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Biomedical subjects

S Satoh

Publications and source records attributed to S Satoh.

At least 19 recordsLinked to original sources
PubMed1999

Role of calcium channels in catecholamine secretion in the rat adrenal gland.

1. We elucidated the contribution of voltage-dependent Ca2+ channels to cholinergic control of catecholamine secretion in the isolated perfused rat adrenal gland. 2. Nifedipine (0.3-3 microM) inhibited increases in noradrenaline output induced by transmural electrical stimulation (1-10 Hz) and acetylcholine (6-200 microM), whereas it only slightly inhibited the adrenaline output responses. Nifedipine also inhibited the catecholamine output response induced by 1, 1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 microM) but not by methacholine (10-300 microM). 3. omega-Conotoxin MVIIC (10-1000 nM) inhibited the catecholamine output responses induced by electrical stimulation but not by acetylcholine, DMPP and methacholine. 4. omega-Conotoxin GVIA (50-500 nM) had no inhibitory effect on the catecholamine output responses. 5. These results suggest that L-type Ca2+ channels are responsible for adrenal catecholamine secretion mediated by nicotinic receptors but not by muscarinic receptors, and that their contribution to noradrenaline secretion may be greater than that to adrenaline secretion. P/Q-type Ca2+ channels may control the secretion at a presynaptic site.

Acetylcholine↗
PubMed1999

Phenotypic consequence of the gene abnormality in the platelet glycoprotein IX gene observed in a patient with Bernard-Soulier syndrome through mammalian cell expression system.

We previously reported a patient with Bernard-Soulier syndrome (BSS) who had a homozygous missense mutation in the GPIX gene Phe55 (TTT) to Ser (TCT) replacement in the leucine-rich motif (LRM) of the GPIX polypeptide, as a probable cause of BSS phenotype. To study the effect of this mutation on the surface expression of the GPIb/IX complex and GPIX, we introduced this mutation into the cDNA of GPIX by site-directed mutagenesis and performed in vitro transfection studies with plasmid for mutant GPIX and other plasmids for GPIb/IX complex. Mutant GPIX could not increase the surface expression of GPIb-alpha, but also surface expression of GPIX itself. Immunostaining of the CHO-K1 cells transfected with two plasmids for mutant GPIX and GPIb-beta showed that mutant GPIX was weakly but certainly detected in the cytoplasm of the transfected cells. These findings indicate that this substitution is responsible for BSS phenotype and that the LRM of GPIX is a critically important element for an efficient expression of the GPIb/IX complex. They also suggest that the failure of GPIb/IX complex expression can be caused by a mechanism other than synthetic defect of the mutant protein.

Animals↗
PubMed1999

Caspase-mediated cleavage of eukaryotic translation initiation factor subunit 2alpha.

Eukaryotic translation initiation factor 2alpha (eIF-2alpha), a target molecule of the interferon-inducible double-stranded-RNA-dependent protein kinase (PKR), was cleaved in apoptotic Saos-2 cells on treatment with poly(I).poly(C) or tumour necrosis factor alpha. This cleavage occurred with a time course similar to that of poly(ADP-ribose) polymerase, a well-known caspase substrate. In addition, eIF-2alpha was cleaved by recombinant active caspase-3 in vitro. By site-directed mutagenesis, the cleavage site was mapped to an Ala-Glu-Val-Asp(300) downward arrowGly(301) sequence located in the C-terminal portion of eIF-2alpha. PKR phosphorylates eIF-2alpha on Ser(51), resulting in the suppression of protein synthesis. PKR-mediated translational suppression was repressed when the C-terminally cleaved product of eIF-2alpha was overexpressed in Saos-2 cells, even though PKR can phosphorylate this cleaved product. These results suggest that caspase-3 or related protease(s) can modulate the efficiency of protein synthesis by cleaving the alpha subunit of eIF-2, a key component in the initiation of translation.

Apoptosis↗
PubMed1999

Evaluation of ammonia and lidocaine clearance, and galactose elimination capacity of xenoperfused pig livers using a pharmacokinetic analysis.

BACKGROUND: We introduced the pharmacokinetic method into the functional evaluation of xenogeneic extracorporeal liver perfusion as an artificial liver assist device, and examined the influence of xenogeneic humoral injury on the metabolic function of xenoperfused pig livers. METHODS: Isolated pig livers were perfused with fresh porcine blood (group 1; n=5) or fresh human blood (group 2; n=5) for 9 hr. Clearance (CL) of ammonia and lidocaine, and galactose elimination capacity (Vmax) were determined at three points during the perfusion using a one-compartment pharmacokinetic model. RESULTS: Concentrations of ammonia and lidocaine decreased exponentially and those of galactose decreased linearly after a bolus injection in both groups. A one-compartment model provided satisfactory curve fittings for these test substances. No decreases of ammonia CL, lidocaine CL, or galactose Vmax were observed until 9 hr in either group. No differences were observed between the two groups with respect to these metabolic functions. In group 1, only slight interlobular edema was observed at 9 hr. In group 2, membrane attack complex was diffusely deposited at 3 hr and severe interlobular damage was histologically observed at 9 hr, although hepatocellular damage was minimal even at 9 hr. Alpha glutathione S-transferase and mitochondrial aspartate aminotransferase were comparable between the two groups. CONCLUSIONS: Pharmacokinetic analysis allowed the evaluation of ammonia CL, lidocaine CL, and galactose Vmax of the perfused pig livers. Despite xenogeneic humoral injuries, the xenoperfused livers maintained these metabolic functions at the same levels as the alloperfused livers for 9 hr.

Ammonia↗
PubMed1999

An inherited prion disease with a PrP P105L mutation: clinicopathologic and PrP heterogeneity.

OBJECTIVE: To clarify a clinical and neuropathologic phenotype of an inherited prion disease associated with a missense mutation at codon 105 in the prion protein (PrP) gene that was originally described as a variant of Gerstmann-Sträussler-Scheinker disease demonstrating spastic paraparesis. METHODS: Two siblings from a Japanese family are described. PrP gene analyses, neuropathologic studies with immunohistochemistry, and Western blot analysis of the PrP were performed. RESULTS: Both patients showed a missense (proline-->leucine) mutation at codon 105 and a methionine/valine polymorphism at codon 129 of the PrP gene. Clinically, Patient 1 presented with progressive spastic paraparesis, ataxia, and dementia. Patient 2, the sister of Patient 1, showed prominent action myoclonus and dementia. Neuropathologically, multiple PrP-positive amyloid plaques and diffuse PrP deposition in the deep cortical layers were found in the cerebral cortex with primarily frontal dominant atrophy in both patients. Tau-positive pathologic structures including neurofibrillary tangles, neuropil threads, and dystrophic neurites around the plaques were abundant in the brain of Patient 2. In contrast, the tau pathology was scarce in Patient 1. Western blot analysis of the brain showed different patterns of detergent-insoluble PrP fragments between the patients. CONCLUSIONS: Despite the identical codon 105 mutation and codon 129 polymorphism of the PrP gene, remarkable clinical and neuropathologic differences, and PrP heterogeneity were present between the affected siblings. The phenotypic variability might be related to PrP heterogeneity.

Amino Acid Substitution↗
PubMed1999

Intrarenal angiotensin converting enzyme inhibition in spontaneously hypertensive rats.

We examined the hypotensive effect of enalapril in relation to the local renin-angiotensin system of the kidney in spontaneously hypertensive rats (SHR). Oral administration of enalapril for 7 days decreased mean arterial blood pressure and renal tissue angiotensin II concentration without affecting plasma angiotensin II concentration in SHR. The enalapril treatment did not affect maximum binding of angiotensin II to renal tubules and glomeruli in SHR. In normotensive Wistar-Kyoto rats, no significant changes in mean arterial blood pressure, renal and plasma angiotensin levels were observed with enalapril treatment. Direct infusion of enalapril into the renal medullary interstitium decreased mean arterial blood pressure in association with the reduction of renal tissue angiotensin II concentration without changes in plasma angiotensin II concentration in SHR. These observations suggest that the inhibition of angiotensin conversion in the kidney is important for the hypotensive action of enalapril.

Administration, Oral↗
PubMed1999

Effect of transforming RNA on the synthesis of a protein with a secretory signal sequence in vitro.

U5 small nuclear RNA itself can act as a clastogenic and transforming agent when transfected into cells. In the previous work, the 3' half of the U5 small nuclear RNA first stem structure (designated RNA3S) was capable of driving normal cells into tumorigenic cells when expressed with a poly(A) tail (RNA3S+). This transformation critically depended upon the polypurine sequence GGAGAGGAA in RNA3S+. In this work, we first examined the pre-beta-lactamase and luciferase (model secretory and nonsecretory proteins) translation with the in vitro synthesized RNA3S in rabbit reticulocyte lysate. The capped RNA3S with a poly(A) tail suppressed the translation. In addition, the polypurine sequence played a crucial role in affecting the secretory protein synthesis, indicating a primary action of RNA3S+. Further studies revealed that the oligodeoxynucleotides, corresponding to the polypurine and its antisense sequences, directly contacted 28 S rRNA in ribosome and 7SL RNA in signal recognition particle, respectively, and differentially affected the nascent chain elongation of secretory protein synthesis. These results suggest that RNA3S+ blocks a physiological regulatory function played by signal recognition particle and the ribosome in the secretory protein synthesis and support the idea that the transformation might result from a repressed cellular activity.

Animals↗
PubMed1999

Effects of NKH477 on renal functions and cyclic AMP production in anesthetized dogs.

The present study was undertaken to evaluate the effects of an adenylate cyclase activator N,N-dimethyl-beta-alanine[3R-(3alpha, 4alphabeta, 5beta, 6beta, 6aalpha, 10alpha, 10abeta, 10balpha)]-5(acetyloxy)-3-ethenyldodecahydro-10, 10b-dihydroxy-3, 4a, 7, 7, 10a-pentamethyl-1-oxo-1H-naphtho [2,1-b] pyran-6-yl ester hydrochloride (NKH477) on renal functions and cyclic AMP production in the dog kidney. The intrarenal arterial infusion of NKH477 (30, 100 and 300 ng kg(-1) min(-1)) increased renal blood flow, glomerular filtration rate, urine flow rate, urinary Na+ and cyclic AMP excretion, fractional Na+ excretion and arterial and renal venous plasma cyclic AMP concentrations in a dose-dependent manner. The intrarenal arterial infusion of rolipram (0.3 microg kg(-1) min(-1)), a cyclic AMP-specific phosphodiesterase inhibitor, also caused the same renal responses as NKH477. The increasing effects of NKH477 on renal blood flow, fractional Na+ excretion and renal venous plasma cyclic AMP concentration were facilitated in the presence of rolipram. NKH477 reduced glomerular filtration rate and filtration fraction in the presence of rolipram. The increasing effects of NKH477 on urine flow rate and urinary Na+ excretion were not affected by rolipram. The present results suggest that NKH477 increases glomerular filtration and suppresses tubular sodium reabsorption through activation of cyclic AMP production, and thereby induces natriuresis. The results also demonstrate that renal cyclic AMP level during the activation of adenylate cyclase is regulated by phosphodiesterase IV in both the vascular and tubular sites.

Anesthesia↗
PubMed1999

Phosphorylation of nonstructural 5A protein of hepatitis C virus: HCV group-specific hyperphosphorylation.

We previously showed that two proteins with molecular weights of 56 and 58 kDa are produced from nonstructural protein 5A (NS5A) derived from hepatitis C virus (HCV)-1b genotype. The 56-kDa protein is phosphorylated at serine residues in NS5A, including those located in the C-terminal region of NS5A, while the 58-kDa protein, the hyperphosphorylated form of the 56-kDa protein, is phosphorylated at serine residues in the central region. This hyperphosphorylation is dependent on the presence of HCV NS4A protein. To clarify whether NS4A-dependent phosphorylation also occurs in other HCV genotypes, phosphorylation of NS5A was analyzed by two-dimensional gel electrophoresis. Here, we report that NS5A from the HCV-2a genotype was phosphorylated. However, hyperphosphorylation of NS5A occurs in the HCV-1b genotype but not in the -2a genotype. This result suggests that modification of NS5A phosphorylation reflects the virological features of HCV and that there are physiological differences in the roles of differently phosphorylated NS5A between HCV genotypes.

Alkaline Phosphatase↗
PubMed1999

Inhibition of IRS-1 phosphorylation and the alterations of GLUT4 in isolated adipocytes from cachectic tumor-bearing rats.

Cellular and molecular mechanisms of insulin resistance in isolated adipocytes from methylcholanthrene-induced sarcoma-bearing rats were investigated by measuring 3-O-[14C]methyl glucose transport activity, glucose transporter-4 (GLUT4) protein in both plasma membrane and low-density microsomes, and insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate-1 (IRS-1). Compared to both pair-fed and freely fed controls, tumor-bearing rats (TBR) had a decreased insulin-stimulated glucose transport activity with a lower Vmax and a higher EC50. GLUT4 protein in low-density microsomes from adipocytes maintained at the basal state was less in TBR than in controls. In insulin-stimulated adipocytes, GLUT4 protein in plasma membranes was also less in tumor-bearing rats than in controls. Insulin-induced tyrosine phosphorylation of IRS-1 was less in TBR than controls, but that of the IR was similar among the three groups. These data suggest that the insulin resistance seen in adipose cells of these tumor-bearing rats was caused in part by a decreased amount of GLUT4 protein in both basal and insulin-stimulated states resulting from the selective inhibition of insulin-stimulated phosphorylation of IRS-1.

Adipocytes↗
PubMed1999

The key amino acid residue of prostaglandin EP3 receptor for governing G protein association and activation steps.

To assess the role of the conserved DPWXY motif of the seventh transmembrane domain in prostanoid receptor-mediated G protein activation, we have mutated the negatively charged Asp-318 in this motif of the Gi-coupled mouse prostaglandin EP3 receptor to uncharged but polar Asn (EP3-D318N) and to the non-polar Leu (EP3-D318L). The EP3 agonist and antagonist showed similar binding affinities for the wild-type and two mutant receptors. The wild-type and EP3-D318N receptors but not EP3-D318L receptor associated with Gi in guanine nucleotide- and pertussis toxin-sensitive manners. On the other hand, the wild-type receptor but not two mutant receptors had the ability to stimulate GTPase activity and to inhibit the adenylate cyclase. These findings demonstrate that the chemical nature of the amino acid residue at position 318 of the seventh transmembrane domain of the EP3 receptor dissociates the step of Gi association from that of subsequent Gi activation in the process of the EP3 receptor-Gi coupling.

Amino Acids↗
PubMed1999

Serologically silent hepatitis B virus coinfection in patients with hepatitis C virus-associated chronic liver disease: clinical and virological significance.

Frequent coinfection of surface antigen-negative hepatitis B virus (silent HBV) in hepatitis C virus (HCV)-associated chronic liver disease (CLD) has been reported. The clinical and virological significance of silent HBV infection was investigated in 65 patients with HCV-associated CLD who subsequently received interferon (IFN) therapy. HBV DNA was detected in 34 (52.3%) patients by a nested polymerase chain reaction (PCR). Virologically, all of the 34 patients were found to have HBV with an eight-nucleotide deletion in the core promoter. Coinfection of silent HBV was more frequent with HCV genotype 1b than in 2a (64.3% vs. 28.6%, P<.01). With HCV genotype 1b, the serum RNA level was significantly higher (> or =10(6) copies per milliliter vs. < or =10(5) copies per milliliter) in patients with silent HBV than those without coinfection (P<.01). Clinically, silent HBV was associated with a higher level of serum alanine aminotransferase (158.5+/-104.8 vs. 121.8+/-78.6 IU/I; mean +/- SD) and a greater histological activity of hepatitis as evaluated by histological activity index score (9.4+/-3.8 vs. 8.6+/-4.5; mean +/- SD), although it was not statistically significant. Silent HBV was also associated with poor efficacy of IFN therapy (P<.01). The results suggest that silent HBV has some promoting effect for HCV replication, at least for HCV genotype 1b, and may affect the histological activity of hepatitis and IFN response in HCV-associated CLD.

Adult↗
PubMed1999

Circulating prostate-specific antigen mRNA during radical prostatectomy in patients with localized prostate cancer: with special reference to neoadjuvant hormonal therapy.

To determine the potential risk of hematogenous dissemination of prostate cancer cells during radical prostatectomy (RP), we investigated the pre- and intraoperative circulating prostate-specific antigen (PSA) mRNA in patients with clinically localized prostate cancer, with special reference to neoadjuvant hormonal therapy (NHT). Using a nested reverse transcriptase (RT) polymerase reaction (PCR) assay, PSA mRNA in the peripheral blood was evaluated pre- and postoperatively in a total of 23 patients, 10 of whom received NHT with antiandrogens. The RT-PCR assay employed detected one LNCaP cell in 10(7) mononuclear blood cells, and showed no positive signal in the blood samples from all 15 healthy controls. Pre- and intraoperative circulating PSA mRNA was positive in 11 (48%) and 18 patients (78%), respectively. All 11 patients with positive preoperative PSA mRNA continued to be positive during RP, and seven (58%) of 12 patients with negative preoperative PSA mRNA had a positive conversion. Although the patients' ages, preoperative serum PSA values and clinical or pathological stages were not associated with the pre- and intraoperative PSA mRNA results, the NHT group showed a significantly lower incidence of preoperative PSA mRNA positivity (2/10) than the group receiving RP alone (9/13) (20% vs 69%, P = 0.036). NHT, however, showed no suppressive effect on either intraoperative positivity or positive conversion of circulating PSA mRNA. The present study suggests that a substantial number of patients receiving RP are at risk of hematogenous dissemination, and NHT with antiandrogens has a minimal or no suppressive effect on the circulating PSA mRNA during surgical manipulation of the prostate. Because the clinical significance of circulating cancer cells remains to be determined, long-term follow-up in association with the circulating cancer cells assessed by the RT-PCR is essential in order to establish the role of molecular staging as well as NHT.

Aged↗
PubMed1999

Adrenoceptor-mediated regulation of myofibrillar Ca2+ sensitivity through the GTP-binding protein-related mechanisms: tension recording in beta-escin-skinned single rat cardiac cells with preserved receptor functions.

To investigate the mechanisms of receptor-mediated regulation of heart muscle contraction, we developed a tension-recording system using beta-escin-skinned single cardiac cells of rats and studied the effects of agonists on myofibrillar Ca2+ sensitivity and Ca2+ release from the sarcoplasmic reticulum (SR). In pCa/tension relations, 1 microM isoproterenol plus 100 microM guanosine 5'-triphosphate (GTP) decreased the myofibrillar Ca2+ sensitivity (pCa50, the [Ca2+] required for half-maximal tension, as an indicator of the sensitivity; from 6.07 to 5.92); this effect was blocked by 1 microM metoprolol or 1 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). Phenylephrine (10 microM) plus 100 microM GTP increased the Ca2+ sensitivity (pCa50; from 6.12 to 6. 28), and this effect was blocked by 1 microM phentolamine or 1 mM GDPbetaS. After Ca2+ loading into the SR, 10 microM phenylephrine plus 100 microM GTP in a low-ethylene- glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 0. 1 mM) relaxing solution induced oscillatory contractions that were attenuated by either 1 microM phentolamine or pre-treatment with 10 microM inositol 1,4,5-trisphosphate. Our results demonstrate that beta1-adrenergic stimulation decreases myofibrillar Ca2+ sensitivity and that alpha1-adrenergic stimulation both increases the Ca2+ sensitivity and activates Ca2+ release from the agonist-sensitive SR through GTP-binding protein-related mechanisms.

Adrenergic Agonists↗
PubMed1999

Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P3) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

Animals↗
PubMed1999

Effects of GABA on noradrenaline release and vasoconstriction induced by renal nerve stimulation in isolated perfused rat kidney.

We examined effects of gamma-aminobutyric acid (GABA) on vasoconstriction and noradrenaline (NA) release induced by electrical renal nerve stimulation (RNS) in the isolated pump-perfused rat kidney. RNS (1 and 2 Hz for 2.5 min each, 0.5-ms duration, supramaximal voltage) increased renal perfusion pressure (PP) and renal NA efflux. GABA (3, 10 and 100 microM) attenuated the RNS-induced increases in PP by 10-40% (P<0.01) and NA efflux by 10-30% (P<0.01). GABA did not affect exogenous NA (40 and 60 nM)-induced increases in PP. The selective GABA(B) agonist baclofen (3, 10 and 100 microM) also attenuated the RNS-induced increases in PP and NA efflux, whereas the RNS-induced responses were relatively resistant to the selective GABA(A) agonist muscimol (3, 10 and 100 microM). The selective GABA(B) antagonist 2-hydroxysaclofen (50 microM), but not the selective GABA(A) antagonist bicuculline (50 microM), abolished the inhibitory effects of GABA (10 microM) on the RNS-induced responses. The selective alpha2-adrenoceptor antagonist rauwolscine (10 nM) enhanced the RNS-induced responses. GABA (3, 10 and 100 microM) potently attenuated the RNS-induced increases in PP by 40-60% (P<0.01) and NA efflux by 20-50% (P<0.01) in the presence of rauwolscine. Prazosin (10 and 30 nM) suppressed the RNS-induced increases in PP by about 70-80%. Neither rauwolscine (10 nM) nor GABA (10 microM) suppressed the residual prazosin-resistant PP response. These results suggest that GABA suppresses sympathetic neurotransmitter release via presynaptic GABA(B) receptors, and thereby attenuates adrenergically induced vasoconstriction in the rat kidney.

Adrenergic alpha-2 Receptor Antagonists↗
PubMed1999

In vivo targeted gene transfer into liver cells mediated by a novel galactosyl-D-lysine/D-serine copolymer.

A novel synthetic polypeptide designed as a DNA binding-molecule for liver-specific, receptor-mediated, gene transfer was used to selectively introduce reporter genes into liver cells in the form of plasmid DNA-ligand complexes. The polypeptide was a D-lysine/D-serine copolymer (Lys/Ser = 33/36 or 53/60) modified with a polyethylene glycol 5000 at the carboxyl-terminus (PLSP). In addition, the lysine epsilon-amino groups were covalently bound to galactose (galactosyl-PLSP), a ligand for the asialoglycoprotein receptor on hepatocytes. Solutions of DNA-ligand complex were prepared by adding solutions of DNA and galactosyl-PLSP in a mixing ratio of DNA:galactosyl-PLSP = 1:3 (w/w). Following injection of the DNA-ligand complex into mice via the tail vein, high levels of luciferase and enhanced green fluorescent protein, which were encoded by the reporter genes, were observed in liver. In contrast, luciferase activity in kidney, spleen, lung and heart was negligible. The high levels of gene expression obtained with DNA/galactosyl-PLSP complexes were achieved without partial hepatectomy or administration of lysosomotrophic agents. Thus, the synthetic Lys/Ser copolymer used in the present study appears to be a promising new tool which enhances the efficacy of receptor-mediated gene transfer into hepatocytes and which may provide another step toward the clinical practice of organ-specific gene therapy.

Animals↗