Biomedical subjects

M Inada

Publications and source records attributed to M Inada.

At least 19 recordsLinked to original sources

PubMed1995

Gene transcription of angiotensin II type 2 receptor is repressed by growth factors and glucocorticoids in PC12 cells.

The rat angiotensin II type 2 receptor (AT2-R) expression was markedly downregulated by the mitogenic action of serum, growth factors and dexamethasone. The regulation by serum or growth factors did not affect the AT2-R mRNA half-life (18 h), whereas the AT2-R half-lives of dexamethasone-treated cells and proliferating cells decreased to 10 h and 15 h, respectively. Nuclear run-off assays indicated the mechanism of repression of AT2-R expression by serum, growth factors and dexamethasone or in proliferating cells to be, in large part, transcriptional. These findings indicate that transcription of the AT2-R gene is regulated in a growth state-dependent manner and suggest that this regulation provides a means by which cells can modulate their responsiveness to the actions of angiotensin II mediated through AT2-R.

Angiotensin II↗

PubMed1995

Identification of a negative cis-regulatory element and trans-acting protein that inhibit transcription of the angiotensin II type 1a receptor gene.

The rat angiotensin II type 1a receptor (AT1a-R) gene is expressed in a cell-specific manner. We demonstrated that the negative regulatory element (NRE) between -489 and -331 is active in PC12 cells (Murasawa, S., Matsubara, H., Urakami, M., and Inada, M. (1993) J. Biol. Chem. 268, 26996-27003). Gel retardation assays confirmed that PC12 cells have a trans-acting factor bound to the NRE. By means of a DNase I footprint assay we identified the core of the NRE as an (A+T)-rich sequence (TAATCTTTTATTTTA) located at nucleotides -456 to -442. Oligonucleotides corresponding to the NRE core sequence bound to nuclear protein. Site-directed mutagenesis at nucleotides -451 to -448 eliminated the specific protein/DNA binding and restored expression of the AT1a-R in transient transfection assays (2.7-fold increase). The NRE did not negatively affect the thymidine kinase promoter. No homology was found with known NREs, suggesting that this is a novel NRE. Southwestern blotting revealed a 53-kDa, specific binding protein in PC12 cells and the rat brain, but not in the liver, spleen, adrenal gland, and kidney. These findings demonstrate that the NRE of the rat AT1a-R is an (A+T)-rich sequence located at nucleotides -456 to -442 and the 53-kDa protein is a specific binding protein, and suggest that this protein may be a trans-acting factor which determines the neuron-specific down-regulation of the AT1a-R gene.

Angiotensin II↗

PubMed1995

Structure of the rat V1a vasopressin receptor gene and characterization of its promoter region and complete cDNA sequence of the 3'-end.

The gene encoding the rat V1a arginine vasopressin (AVP) receptor was isolated, and its structural organization and 5'-flanking region were characterized. In addition, the complete cDNA sequence of the major transcript of the rat V1a receptor gene was determined. Southern blots demonstrated a single copy of the V1a receptor gene in the rat genome, spanning a region of 3.8 kilobases (kb) and consisting of two exons and one intron (1.8 kb). The location of the intron was unique among G protein-coupled receptor genes in that the first exon encodes six of the seven transmembrane regions, the seventh region being encoded by the second exon. Primer extension, RNase protection, and rapid amplification of the 5'-end of the cDNA identified three transcriptional initiation sites (-405, -243, and -237), the major transcription initiation sites being mapped to positions -243 and -237 base pairs (bp) upstream of the ATG initiation codon (+1 bp). This portion of the 5'-flanking region has neither a TATA nor a CCAAT box, is GC-rich but has no GC box motif, and has features of promoters seen in housekeeping genes. Chimeras containing 2.2 kb of the 5'-flanking region and deletion analyses using the chloramphenicol acetyltransferase gene indicated that a "minimal" region, exhibiting promoter activity and tissue specificity, is located between nucleotides -296 and -221, when transfected into vascular smooth muscle cells. Gel mobility shift assay and Southwestern blotting suggested that approximately 30- and approximately 28-kDa nuclear proteins specifically bind to this region. Rapid amplification of the 3'-end of the cDNA showed that the major transcript terminates 442 bp downstream of the stop codon, in agreement with the mRNA size (2.1 kb). This study demonstrated a distinctive feature in the structural organization of the AVP-oxytocin receptor family genes, and characterization of the 5'-flanking region reported here will lead to a better understanding of the mechanism of transcriptional regulation of the rat V1a AVP receptor gene.

3T3 Cells↗

PubMed1995

Two vasopressin type 2 receptor gene mutations R143P and delta V278 in patients with nephrogenic diabetes insipidus impair ligand binding of the receptor.

We recently identified vasopressin type 2 receptor gene mutations in two unrelated Japanese families with X-linked nephrogenic diabetes insipidus, which were a missense mutation from Arg143 to Pro (R143P) and a single amino acid deletion of Val278 or 279 (delta V278). To investigate the mechanism by which the mutations cause arginine vasopressin (AVP) resistance in this disorder, we expressed them in mammalian cells and analyzed their functional properties. Stable expression study with Chinese hamster ovary (CHO) cells demonstrated that the R143P mutation reduced receptor binding capacity to 10% of normal. However, the R143P mutant itself had a normal affinity for AVP and stimulated adenylyl cyclase production at up to 50% of the wild-type level, suggesting that the mutant receptors could function normally despite their reduced surface expression. In contrast, the delta V278 mutation totally abolished receptor-ligand binding and subsequent adenylyl cyclase stimulation, indicating that delta V278 mutant is virtually nonfunctional receptor. Northern blotting revealed that mutant CHO cell lines produced levels of receptor mRNA similar to the wild-type cell line. Our results suggest that the two mutations impair binding activity of the receptor without affecting mRNA accumulation, thereby causing AVP resistance through a posttranscriptional mechanism.

Amino Acid Sequence↗

PubMed1995

Characterization of glucocorticoid response element of rat angiotensin II type 1A receptor gene.

The responsiveness of the rat angiotensin II type 1a and type 1b receptor (AT1a-R and AT1b-R) genes to glucocorticoid was examined in rat vascular smooth muscle cells (VSMCs) and the glucocorticoid response element (GRE) of the AT1a-R gene was characterized. Glucocorticoid induced an increase in AT1a-R mRNA levels, whereas AT1b-R mRNA levels were unaffected. The nuclear run-off assay indicated that the transcription of the AT1a-R gene, but not that of the AT1b-R gene, was increased by glucocorticoid. The mRNA stability of AT1a-R was unchanged by glucocorticoid. Promoter/chrolamphenicol acetyltransferase reporter analysis demonstrated that the 5'-flanking region of the AT1a-R gene was functional in rat VSMCs and established that the GRE motif between -770 to -756 could confer glucocorticoid responsiveness on the AT1a-R gene.

Angiotensin II↗

PubMed1995

Regulation by basic fibroblast growth factor of glycosaminoglycan biosynthesis in cultured vascular endothelial cells.

The alteration of glycosaminoglycans (GAGs) in cultured bovine aortic endothelial cells after exposure to basic fibroblast growth factor (bFGF) was investigated. It was found that the incorporation of [3H]glucosamine into GAGs was markedly increased by bFGF in both the cell layer and the conditioned medium; however, that of [35S]sulfate was not changed by the growth factor. These results indicated that bFGF enhanced the sugar-chain formation but did not affect their sulfation in endothelial GAG production. Similar changes were observed in either bovine aortic smooth-muscle cells and human fibroblastic IMR-90 cells to greater and lesser degrees, respectively. Characterization of GAGs in the endothelial cell layer and the conditioned medium revealed that bFGF enhanced both heparan sulfate and the other GAGs to a similar degree. The present data suggest that bFGF may be involved in the regulation of the blood coagulation system via altering GAGs of the vascular tissue when the endothelium was damaged.

Animals↗

PubMed1995

No detectable cytomegalovirus and Epstein-Barr virus genomes in the pancreas of recent-onset IDDM patients.

Viral infection is assumed to trigger or exacerbate autoimmune responses against pancreatic beta cells leading to the development of insulin-dependent diabetes mellitus (IDDM). We therefore examined by polymerase chain reaction the presence of two candidate viruses, cytomegalovirus and Epstein-Barr virus, in IDDM pancreases. Pancreas tissues were obtained by biopsy under laparoscopy from 16 recent-onset IDDM patients: age 17-53 years; disease duration 0-7 months; six had flu-like symptoms before onset. Frozen sections were made and subjected to DNA amplification. DNA samples were prepared from the frozen sections and polymerase chain reaction was performed using primers specific to cytomegalovirus, Epstein-Barr virus and control gene for HLA-DP. Cytomegalovirus- and Epstein-Barr virus-infected cells were used for positive control. Southern blot analysis could detect cytomegalovirus DNA from as few as 2 x 10(-1) cytomegalovirus-infected cells and Epstein-Barr virus DNA from two Epstein-Barr virus-infected cells. This highly sensitive analysis, however, could not detect cytomegalovirus or Epstein-Barr virus genomes in pancreases of recent-onset IDDM. A single copy human gene (HLA-DP) was amplified from all IDDM pancreases indicating that DNA amplification was performed without inhibition. We conclude that cytomegalovirus or Epstein-Barr virus genomes are unlikely to exist in pancreas biopsy specimens of recent-onset IDDM patients.

Adolescent↗

PubMed1995

Altered lipid composition and differential changes in activities of membrane-bound enzymes of erythrocytes in hepatic cirrhosis.

Lipid composition, fluidity, and Na+,K(+)-adenosine triphosphatase (ATPase), Mg(2+)-ATPase, and acetylcholinesterase (AChE) activities of erythrocyte membranes were examined in comparison to plasma lipid composition and lecithin:cholesterol acyltransferase (LCAT) activities in 39 patients with hepatic cirrhosis due to viral hepatitis (Child-Pugh class A, n = 12; class B, n = 13; and class C, n = 14). Plasma LCAT activities decreased and the plasma free-cholesterol to phospholipid molar ratio (C/PL) increased with progressive severity of hepatic cirrhosis. C/PL and fluorescence polarization (inverse of fluidity) of erythrocyte membranes also increased with disease progression (C/PL: Child-Pugh A, 0.911 +/- 0.010; B, 0.941 +/- 0.011; C, 0.979 +/- 0.028; and normal, 0.798 +/- 0.010; fluorescence polarization: Child-Pugh A, 0.348 +/- 0.002; B, 0.351 +/- 0.002; C, 0.355 +/- 0.002; and normal, 0.340 +/- 0.002). There was a correlation between C/PL and fluorescence polarization of erythrocyte membranes (r = .629, P < .001). Na+,K(+)-ATPase activity of erythrocyte membranes did not differ between cirrhotic patients and normal subjects. On the other hand, Mg(2+)-ATPase activity decreased in Child-Pugh C cirrhosis. AChE activity was decreased in Child-Pugh A cirrhosis, and decreased further in Child-Pugh B and C cirrhosis. AChE and Mg(2+)-ATPase activities correlated inversely with fluorescence polarization (r = -.652, P < .001 and r = -.381, P < .01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

PubMed1995

Expression studies of two vasopressin V2 receptor gene mutations, R202C and 804insG, in nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is a rare X-linked disorder associated with renal tubule resistance to arginine vasopressin (AVP). To understand the mechanisms of AVP resistance underlying this disorder, we have analyzed the vasopressin V2 receptor gene in two unrelated Japanese kindreds with NDI and expressed the mutants to characterize their functional properties. Direct sequencing revealed two V2 receptor gene mutations: a missense mutation from Arg202 to Cys in the third extracellular domain (R202C) and a single base insertion (G) in two consecutive GGG triplets (nucleotide 804 to 809) in the third cytoplasmic domain, resulting in a frame shift with premature termination at codon 258 (804insG). Transient expression study with COS-7 cells showed that R202C mutation reduced both binding affinity (15%) and capacity (30%), while 804insG mutation abolished binding ability. For further evaluation of the binding ability of the R202C mutant, we expressed the mutants in Chinese hamster ovary (CHO) cells. Although the mutant cell lines produced V2 receptor mRNA comparable levels to the wild-type receptor cell lines, R202C mutant cell lines had no binding ability. Our results suggest an introduction of a new cysteine residue in the extracellular domain and a receptor truncation removing one third of the carboxyl terminus could impair ligand binding activity of the V2 receptor through a post-transcriptional mechanism, thereby causing AVP resistance in the NDI patients.

Adult↗

PubMed1995

Monitoring urine oxygen tension during acute change in cardiac output in dogs.

To evaluate whether renal blood flow (RBF) can be monitored during acute change in cardiac index, ureter urine oxygen tension (PuO2) and bladder urine oxygen tension (PbO2) were measured in six mongrel dogs. PuO2, cardiac index, and RBF increased after dobutamine infusion and decreased after propranolol infusion. PuO2 had an excellent correlation with RBF (r = 0.94) and a fair correlation with cardiac index (r = 0.50) and mean blood pressure (r = 0.56); RBF had a fair correlation with mean blood pressure (r = 0.52, P < 0.05) but was not related to cardiac index. With multiple-regression analysis, PuO2 was found to be the significant factor related to RBF. PbO2 had a good correlation with PuO2 (r = 0.94) at control levels. Furthermore, when two dogs were added to evaluate relationships among PbO2, PuO2, and RBF, PbO2 had an excellent correlation with PuO2 (r = 0.92) and RBF (r = 0.91). These data indicate that PuO2 is a more sensitive predictor of RBF than cardiac index and mean blood pressure and that PbO2 can be a noninvasive indicator reflecting RBF during acute circulatory change in dogs.

Animals↗

PubMed1995

Effect of atrial and ventricular activation interval on hemodynamics during atrioventricular and ventriculoatrial pacing: determination from pressure-volume loops in dogs.

To determine the effect of relative timing of atrial and ventricular activation on hemodynamic indices, we evaluated pressure-volume loops during pacing at 160 beats/min with various atrioventricular intervals in dogs. End-systolic pressure, end-diastolic volume, and stroke volume were reduced during pacing at atrioventricular intervals of 0 ms, compared with 80 ms. These changes were more significant during pacing at atrioventricular intervals of -50 and -100 ms than at 0 ms. During tachycardia, the abnormal timing of atrial contraction leads to unfavorable hemodynamic change and the degree of the changes are determined by the ventriculoatrial activation interval.

Animals↗

PubMed1995

Clinical significance of S-T segment elevation in lead aVR in anterior myocardial infarction. Assessment by thallium-201 exercise scintigraphy.

The significance of exercise-induced S-T elevation in aVR was studied in 57 patients with recent anterior infarction and single-vessel disease. S-T elevation in aVR was found at peak exercise in 24 patients. Although the initial defect area was similar in the groups with and without S-T elevation in aVR, the redistribution area was larger in the former group (p < 0.01). When three electrocardiographic criteria were used in the multivariate analysis, S-T elevation in aVR was the significant variable related to redistribution in the anterior wall. Thus, S-T elevation in aVR may indicate ischemia of the anterior wall.

Adult↗

PubMed1995

Glucocorticoids regulate V1a vasopressin receptor expression by increasing mRNA stability in vascular smooth muscle cells.

Enhancement of vascular responsiveness is considered to be one of the major contributing factors observed in glucocorticoid-induced hypertension. We examined the effects of glucocorticoids on V1a arginine vasopressin receptor mRNA and protein levels in vascular smooth muscle cells. Dexamethasone (1 mumol/L) produced a 1.8-fold increase in V1a receptor density without changing its affinity. Steady-state values of V1a receptor mRNA, analyzed by Northern blotting, increased 2.7-fold after a 12-hour exposure to dexamethasone. This effect of dexamethasone was blocked by the glucocorticoid antagonist RU38486 and did not occur in the presence of the protein synthesis inhibitor cycloheximide. The V1a receptor gene transcription rate, determined by nuclear run-off assays, was unchanged in cells treated with dexamethasone for 12 hours. Dexamethasone increased the half-life of V1a receptor mRNA by 2.2-fold. These findings suggest that dexamethasone upregulates the expression of the V1a receptor by increasing mRNA stability rather than by gene transcription and that de novo protein synthesis is involved in this regulation.

Animals↗

PubMed1995

Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type 2 receptor gene mutations in unrelated X-linked NDI families, including R143P, delta V278, R202C, and 804insG. All these mutations reduced ligand binding activity to < 10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized (50-55 kD) and intracellular proteins (40 and 75 kD), similar to the wild-type AVPR2 clone, whereas the R143P and delta V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 804insG mutant cell produced no proteins despite an adequate mRNA level. Immunofluorescence staining confirmed that the R202C mutant reaches the cell surface, whereas the R143P and delta V278 mutants are retained within the cytoplasmic compartment. Thus, R202C, R143P/delta V278, and 804insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI. This phenotypic classification will help understanding of molecular pathophysiology of this disorder.

Amino Acid Sequence↗

PubMed1995

Bioactivity of thyrotropin (TSH) in patients with central hypothyroidism: comparison between in vivo 3,5,3'-triiodothyronine response to TSH and in vitro bioactivity of TSH.

To investigate the cause(s) of central hypothyroidism with normal or elevated TSH concentrations, we evaluated the bioactivity of serum TSH as well as pituitary and thyroid function. Seven hypothyroid patients had documented deficiencies of anterior pituitary hormones other than TSH. Basal TSH concentrations ranged from 2.2-14.8 microU/mL. Six patients had low T4 and free T4 concentrations; the remaining patient had a low free T4 and a low normal T4 level with an elevated TSH concentration of 14.4 microU/mL. The mean increment in TSH 30, 60, and 90 min after TRH administration (mean delta TSH) in these patients was 13.5 +/- 9.1 microU/mL (mean +/- SD), which was not significantly different from the value in controls (9.2 +/- 3.5 microU/mL). However, the ratio of the T3 increment at 120 min (delta T3) to mean delta TSH (delta T3/mean delta TSH) in patients was 53.9 +/- 29.3 ng/microU, significantly lower than the control value of 239.5 +/- 97.5 ng/microU (P < 0.01), suggesting that the thyroid response to endogenous TSH was blunted. The serum T4 concentration correlated with the mean delta TSH in these patients (r = 0.78; P < 0.05), suggesting that hypothyroidism is dependent on conserved pituitary function. The mean bioactivity to immunoreactivity ratio of basal TSH in patients was 0.97 +/- 0.27 and was not significantly different from the normal value of 1.05 +/- 0.22. One of the two patients with high basal TSH (> 10 microU/mL) had a ratio of 0.59, which is just below the mean +/- SD of normal subjects (0.61), suggesting that most patients had normal TSH bioactivity in vitro. Our findings suggest that in vivo bioactivity of TSH is decreased because of a pituitary disorder, but in vitro bioactivity of TSH is variable in patients with central hypothyroidism.

Adult↗

PubMed1995

Thyroid stimulating antibody in sera of Graves' ophthalmopathy patients as a possible marker for predicting the efficacy of methylprednisolone pulse therapy.

Nine patients with Graves' ophthalmopathy (GO) were treated with intravenous methylprednisolone pulse therapy and followed up by ophthalmological assessment, magnetic resonance imaging, and thyroid-associated autoantibody (thyroid stimulating antibody (TSAb), TSH binding inhibitor immunoglobulins (TBII), and anti-eye muscle antibody (EMAb)). Ophthalmological assessment was performed by the ophthalmopathy index (OI) which was made on the basis of the system recommended by the American Thyroid Association Committee. EMAb was expressed as the ratio of density of the 64 kDa band of eye muscle membrane to that of 92 kDa non-specific band found with all normal sera when assessed by western blotting. Five patients with mild ophthalmopathy (OI < 4) did not show progressive improvement in OI. Three of 4 patients with severe eye disease (OI > 4) showed a progressive and distinct improvement in OI. These 3 patients had high TSAb levels before methylprednisolone pulse therapy. One patient with severe ophthalmopathy did not respond to this pulse therapy; this patient's TSAb was negative. A significant positive correlation was observed between the activity of TSAb before treatment and the improvement in OI (delta OI) (r = 0.86, P < 0.01, n = 9). The relationship between delta OI and EMAb did not reach significance. These results suggest that TSAb in sera of GO patients can be a useful marker for predicting the efficacy of methylprednisolone pulse therapy.

Adolescent↗

PubMed1995

[Clinical significance of abnormal high signal intensity of left ventricular myocardium by gadolinium-diethylenetriaminepenta-acetic acid enhanced magnetic resonance imaging in hypertrophic cardiomyopathy].

The significance of abnormal high signal intensity observed in left ventricular myocardium by gadolinium-diethylenetriaminepenta-acetic acid (Gd-DTPA) enhanced magnetic resonance (MR) imaging in hypertrophic cardiomyopathy (HCM) patients was assessed by comparison with T1-weighted MR imaging, thallium-201 (201Tl) myocardial scintigraphy, radionuclide angiocardiography, M-mode echocardiography, electrocardiography, and chest radiography. The 16 patients were divided into three groups: 8 patients (group I) with abnormal high signal intensity before and after Gd-DTPA enhancement, 4 (group II) with abnormal high signal intensity only after enhancement and 4 (group III) without abnormal high signal intensity. Thallium-201 myocardial single photon emission computed tomography (SPECT) showed 4 of the 8 patients in group I, 3 of the 4 patients in group II and only 1 of the 4 patients in group III had abnormalities of regional 201Tl uptake in the left ventricular myocardium. No significant difference in left ventricular ejection fraction (LVEF) was seen between groups I, II, and III (64 +/- 13%, 67 +/- 17% and 71 +/- 7%, respectively) although three patients of group I had LVEF of less than 55%. Left ventricular peak filling rates (PFR) of groups I and II were significantly lower than that of group III (1.90 +/- 0.44, 2.41 +/- 0.43 and 3.37 +/- 0.48 EDV/sec, respectively). Group I had larger end-diastolic left ventricular dimension (LVDd), significantly larger end-systolic left ventricular dimension (LVDs), and smaller % fractional shortening (%FS) than group III (49 +/- 4 vs 42 +/- 6 mm, 31 +/- 5 vs 22 +/- 4 mm, and 38 +/- 8 vs 49 +/- 4%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1994

Clinical significance of right ventricular dilatation in patients with right ventricular infarction.

BACKGROUND: Right ventricular infarction can be accurately diagnosed by ST-segment elevation in the right precordial leads. However, the clinical outcome of right ventricular infarction encompasses a wide spectrum, ranging from no hemodynamic compromise to cardiogenic shock. The present study examined the clinical significance of echocardiographic right ventricular dilatation in patients with right ventricular infarction. METHODS: We studied 60 consecutive patients with ECG evidence of right ventricular infarction (at least 1 mm ST-segment elevation and QS or QR in V4R) after their first acute Q-wave inferior infarction. They had been admitted to the coronary care unit within 24 h of the onset of chest pain. The presence of right ventricular dilatation was diagnosed when the end-diastolic ratio between right and left ventricle was more than 0.5 on two-dimensional echocardiogram. RESULTS: Of the 60 patients with ECG evidence of right ventricular infarction, 29 had right ventricular dilatation (group 1) and 31 did not (group 2). We used four clinical variables in multivariate analysis to determine the significant factors related to right ventricular infarction. Mean right atrial pressure and number of left ventricular segments with advanced asynergy were found to be the important factors. Furthermore, a significantly higher incidence of major complications (cardiogenic shock and need for temporary pacing) was observed in group 1 than in group 2. Right ventricular dilatation was found to be the significant factor related to major complications. CONCLUSION: Echocardiographic right ventricular dilatation is an important non-invasive sign obtained on admission in patients with right ventricular infarction, because it is associated with larger left ventricular infarct size and increased risk of major complications.

Aged↗