). PA was also formed by the sequential reactions of PC-PLC and DAG kinase in the PDGF-BB-stimulated VSMC, and these sequential reactions were not affected by PKC depletion. In contrast, PDGF-AA stimulation did not result in increased PA synthesis as neither PLD nor DAG kinase activities were affected. PA may be a significant second messenger in the activation of DNA synthesis by PDGF-BB. These results indicate that signaling mechanisms of the PDGF-alpha and -beta receptors in VSMC are distinctly different in signal transduction in VSMC and that the alpha receptor promotes cellular hypertrophy (but not hyperplasia), whereas a mitogenic response is mediated only through the beta receptor.
Animals↗
Glucocorticoid induction of rat angiotensin II type 1A receptor gene promoter.
Promoter/luciferase reporter analysis indicated that the 5'-flanking region of rat angiotensin II type 1A receptor gene was functional, both in the rat aortic smooth muscle cells (RASMCs) and in Y1 cells derived from mouse adrenal cortex. In response to dexamethasone (Dex), transcriptional activity of promoter increased in transfected RASMCs, suggesting the functional cis-action of a glucocorticoid responsive elements (GRE). Furthermore, the expression of the rat AT1A gene was also up-regulated by Dex at the levels of both mRNA and protein in RASMCs.
Adrenal Cortex Neoplasms↗
The genomic organization of the rat angiotensin II receptor AT1B.
Expression of two subtypes of angiotensin type 1 receptor (AT1A and AT1B) seems to be controlled by different mechanisms. As a step toward the elucidation of difference in the mechanism in their gene expression, the genomic organization of rat angiotensin II type 1B receptor was investigated. Rat genomic DNA fragments were cloned by screening a genomic library with fragments from two cDNA clones (2.3 and 2.2 kb) encoding the rat AT1B receptor. Three lambda clones were isolated, and the hybridizing restriction fragments were sequenced. Comparison of the genomic clone DNA and cDNA sequences revealed that the rat AT1B receptor gene contains three exons and two introns. Two of the exons encoded 5' untranslated sequences while the third exon contained the entire coding region. The 5'-flanking region contained the typical sequence motifs found in many eukaryotic promoters including a TATA box and a potential SP1 binding site. The transcription initiation site was located at -8 bp upstream the 5' end of the cDNA in the 5'-flanking region.
Animals↗
Molecular cloning and expression of the gene encoding human angiotensin II type 2 receptor.
The gene of human angiotensin II type 2 (AT2) receptor was isolated from a genomic DNA library prepared from human placenta. The coding region of the human AT2 receptor gene was contained in a single exon coding segment of the gene indicating an intronless structure of the coding region. The amino acid sequence of human AT2 receptor deduced from its nucleotide sequence has 363 amino acids and shows a high degree of sequence identity to rat and mouse receptor sequences. Specific binding of [125I]Sar1Ile8-angiotensin II was demonstrated in COS-7 cells transfected with a plasmid containing the human AT2 sequence. Scatchard analysis and ligand displacement profile were typical of the AT2 receptor. Reverse transcription-polymerase chain reaction analysis showed that AT2 receptor mRNA was expressed in adult uterus and pheochromocytoma.
Amino Acid Sequence↗
Identification of two cardiac glycosides as Na(+)-pump inhibitors in rat urine and diet.
Endogenous Na(+)-pump specific inhibitors are present in the plasma, urine, and tissues of humans and animals. To date, the source of these inhibitors has not been rigorously defined. In the present study, large amounts of several Na(+)-pump specific inhibitors have been demonstrated to exist in the urine of rats raised on a regular chow diet and tap water. All of the inhibitor levels have been found to increase 1.5-8-fold by the surgical preparation of reduced renal mass (RRM) and one-kidney, one-clip (IK, IC) hypertension. These urinary inhibitors, however, except for the ouabain-like inhibitor which eluted from a high performance liquid chromatography C18 column at the same retention time as [3H]ouabain, disappeared within a week after switching the diet from regular diet (number 5001, PMI Feeds, Inc.) to pure synthetic diet (number 5755). The urinary level of the ouabain-like inhibitor decreased to only one-half of the level in the control rat raised on a regular diet. Two of these inhibitors were purified from both urine and diet by a combination of Amberlite XAD-2 adsorption chromatography, reverse phase low pressure liquid chromatography, and several high performance liquid chromatographies. Reverse phase high performance liquid chromatography, liquid secondary ion and gas-liquid mass spectrometries, and proton nuclear magnetic resonance spectroscopy identified these inhibitors as a stereoisomer of convalloside, probably neoconvalloside, and a mono-rhamnoside of periplogenin or its stereoisomer. These cardiac glycosides exhibited inhibitory potencies comparable to ouabain against ouabain-displacement from Na+,K(+)-ATPase and against 86Rb uptake into human erythrocytes, and they also exhibited cross-reactivity to anti-ouabain antibodies and anti-digoxin antibodies. These results clearly demonstrate that the principal source of most of the inhibitors in rat urine is the diet. The results suggest that the ouabain-like inhibitor may be derived from an endogenous origin.
Analysis of Variance↗
The genomic organization of human angiotensin II type 1 receptor.
As a step toward the elucidation of human Ang II type 1 receptor gene expression, the genomic organization of the human AT1 receptor was investigated. Comparison of the genomic DNA and cDNA sequences revealed that it consists of at least five exons. The length of the AT1 receptor gene is greater than 55 kb, and the size of the exons ranges from 59 to 2,014 base pairs. Four of the exons encoded 5'-untranslated sequences. Multiple transcription initiation sites were observed by primer extension experiment. The promoter function was examined by using luciferase as a report gene in transfected human vascular smooth muscle cells.
Angiotensin II↗
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↗