[On the mechanism of beta-adrenergic activation of cardiac outward currents--chloride and delayed rectifier potassium conductance].
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Action Potentials↗
Biomedical subjects
M Horie
Publications and source records attributed to M Horie.
Effect of a novel squalene epoxidase inhibitor, NB-598, on the regulation of cholesterol metabolism in Hep G2 cells.
We have reported previously that NB-598 competitively inhibits human squalene epoxidase and strongly inhibits cholesterol synthesis from [14C]acetate in cultured cells. Furthermore, multiple oral administration of NB-598 decreased serum cholesterol levels in dogs (Horie, M., Tsuchiya, Y., Hayashi, M., Iida, Y., Iwasawa, Y., Nagata, Y., Sawasaki, Y., Fukuzumi, H., Kitani, K., and Kamei, T. (1990) J. Biol. Chem. 265, 18075-18078). In the present study, the effects of NB-598 on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and low-density-lipoprotein (LDL) receptor were examined using a human hepatoma cell line Hep G2. Incubation of Hep G2 cells with NB-598 for 18 h increased HMG-CoA reductase activity in a dose-dependent manner. However, the increase in activity induced by NB-598 was lower than that induced by L-654,969 (a potent HMG-CoA reductase inhibitor), although NB-598 inhibited cholesterol synthesis more potently than L-654,969. On the other hand, HMG-CoA reductase mRNA was increased to the same extent by both inhibitors. These results demonstrate that NB-598 does not inhibit the synthesis of non-sterol derivative(s) of mevalonate, which regulate HMG-CoA reductase activity at the post-transcriptional level. NB-598 increased the binding of 125I-LDL to Hep G2 cells. LDL receptor mRNA was also induced by NB-598. In the presence of LDL or cycloheximide, NB-598 did not increase LDL receptor activity. These results demonstrate that the induction of LDL receptor activity by NB-598 is due to increases in mRNA and protein through the inhibition of cholesterol synthesis at the squalene epoxidase step. From these observations, squalene epoxidase inhibitor is expected to be highly effective in the treatment of hypercholesterolemia and also is very useful as a research tool for studying the regulation of cholesterol metabolism.
Acetates↗
Effects of YM264, a novel PAF antagonist, on puromycin aminonucleoside-induced nephropathy in the rat.
We investigated the effects of YM264, WEB2086, methylprednisolone and ticlopidine on puromycin-induced nephropathy in the rat. Puromycin produces marked proteinuria, hypercholesterolemia, and hypoalbuminemia. The structurally differing PAF antagonists YM264 and WEB2086 inhibited proteinuria and improved hypercholesterolemia and hypoalbuminemia. Methylprednisolone also exhibited a beneficial effect on these variables. However, ticlopidine, a platelet inhibitor, showed no inhibitory effect on nephropathy. These results indicate that PAF may play a major role in puromycin-induced nephropathy in the rat, and that PAF antagonists may prove of therapeutic value in the treatment of nephropathy in humans.
Animals↗
Simultaneous determination of residual synthetic antibacterials in fish by high-performance liquid chromatography.
A simple and rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of sulphamonomethoxine (SMMX), sulphadimethoxine (SDMX), sulphisozole (SIZ), nalidixic acid (NA), oxolinic acid (OXA), piromidic acid (PMA), furazolidone (FZ) and sodium nifurstyrenate (NFSA) in cultured fish was developed. The drugs were extracted with 0.2% metaphosphoric acid-methanol (6:4), followed by a Bond Elut C18 clean-up procedure. The HPLC separation was carried out on an Inertsil ODS column (150 x 4.6 mm I.D.) using 5 mM aqueous oxalic acid-acetonitrile (55:45) as the mobile phase with detection at 265 nm (0.04 a.u.f.s.). The calibration graphs were rectilinear from 1 to 20 ng for OXA, from 2 to 50 ng for SMMX, SDMX, SIZ, NA, PMA and FZ and from 5 to 100 ng for NFSA. The recoveries of each drug added to fish were 65.0-89.5%. The detection limits were 0.02 micrograms/g for OXA, 0.05 micrograms/g for SMMX, SDMX, SIZ, NA, PMA and FZ and 0.1 micrograms/g for NFSA.
Anti-Infective Agents↗
Analysis of Marek's disease tumor-associated surface antigen on MDCC-MSB1-clo.18 cells.
The physico-chemical nature of Marek's disease tumor-associated surface antigen (MATSA) on Marek's disease (MD) lymphoblastoid tumor cell line (MDCC-MSB1-clo.18) was examined by the cellular enzyme-linked immunosorbent assay (CELISA) and the sandwich enzyme-linked immunosorbent assay (ELISA) using an anti-MATSA immune serum or a monoclonal antibody (MAb) 2B9 developed against MATSA. Our results indicate that MATSA is a glycoprotein and 2B9 recognizes an antigenic site in the protein moiety of MATSA. MATSA was solubilized from MSB1-clo.18 cells by treatment with 0.5% Nonidet P-40, and purified by affinity chromatography coupling with 2B9 and further by ion exchange chromatography on diethylaminoethylcellulose (IECD). MATSA was eluted with 0.2 to 0.3 M KCl in IECD and the purity of MATSA was increased about 2,500-fold. The purified MATSA was shown to have a molecular weight (Mr) of 70,000 by SDS-PAGE. The reactivity of purified MATSA with anti-thymus cell serum was examined. MATSA was detectable by anti-thymus cell serum, although 2B9, which was used to purify MATSA from MSB1-clo.18 cells, was not reactive to cells prepared from the thymus. However, MATSA was no longer detectable after the absorption of anti-thymus cell serum by chicken bursa cells. The absorption of anti-thymus cell serum by chicken red blood cells (RBC) had no effect on the reactivity against MATSA. These results suggest that MATSA may be a lymphocyte-specific antigen modified during leukemogenesis by Marek's disease virus (MDV).
Animals↗
A role for the interferon response DNA sequence in directing transcription of the T18d Tla gene.
T18d of BALB/c mice is a member of the Tla category of class I genes of the major histocompatibility complex of the mouse and is highly restricted in expression. Deletion analysis implies that an element essential to T18d expression resides within the region -4 to +54. The homologous region of T3d, a Tla gene which normally is not expressed in BALB/c mice, also has promoter activity. Thus the expressibility of T18d vs T3d is unlikely to be due to sequence differences in this region. A DNA-binding protein, factor VI, was found to bind to the region -33 to +54. DNase I footprinting analysis indicated that the DNA fragment 5'-ACTATAGTTTCACTTTTT-3' (+3 to +20) was protected by factor VI. This region includes the interferon response sequence (IRS). Homologous DNA segments of other class I genes, Ld and Dd, competed for factor VI in DNA-protein binding assay with lower affinity as compared with T18d. In mutation analysis, the 3' portion of the IRS is more important than the 5' portion with respect to binding affinity of factor VI and to transcriptional activity in transfected cells. This result signifies a role of IRS in T18d transcription and suggests that the mechanism of T18d transcription might be unusual.
Animals↗
Properties of the promoter region of the T18d (T13c) Tla gene.
The T18d (formerly T13c) gene of BALB/c mice belongs to the category of Tla genes which is expressed by both thymocytes and TL+ T-cell leukemias. To elucidate the regulation of T18d, different restriction fragments of the 5' flanking region between -457 and +146 were linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into TL+ and TL- cells. By comparison of transiently expressed CAT activity among cells transfected with different CAT constructs, the results suggest that determination of TL+ vs TL- phenotypes is located within the region -105 to -33, and that an element essential to T18d expression resides within the region -33 to +54. Putative DNA-binding factors characterizing particular cell types and displaying selective affinity for particular T18d restriction fragments were identified by electrophoretic mobility shift assays with nuclear extracts (NEs). Two factors (or complexes) which bound specifically to the T18d fragment -105 to -33 were expressed preferentially in TL+ cells and thus may be involved in determining the tissue-selective expression of T18d. The close proximity of negative and positive cis-acting elements within the promoter region is consistent with regulation of T18d gene expression by a variety of trans-acting factors whose production is attuned to development and differentiation. The data provided may serve as a guide to study the regulation of other categories of Tla genes that are normally silent in thymocytes but may become expressed by leukemia cells.
Animals↗
Hypolipidemic effects of NB-598 in dogs.
NB-598, a new inhibitor of mammalian squalene epoxidase, was found to be a potent inhibitor of microsomal squalene epoxidase from dog liver. Hypolipidemic effects of NB-598 were compared with those of simvastatin (MK-733, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) in dogs. NB-598 was found to decrease serum total cholesterol levels and increase serum squalene levels in a dose-dependent manner. MK-733 decreased serum total cholesterol and squalene levels. Both NB-598 and MK-733 decreased all classes of lipoprotein cholesterol, and they decreased low density lipoprotein cholesterol most potently. Both drugs decreased phospholipid levels in parallel with cholesterol levels. NB-598 also decreased triacylglycerol levels. After termination of drug administration, these levels returned to the control levels. The potency of NB-598 is thought to be as great or greater than that of MK-733. Moreover, NB-598 increased squalene concentrations in the feces and gallbladder bile, but it did not affect neutral sterol and bile acid concentrations. NB-598 did not affect the lithogenic index.
Animals↗
Mechanisms of endothelin-1 mRNA and peptides induction by TGF-beta and TPA in MDCK cells.
Mechanisms of prepro-ET-1 mRNA expression and mature endothelin-1 (ET-1) peptide secretion in MDCK cells (dog collecting duct origin) were investigated. MDCK cells constitutively expressed prepro-ET-1 mRNA (approximately 2.3 kb). TGF-beta time-dependently increased prepro-ET-1 mRNA levels between 30 min and 6 h. Induction of the mRNA 6 h following TGF-beta addition was dose-dependent with a half-maximal concentration of 10 pM. The half-life of prepro-ET-1 mRNA was 15 min in controls when the cells were treated with 10 micrograms/ml of actinomycin D, whereas it was extended to 30-45 min by TGF-beta treatment. Prepro-ET-1 mRNA was rapidly (15-30 min) induced by 0.5 microM TPA, one of the phorbol esters, but downregulated below baseline after 1 h. Our data show that MDCK cells constitutively secrete ET-1 and increase its production in response to TGF-beta. An axis of TGF-beta-ET-1-collecting duct may play an important role in regulation of electrolyte transport and cell growth of the renal tubules.
Animals↗
Polarized secretion of endothelin-1 and big ET-1 in MDCK cells is inhibited by cell Na+ flux and disrupted by NH4Cl.
We recently found that TGF-beta increases ET-1 secretion in MDCK, a renal tubular cell line. The secretion of ET-1, by a confluent monolayer of MDCK cells grown on a filter, to the basolateral side of the epithelium was two times greater than that to the apical side. However, TGF-beta-increased ET-1 and big ET-1 secretion were exclusively released to the basolateral side. We investigated the mechanism of polarized secretion of ET-1 and big ET-1 in MDCK cells. After 24 h of incubation with 80 pM TGF-beta, basolateral secretion of both ET-1 and big ET-1 increased two to threefold without a significant increase on the apical side. TGF-beta-stimulated ET-1 and big ET-1 secretion in the basolateral bath were inhibited when 10 microM amiloride (Na+ channel blocker) or 1 microM ouabain (Na+, K(+)-ATPase inhibitor) was added for 3 h. Polarized secretion of ET-1 and big ET-1 was not affected. In contrast, 10 mM NH4Cl or 0.2 mM chloroquine (both lysosomal function inhibitors) reduced TGF-beta-stimulated ET-1 secretion in the basolateral bath whereas big ET-1 secretion in the apical bath increased two times. Thus, the polarity of big ET-1 secretion was reversed. In addition, the conversion rate from big ET-1 to ET-1 was significantly decreased from 80 to 55% by inhibiting lysosomal function. Prepro-ET-1 mRNAs 3 h after these perturbations were virtually unaltered. Our data show that ET-1 and big ET-1 secretion may be regulated by cell Na+ flux and that lysosomal functions may play some roles in the conversion of big ET-1 to mature ET-1 and in polarized secretion of big ET-1. Translational or posttranslational regulation may play an additional role in ET-1 secretion as well as the mRNA level.
Ammonium Chloride↗
Magnetic resonance imaging in children with spastic diplegia: correlation with the severity of their motor and mental abnormality.
Magnetic resonance imaging (MRI) findings for 34 children with spastic diplegia, examined between two and 10 years of age, were analysed. Dilatation of the trigone, atrophy of the peritrigonal white matter and prominent deep cortical sulci were seen. On T2-weighted images, periventricular high-intensity areas in the white matter adjacent to the trigones and bodies of the lateral ventricles were seen in many children. These MRI features may reflect the pathological changes of periventricular leukomalacia in children with spastic diplegia. Among the MRI findings, only the amount of white matter correlated with severity of disability: white matter reduction corresponded to the more severe motor disabilities.
Brain↗
Nicorandil reduces the basal level of cytosolic free calcium in single guinea pig ventricular myocytes.
Using fluorescent Ca2+ indicator fura-2 and whole-cell patch-clamp techniques, we examined the effect of 2-nicotinamidoethyl nitrate (nicorandil) on the intracellular free Ca2+ concentration ([Ca2+]i) and electrical properties in single guinea pig ventricular myocytes. Nicorandil (10 nM approximately 1 mM) reduced the resting level [Ca2+]i monitored as fura-2 fluorescence ratio in a concentration-dependent manner. Dibutyryl guanosine 3':5'-cyclic monophosphate (cyclic GMP), a membrane permeable cyclic GMP analogue, mimicked the nicorandil action. Neither application of caffeine (10 mM) nor deprivation of extracellular Na+ ions could prevent the nicorandil action on [Ca2+]i. In contrast, the nicorandil effect was virtually blocked by sodium orthovanadate (40 microM), a Ca2+ pumping ATPase inhibitor. During electrophysiological experiments, nicorandil shortened action potential durations (205 +/- 80 ms to 153 +/- 76 ms) by increasing a glibenclamide-sensitive outward K+ conductance. However, the drug produced little hyperpolarization (approximately 2 mV) because the resting potential of ventricular myocytes was close to the K+ equilibrium potential. The involvement of voltage-dependent Ca-channel current and Na-Ca exchanger was considered to be minimal under physiological conditions. It is thus concluded that nicorandil decreases basal [Ca2+]i via cyclic GMP-mediated activation of the plasma membrane Ca2+ pump in guinea pig ventricular myocytes.
Animals↗
[Metabolic fate of bufalin in rats].
In an attempt to evaluate the metabolic fate of "Kyushin", which is a traditional medicine containing Toad venom, a 3H-labeled compound of bufalin, which is one of the main active components, has been synthesized. The metabolic fate of the 3H-bufalin was studied after its single and repeated oral administration in rats. After a single administration of the 3H-bufalin (20 micrograms/kg), the radioactivity in the blood reached a maximum level at 15 min. The radioactivity in the blood declined in a triphasic manner with half-life times of 18 min, 2.6 and 86 h. Within 24 h after the single administration, the excretion of the radioactivity into the urine and feces amounted to 1.3% and 81% of the administered dose, respectively. Tissue radioactivity was higher in the stomach, small intestine, liver, lung, kidney and pancreas, while the radioactivity in other tissues was lower than that in blood. Radioactivity disappeared rapidly from any tissues. In case of repeated administration for 14 d, the disposition of radioactivity was almost same as the result after a single dosing, and radioactivity scarcely remained in any tissues after the last administration.
Administration, Oral↗
[Metabolic fate of bufalin and cinobufagin].
In the course of study of the metabolic fate of "Kyushin", a traditional medicine containing toad venom, the metabolic fates of bufalin and cinobufagin, main constituents of toad venom, have been studied. Six metabolites were detected in the extracts from incubation mixture of rat liver slice with bufalin, and one main metabolite shown by mass spectroscopy and high performance liquid chromatography (HPLC) to be 3 alpha-bufalin. Serum levels of bufalin and 3 alpha-bufalin were determined by HPLC after oral administration of 2000 micrograms/kg of bufalin, and both compounds appeared in the rat serum. On the other hand, only 3 alpha-bufalin appeared after administration of 20 or 200 micrograms/kg. 3 alpha-Bufalin levels increased dose dependently. Serum levels of cinobufagin and its metabolites and digitoxin were compared after repeated intravenous administration (5 h interval) of cinobufagin or digitoxin. Although digitoxin was accumulated in the rat serum, cinobufagin and its metabolites were not. Inhibitory activities of metabolites of bufalin and cinobufagin on (Na+ + K+)-adenosine triphosphatase were less than those of original compounds.
Administration, Oral↗
NB-598: a potent competitive inhibitor of squalene epoxidase.
NB-598, (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bith iophen-5-yl)methoxy]benzene-methanamine, was found to inhibit human microsomal squalene epoxidase (from Hep G2 cells) in a competitive manner. NB-598 inhibited cholesterol synthesis from [14C]acetate dose dependently in Hep G2 cells and increased the intracellular radioactivity of squalene. A single oral administration of NB-598 inhibited cholesterol synthesis from [14C]acetate in rats. Moreover, multiple oral administration of NB-598 to dogs decreased serum total and low density lipoprotein cholesterol levels and increased serum squalene levels. After termination of treatment, the reduced serum cholesterol and increased squalene levels returned to their control values.
Animals↗
Atrial natriuretic peptide reduces the basal level of cytosolic free Ca2+ in guinea pig cardiac myocytes.
The cytosolic free Ca2+ concentration ([Ca2+]i) was monitored in quiescent atrial and ventricular myocytes isolated from guinea-pig hearts by the fura-2 fluorescence ratio technique. Recombinant human atrial natriuretic peptide (ANP) was found to reduce their basal [Ca2+]i level in a dose-dependent manner. Dibutyryl-cGMP mimicked the effect of ANP. Neither the prior application of caffeine nor removal of extracellular Na+ impaired the ANP effect. ANP had no inhibitory effect on voltage-gated Ca2+ currents measured by a whole-cell patch clamp technique. The ANP-induced [Ca2+]i decrease was abolished by orthovanadate. Thus, it is concluded that ANP reduces the basal [Ca2+]i presumably through the cGMP-mediated activation of the plasma membrane Ca2(+)-pump in cardiac myocytes.
Animals↗
Identification and determination of sulphamethazine and N4-acetylsulphamethazine in meat by high-performance liquid chromatography with photodiode-array detection.
A simple and selective method for the determination of sulphamethazine (SMT) and its metabolite, N4-acetylsulphamethazine (N4-AcSMT), in meat by high-performance liquid chromatography (HPLC) with photodiode-array detection was developed. The drugs were extracted from meat with 0.2% metaphosphoric acid-methanol (6:4), followed by a Bond-Elut C18 clean-up procedure. The HPLC separation was carried out on a Supersphere RP-18e column (125 X 4.0 mm I.D.) using 0.05 M sodium dihydrogenphosphate (pH 4.5)-acetonitrile (8:2) as the mobile phase at a flow-rate of 0.5 ml/min, and monitored with a photodiode-array detector. The recoveries of SMT and N4-AcSMT from meat fortified at 0.5 micrograms/g were 90.1-93.3 and 93.0-94.4%, respectively, with coefficients of variation of 1.9-3.2 and 1.5-2.7%. The limits of detection were 0.02 micrograms/g for each drug. SMT was found in ten samples of imported meat (12.5%) at levels ranging from 0.05 to 1.05 micrograms/g.
Animals↗
Kinetic analyses of creatine kinase release patterns in patients with acute myocardial infarction undergoing emergency coronary arteriography.
To quantify the effects of early reperfusion on the size of infarcts, an enzyme indicator was developed: myocardial creatine kinase (CK) release rate (kr), based on a compartmental kinetics model. In 59 patients with acute myocardial infarction (MI) who received intracoronary thrombolysis therapy in the acute phase, the kr showed a good correlation with the flow condition of infarct-related coronary artery and the time required to reach peak enzyme activity. Apparent serum CK disappearance rate (kd') was estimated by using the method of Norris. The kd' was significantly underestimated in patients without reperfusion, suggesting the presence of prolonged enzyme release from the infarcted area. In 22 of 59 patients, who had a first acute anterior MI (left anterior descending arterial lesion), the correction of cumulative enzyme release by myocardial enzyme release (kr) resulted in a closer correlation with chronic phase left ventricular function. Thus, kinetic analyses of serum enzyme release provide a useful means to estimate the infarct size during intracoronary thrombolysis therapy.
Adult↗