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Biomedical subjects

M Inoue

Publications and source records attributed to M Inoue.

At least 181 records · Page 10Linked to original sources

Activation of Ca(2+)-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells.

The effects of cyanide (CN) on whole cell current measured with the perforated-patch method were studied in adrenal medullary cells. Application of CN produced initially inward and then outward currents at -52 mV or more negative. As the membrane potential was hyperpolarized, amplitude and latency of the outward current (Io) by CN became small and long, respectively. A decrease in the external Na+ concentration did not affect the latency for CN-induced Io but enhanced the amplitude markedly. The CN Io reversed polarity at -85 mV, close to the Nernst potential for K+, and was suppressed by the K+ channel blockers curare and apamin but not by glibenclamide, suggesting that Io is due to the activation of Ca(2+)-dependent K+ channels. Consistent with this notion, the Ca(2+)-mobilizing agents, muscarine and caffeine, also produced Io. Exposure to CN in a Ca(2+)-deficient medium for 4 min abolished caffeine- or muscarine-induced Io without development of Io, and addition of Ca2+ to the CN-containing solution induced Io. We conclude that exposure to CN produces Ca(2+)-dependent K+ currents in an external Ca(2+)-dependent manner, probably via facilitation of Ca2+ influx.

Adrenal Medulla

Nitric oxide regulates energy metabolism and Bcl-2 expression in intestinal epithelial cells.

Nitric oxide (NO) inhibits the respiration of mitochondria and enteric bacteria, particularly under low O2 concentration, and induces apoptosis of various types of cells. To gain insight into the molecular role of NO in the intestine, we examined its effects on the respiration, Ca2+ status, and expression of Bcl-2 in cultured intestinal epithelial cells (IEC-6). NO reversibly inhibited the respiration of IEC-6 cells, especially under physiologically low O2 concentration. Although NO elevated cytosolic Ca2+ as determined by the fura 2 method, the cells were fairly resistant to NO. Kinetic analysis revealed that prolonged exposure to NO elevated the levels of Bcl-2 and suppressed the NO-induced changes in Ca2+ status of the cells. Because Bcl-2 possesses antiapoptotic function, toxic NO effects might appear minimally in enterocytes enriched with Bcl-2. Thus NO might effectively exhibit its antibacterial action in anaerobic intestinal lumen without inducing apoptosis of Bcl-2-enriched mucosal cells.

Animals

Pepsinogen secretion: coupling of exocytosis visualized by video microscopy and [Ca2+]i in single cells.

Conventional in vitro studies of pepsinogen secretion have measured secretion into the bulk medium and have demonstrated the critical role of Ca2+ in the process. The present study was undertaken to obtain further details of the process of secretion and its relation to Ca2+ changes over very short time periods. The relation between Ca2+ mobilization and exocytosis in an isolated individual peptic cell of the bullfrog was investigated by a method to measure both intracellular Ca2+ ([Ca2+]i), using a fluorescent Ca2+ indicator, fura 2, and exocytosis from single cells using a video microscope analyzing system. Bombesin (3.2 x 10(-7) M) and bethanechol (3.2 x 10(-4) M) caused a rapid increase in [Ca2+]i (initial peak) and a corresponding high frequency of initial exocytosis. After the initial peak, [Ca2+]i was maintained at a somewhat elevated level over the baseline (sustained phase), with a corresponding low frequency of exocytosis. Both the sustained phase of elevated [Ca2+]i and the related exocytosis were eliminated by the depletion of extracellular Ca2+. Low concentrations of bombesin (3.2 x 10(-10) M) and bethanechol (3.2 x 10(-7) M) caused sustained low-amplitude Ca2+ oscillations with correspondingly low frequencies but also caused sustained exocytosis. These data show that 1) cellular response differs between high and low concentrations of stimulus, 2) there is a close relation between [Ca2+]i and exocytosis, 3) exocytosis follows elevation of [Ca2+]i by 14-45 s (n = 6), and 4) there is a significant positive correlation between the peak [Ca2+]i and the number of exocytoses.

Animals

Detection of human papillomavirus type 2 related sequence in oral papilloma.

Oral papilloma is a benign tumourous lesion. Part of this lesion is associated with human papillomavirus (HPV) infection. We analysed the genetical and histopathological evidence for HPV type 2 infection in three oral papillomas. Southern blot hybridization showed HPV 2a sequence in one lesion. Cells of the positive specimen appeared to contain high copy numbers of the viral DNA in an episomal state. In situ staining demonstrated virus capsid antigen in koilocytotic cells and surrounding cells in the hyperplastic epithelial layer. Two other specimens contained no HPV sequences by labeled probe of full length linear HPVs 2a, 6b, 11, 16, 18, 31 and 33 DNA under low stringency hybridization conditions. These results showed the possibility that HPV 2 plays a role in oral papilloma.

Adult

Granulocyte macrophage-colony stimulating factor reciprocally regulates alphav-associated integrins on murine osteoclast precursors.

The integrins alphavbeta3 and alphavbeta3 are expressed reciprocally during murine osteoclastogenesis in vitro. Specifically, immature osteoclast precursors, in the form of bone marrow macrophages, contain exclusively alphavbeta5, surface expression of which declines with commitment to the osteoclast phenotype, while levels of alphavbeta3 increase concomitantly. The distinct functional significance of alphavbeta5 is underscored by the integrin's capacity, unlike alphavbeta3, to mediate both attachment and spreading on ligand, of marrow macrophages, suggesting alphavbeta3 negotiates initial recognition, by osteoclast precursors, of bone matrix. Northern analysis demonstrates changes in the two beta-subunits, and not alphav, are responsible for these alterations. Treatment of early precursors with granulocyte-macrophage colony stimulating factor (GM-CSF) leads to alterations in beta3 and beta5 mRNA and alphavbeta5 and alphavbeta3, paralleling those occurring during osteoclastogenesis. Nuclear run-on and message stability studies demonstrate that while GM-CSF treatment of precursors alters beta5 transcriptionally, the changes in beta3 arise from prolonged mRNA t1/2. Similar to GM-CSF treatment, the rate of beta5 transcription falls during authentic osteoclastogenesis. In contrast to cytokine-induced alphavbeta3, however, that attending osteoclastogenesis reflects accelerated transcription of the beta3-subunit. Thus, while GM-CSF may participate in modulation of alphavbeta5 during osteoclast differentiation, signals other than those derived from the cytokine must regulate expression of alphavbeta3.

Animals

Enhancement of serum nitric oxide by Shichimotsu-koka-to (Kampo medicine).

The Kampo medicine Shichimotsu-koka-to (SKT) is used to treat hypertension and atherosclerosis in Japan. The action of SKT was studied, focusing on nitric oxide, which is intimately involved in regulation of blood pressure and cell functions associated with atherogenesis and inflammation. Oral administration of SKT enhanced serum nitric oxide (NOx) levels dose-dependently and 3 d administration was enough to detect its effect. The maximal level of serum NOx was maintained at around 27 microM, a concentration which did not result in harmful effects on cells. On the other hand, L-arginine, the substrate of NO synthase (NOS), was also increased by SKT administration. When the source of L-arginine was studied, only 12.7 mg of L-arginine was contained in 1 g of SKT and this amount of L-arginine could not explain the increased L-arginine levels in serum. These results suggest that SKT may enhance serum L-arginine by acting on L-arginine metabolism, but not by supplying L-arginine itself, resulting in enhancement of serum NOx. In conclusion, the antihypertensive and antiatherosclerotic action shown by SKT may be in part due to enhanced serum NOx, thus suggesting that SKT may become a unique orally active drug for cardiovascular diseases as a new NO donor.

Alanine Transaminase

Antihypercholesterolemic action of a traditional Chinese medicine (Kampo medicine), Ogi-Keishi-Gomotsu-To-Ka-Kojin.

The effect of Ogi-Keishi-Gomotsu-To-Ka-Kojin (OKGK), a traditional Chinese herbal medicine (Kampo medicine), on cholesterol metabolism was studied in male Sprague-Dawley rats. Intake of OKGK at doses of 1.38 g/kg for 4 weeks significantly reduced total cholesterol levels in the serum and liver of hypercholesterolemia rats fed a cholesterol-enriched diet. OKGK suppressed cholesterol absorption through the intestine and stimulated excretion of cholesterol into feces as bile acids. Biochemical study indicated that OKGK treatment enhanced cholesterol 7alpha-hydroxylase activity the rate limiting enzyme of cholic acid synthesis, in the liver without any effect on the rate limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Further, cholesterol-enriched diet containing cholic acid suppressed cholesterol 7alpha-hydroxylase activity, whereas OKGK administration reversed the suppression. In conclusion, these results supported the idea that OKGK may be an effective agent for treatment of patients with hypercholesterolemia.

Animals

Induction of cytokines by a phenylpropanoid glycoside acteoside.

A phenylpropanoid glycoside acteoside was found to induce interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in macrophage-like cell line J774.A1 at 1-100 ng/ml. In addition, when the stimulatory action of acteoside was studied using the bovine glomerular endothelial cell line GEN-T, it stimulated IL-6 production. These stimulatory activities were not abolished by treatment with polymixin B, which inactivates lipopolysaccharide (LPS), indicating that the action was not a contamination of LPS.

Animals

Long-term administration of "sho-saiko-to" increases cytochrome P-450 mRNA level in mouse liver.

Simplified differential display of mRNA was applied to isolate and identify genes transcriptionally regulated in mouse liver by sho-saiko-to administration. A cDNA fragment up-regulated by sho-saiko-to was isolated and characterized. cDNA sequencing and subsequent database analysis revealed that the fragment showed significant sequence similarity with mouse testosterone 16-alpha-hydroxylase (cytochrome P-450[16alpha]) cDNA. The increased level of mRNA expression of cytochrome P-450(16alpha) in association with sho-saiko-to administration suggests the molecular mechanism of the chemopreventive effect of sho-saiko-to. This result indicates the usefulness of the mRNA differential display technique to investigate the molecular mechanism of Kampo medicine.

Animals

Formation of reactive oxygen intermediates might be involved in the trypanocidal activity of gallic acid.

We investigated the mechanism of the trypanocidal activity of gallic acid (GA). GA-induced trypanocidal activity was significantly reduced by pretreatment with superoxide dismutase (SOD) and/or catalase. The ESR technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent revealed that a DMPO-OH adduct was detected in culture medium containing GA. The intensity of ESR signals of the DMPO-OH adduct was increased in a time dependent manner. SOD also inhibited the formation of GA-induced DMPO-OH adducts. Furthermore, GA enhanced DNA single-strand breaks induced by Fenton reagent. These results suggest the possibility that GA acts as pro-oxidant for trypanocidal activity.

Animals

Induction of apoptotic cell death in HL-60 cells by acteoside, a phenylpropanoid glycoside.

Acteoside, a phenylpropanoid glycoside with anti-oxidative activity, induced cell death in promyelocytic leukemia HL-60 cells with an IC50 value of 26.7/microM. Analysis of extracted DNA on agarose gel electrophoresis revealed that acteoside induced the internucleosomal breakdown of chromatin DNA characteristic of apoptosis. Apoptosis-specific DNA fragmentation was clearly detectable 4 h after treatment with acteoside and was independent of the cell cycle phase. These data indicate that acteoside induces apoptosis in HL-60 cells.

Antioxidants

Synthesis of pyridinium dinitrobenzyl sulfates and potassium (dinitrobenzyl beta-D-glucopyranosid)uronates.

Sulfates and glucuronides of 2,4-dinitrobenzyl alcohol 1a and 2,6-dinitrobenzyl alcohol 1b, which are major or putative metabolites of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT), were synthesized from 1a and 1b by reaction with pyridinium sulfonate and methyl (2,3,4-tri-O-acetyl-alpha-D-glucopyranosyl)uronate bromide 3, respectively, as their pyridinium salts (2a, 2b) and potassium salts (6a, 6b). These conjugates are important for the study of the carcinogenicity of 2,4-DNT and 2,6-DNT.

Benzyl Alcohols

Redistribution in thallium-201 myocardial imaging soon after successful coronary stenting--tomographic evaluation during coronary hyperemia induced by adenosine.

To evaluate the clinical significance of reversible perfusion defects that were observed soon after the successful deployment of a coronary stent, 47 patients underwent thallium-201 myocardial scintigraphy and radionuclide angiography in conjunction with adenosine-induced coronary hyperemia before and after complete revascularization. Coronary angiography showed a significant decrease in the percent diameter stenosis (from 87+/-11% before stenting to -1+/-5% after stenting, p<0.01) with no major dissection, residual stenosis, or intra-stent formation of thrombus. Even after the angiographically successful procedure, reversible perfusion defects were present in 17 (36%) of the 47 patients, none of whom showed any wall motion abnormalities during the infusion of adenosine. Disease duration was significantly longer and collateral vessels were more common in the patients with than in those without thallium redistribution, whereas the other clinical, pre- and post-stent angiographic and hemodynamic factors were similar. In conclusion, reversible perfusion defects without wall motion abnormalities were demonstrated during the infusion of adenosine in approximately one-third of patients soon after coronary stenting, and were not consistently related to acute unfavorable outcomes of stent placement.

Adenosine

[Inhibition of cytochrome P450 by nitric oxide].

Nitric oxide (NO) reacts with iron, superoxide, thiols and oxygen. Although NO reversibly interacts with the heme-iron of P450, the pathophysiological role of this interaction remains to be elucidated. We found that hepatic levels of P450 markedly decreased in endotoxemic rats, particularly when the rate of NO generation was increased. To determine the possible role of NO in the modulation of the structure and function of P450, changes in the levels and activities of P450 isozymes were determined in liver microsomes from normal and endotoxemic rats. Electron spin resonance analysis revealed that incubation of microsomes with the NO donor NOC-7 rapidly generated NO-P450 adducts. Microsomal levels of NO-P450 adducts increased and peaked at 10 min after incubation and decreased thereafter; it disappeared completely within 60 min. In contrast, microsomal levels of the low-spin ferric form and CO-differential spectrally detectable P450 rapidly decreased during the initial 10 min; the signal intensity for P450 recovered thereafter. Western blot analysis using specific antibodies against CYP3A2 and CYP2C11 isozymes revealed no detectable degradation of these isoforms. Effect of NO on the catalytic activity of the enzymes was also determined by using testosterone as the substrate. The hydroxylation activity in microsomes rapidly decreased during the initial 10 min and disappeared slowly thereafter. These results suggested that NO might form dissociable complexes with the heme moiety of P450 and irreversibly inactivate them. The mechanism for P450 inactivation by NO and the role of NO-P450 interaction in the pathogenesis of liver injury in endotoxemia are discussed.

Animals

Idiopathic scoliosis in twins studied by DNA fingerprinting: the incidence and type of scoliosis.

We investigated 21 pairs of twins for zygosity and idiopathic scoliosis. DNA fingerprinting confirmed that 13 pairs were monozygotic and eight were dizygotic. There was concordance for idiopathic scoliosis in 92.3% of monozygotic and 62.5% of dizygotic twins. Of the 12 pairs of monozygotic twins concordant for idiopathic scoliosis, six showed discordant curve patterns but eight had differences in Cobb angle of less than 10 degrees. Seven of the ten pairs of monozygotic twins had similar back shapes. Our findings suggest that there is a genetic factor in the aetiology of idiopathic scoliosis; they also indicate that there is a genetic factor in both the severity of the curve and the general shape of the back.

Adolescent

Effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa in vitro.

The effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa were investigated in vitro. The addition of taurine, within the range of 0-5 mmol l(-1), did not appreciably affect the motility of intact fowl spermatozoa. Motility remained almost negligible at 40 degrees C, while vigorous movement was observed at 25 degrees C. Even with the addition of Ca2+ before the addition of taurine, neither stimulation nor inhibition of motility was observed compared with the control (no addition of taurine). Similar results were obtained by the addition of taurine and calyculin A, a specific inhibitor of protein phosphatases. There were no changes in intracellular free Ca2+ concentrations, measured by a fluorescent Ca2+ indicator, fura-2, in taurine-treated spermatozoa. These results suggest that taurine is not involved in the regulation of fowl sperm motility and metabolism by intracellular Ca2+ mobilization in vitro.

Animals

Uniqueness of pilsicainide in class Ic antiarrhythmics.

Pilsicainide, a class Ic agent, is known to be an effective drug particularly for treating atrial tachyarrhythmias. However, its electrophysiological effects on the atrium have not been well studied. To characterize the electrophysiologic effects of pilsicainide on atrial myocytes in class Ic drugs, we examined the effects of this drug on membrane currents in single rabbit atrial myocytes using the tight-seal whole cell voltage-clamp technique. Under the current-clamp condition, pilsicainide did not affect the action potential duration at therapeutic ranges (< or = 3 microM) and slightly shortened it at higher concentrations (> or = 10 microM). These observations were quite different from those with other class Ic agents including flecainide and propafenone which prolong the atrial action potential duration. The drug did not affect the resting membrane potential. Under the voltage-clamp condition, pilsicainide inhibited the transient outward current (Ito) that is more prominent in the atrium than in the ventricle in a concentration-dependent manner. However, in contrast to other class Ic agents, the inhibition to Ito by pilsicainide was observed only at much higher concentrations (IC50-300 microM) and did not affect the inactivation time-course of Ito. Moreover, the drug (10 microM) did not significantly affect the Ca2+, delayed rectifier K+, inward rectifying K+, acetylcholine-induced K+ or ATP-sensitive K+ currents. From these results pilsicainide could be differentiated as a pure Na+ channel blocker from other class Ic agents with diverse effects on membrane currents and should be recognized accordingly in clinical situations.

Action Potentials

Ontogenetic changes in responsiveness to benzodiazepine receptor ligands on ultrasonic vocalizations in rat pups.

The ontogenetic changes in responsiveness to benzodiazepine receptor ligands on ultrasonic vocalizations in rat pups from the age of day 3 to day 12 were evaluated. Rat pups, while separated from their dam and littermates and placed in a cold environment, emit ultrasonic vocalizations. These ultrasonic calls became attenuated dose-dependently in number and power after administration of the anxiolytic diazepam (0.25-1.0 mg/kg, s.c.), but the inhibitory effect of diazepam at the highest dose was less on day 6 and day 9. Moreover, type 1 benzodiazepine receptor ligands, Ro16-6028 and Ro23-0364 (0.5-2.0 mg/kg, s.c.), also dose-dependently attenuated the ultrasonic vocalizations 30-60 min after injection. The inhibitory effects of these drugs became more pronounced with the increasing age of the pup, and they were equivalent on day 12 to those in adult rats. These results suggest that different ontogenetic changes in development of two subtypes of central benzodiazepine receptors of pups might be related in the psychopharmacological mediation of the ultrasonic vocalization.

Animals