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Biomedical subjects

M Horie

Publications and source records attributed to M Horie.

At least 109 records · Page 6Linked to original sources
PubMed1994

Inhibition of the cardiac protein kinase A-dependent chloride conductance by endothelin-1.

Endothelin-1 is a peptide hormone constitutively secreted by vascular and endocardial endothelial cells. Secretion of endothelin-1 is increased under certain pathophysiological conditions, including coronary vasospasm, cardiac ischaemia and myocardial infarction. We have examined the effect of endothelin-1 on the protein kinase A (PKA)-dependent chloride current in voltage-clamped guinea pig ventricular myocytes. This conductance, induced by catecholamines through beta-adrenergic receptors, counteracts the simultaneously increased L-type calcium current by shortening the action potential duration. We report here that endothelin-1, acting through ETA (endothelin-1-selective) receptors, inhibited the current through a pertussis toxin-sensitive mechanism, analogous to muscarinic receptors, by reducing the intracellular cyclic AMP concentration. This effect of endothelin-1 should help protect the ventricle against potentially arrhythmogenic shortening of the action potential during ischaemia when the circulating levels of catecholamines are increased.

Animals↗
PubMed1994

Determination of mirosamicin in animal tissues by high-performance liquid chromatography.

A simple and rapid method using high-performance liquid chromatography (HPLC) for the determination of mirosamicin in animal tissues has been developed. The drug was extracted with 0.3% metaphosphoric acid-methanol (7:3, v/v), and the extracts were cleaned on a Bond Elut SCX (500 mg) cartridge. The HPLC separation was performed on a Puresil 5C18 column (150 x 4.6 mm I.D.) with 0.05 M phosphate buffer (pH 2.5)-acetonitrile (70:30) as the mobile phase at a flow-rate of 0.5 ml/min; the drug was detected at 230 nm with 0.04 AUFS. The calibration graph was linear from 5 to 100 ng. The recoveries of microsamicin from various animal tissues fortified at 1.0 microgram/g were 83.7-88.6% with a relative standard deviation (R.S.D.) of 2.0-5.7%. The detection limit was 0.05 microgram/g.

Animals↗
PubMed1994

Determination of urinary excretion of histamine and 1-methylhistamine by liquid chromatography.

Urinary histamine (Him) and 1-methylhistamine (MH) were determined by liquid chromatography (LC) using on-column derivatization coupled with a column-switching technique. An intact urine sample without any purification was applicable to the LC system because all steps for purification and fluorescence derivatization were fully automated. It was observed that the concentrations of Him and MH increased after hydrolysis of the urine, suggesting the presence of conjugated Him and MH. The level of total/free Him in urine was significantly higher in cancer patients than in normal subjects. Further, a significant correlation between Him and MH was observed in the hydrolysed urine of both normal subjects and cancer patients.

Adolescent↗
PubMed1994

Simultaneous determination of benofloxacin, danofloxacin, enrofloxacin and ofloxacin in chicken tissue by high-performance liquid chromatography.

A simple, rapid and reliable high-performance liquid chromatographic (HPLC) method for the simultaneous determination of residual fluoroquinolones (benofloxacin, danofloxacin, enrofloxacin and ofloxacin) in chicken has been developed. The drugs were extracted with 0.2% metaphosphoric acid-acetonitrile (7:3, v/v), followed by a Bond Elut C18 clean-up procedure. The HPLC separation was carried out on a Wakosil II 5C18-HG column (150 x 4.6 mm I.D.) with 0.05 M phosphate buffer (pH 2.4)-acetonitrile (80:20, v/v) containing 2.5 mM 1-heptanesulfonic acid as the mobile phase. A fluorescence detector was used at an excitation wavelength of 295 nm and an emission wavelength of 455 nm. The calibration graphs were linear from 0.1 to 10 ng for danofloxacin and from 1 to 100 ng for benofloxacin, enrofloxacin and ofloxacin. The recoveries of the drugs from tissues fortified at a level of 0.2 microgram/g were 81.1-89.6%, and the detection limits were 0.01 microgram/g for ofloxacin, danofloxacin and enrofloxacin and 0.02 microgram/kg for benofloxacin. The time needed per sample was less than 60 min.

Animals↗
PubMed1994

Reduced sensitivity of dihydroxyacetone on ATP-sensitive K+ channels of pancreatic beta cells in GK rats.

In the GK (Goto-Kakizaki) rat, a genetic model of non-insulin-dependent diabetes mellitus, glucose-induced insulin secretion is selectively impaired. In addition, it has been suggested by previous studies that impaired glucose metabolism in beta cells of the GK rat results in insufficient closure of ATP-sensitive K+ channels (KATP channels) and a consequent decrease in depolarization, leading to a decreased insulin release. We have recently reported that the site of disturbed glucose metabolism is probably located in the early stages of glycolysis or in the glycerol phosphate shuttle. In the present study, in order to identify the impaired metabolic step in diabetic beta cells, we have investigated insulin secretory capacity by stimulation with dihydroxyacetone (DHA), which is known to be directly converted to DHA-phosphate and to preferentially enter the glycerol phosphate shuttle. In addition, using the patch-clamp technique, we also have studied the sensitivity of DHA on the KATP channels of beta cells in GK rats. The insulin secretion in response to 5 mmol/l DHA with 2.8 mmol/l glucose was impaired, and DHA sensitivity of the KATP channels was reduced in beta cells of GK rats. From these results, we suggest that the intracellular site responsible for impaired glucose metabolism in pancreatic beta cells of GK rats is located in the glycerol phosphate shuttle.

Adenosine Triphosphate↗
PubMed1994

Increased calcium-channel currents of pancreatic beta cells in neonatally streptozocin-induced diabetic rats.

Using a whole-cell patch-clamp technique, voltage-dependent Ca(2+)-channel activities were found to be increased in cultured single beta cells isolated from neonatally streptozocin-induced diabetic rats (NSZ rats). The current-voltage relationship and inactivation time course of Ba2+ currents via L-type Ca2+ channels were indistinguishable between NSZ and control rats. However, the current density observed in NSZ rats was significantly greater than that in control rats. Ba2+ currents via T-type Ca2+ channels were also found to be enhanced in NSZ beta cells. The insulin-secretory capacity of cultured pancreatic islets in response to a depolarizing stimulus (20 mmol/L arginine or 30 mmol/L KCl) in the presence of 11.1 mmol/L glucose was augmented in NSZ rats, whereas that in response to 11.1 and 16.7 mmol/L glucose alone was significantly reduced. It is concluded that the impaired insulinotropic action of glucose in beta cells in NSZ rats is not due to reduced activity of voltage-dependent Ca2+ channels. The fact that insulin secretion induced by a depolarizing stimulus was enhanced in NSZ rats may be related to the augmented activity of the voltage-dependent calcium current found in NSZ beta cells.

Animals↗
PubMed1994

Nitric oxide opens ATP-sensitive K+ channels through suppression of phosphofructokinase activity and inhibits glucose-induced insulin release in pancreatic beta cells.

Nitric oxide (NO) is known to be a potent messenger in the intracellular signal transduction system in many tissues. In pancreatic beta cells, NO has been reported to be formed from L-arginine through NO synthase. To elucidate the effect of NO on insulin secretion and to investigate the intracellular mechanism of its effect, we have used sodium nitroprusside (SNP) as a NO donor. SNP inhibited glucose-induced insulin secretion in a dose-dependent manner, and its effect was reversed by hemoglobin, a known NO scavenger. However, glyceraldehyde-induced insulin secretion was not affected by SNP. Since the closure of ATP-sensitive K+ channels (KATP channel) has been established as a key step in glucose-induced insulin secretion, we have directly assessed the effect of SNP on KATP channel activity using the patch clamp technique. The KATP channel activity reduced by glucose was found to be reversibly activated by the addition of SNP, and this activation was able to be similarly reproduced by applying S-Nitroso-N-acetyl-DL-penicillamine (SNAP), another NO generator. Furthermore, these activating effects were completely eliminated by hemoglobin, in accordance with the reversibility in inhibition of glucose-induced insulin release. However, SNP could not affect the KATP channel suppression by ATP applied to the inside of the plasma membrane. The activation of the KATP channel by NO, therefore, seems to be due to the decreased ATP production attributable to impairment of glucose metabolism in beta cells. Since SNP exhibited no effect on glyceraldehyde-induced KATP channel inhibition, NO may disturb a glycolytic step before glyceraldehyde-3-phosphate. The KATP channel activation by 2-deoxyglucose through presumable ATP consumption due to its phosphorylation by glucokinase was, however, not affected even in the presence of SNP. But in the permeabilized beta cells made by exposure to a low concentration (0.02 U/ml) of streptolysin O (open cell-attached configuration), SNP reopens KATP channels which have been eliminated by fructose-6-phosphate, while this effect was not observed in the KATP channels inhibited by fructose-1,6-bisphosphate. On the other hand, in rat ventricular myocyte KATP channels were not activated by SNP even under a low concentration of glucose. From these observations, the inhibition of phosphofructokinase activity is probably the site responsible for the impairment of glucose metabolism induced by NO in pancreatic beta cells. NO, therefore, seems to be a factor in the deterioration of glucose-induced insulin secretion from pancreatic beta cells through a unique intracellular mechanism.

Adenosine Triphosphate↗
PubMed1994

Extensive proliferative capacity of single isolated CD34 human cord blood cells in suspension culture.

Nonadherent, low-density T-lymphocyte-depleted (NALT-) CD34 cells from normal human cord blood were assessed in suspension culture for the effects of recombinant cytokines on their proliferation, differentiation, and generation of myeloid progenitor cells. In this cell population, 82% of cells expressed c-kit protein as assessed by in situ hybridization, and their cloning efficiency was 85% when cells were plated at low cell numbers with combinations of growth factors. CD34 cells were sorted as 1, 5, or 10 cell(s) per well and also at 5000 cells per dish to initiate stromal-free suspension cultures in the presence of steel factor (SLF), interleukin (IL)-1 alpha, and IL-3. Forty-eight percent of the wells started with a single CD34 cell were positive for growth after 14 days, and the wells contained greater than 5 x 10(3) cells by 21-28 days. Progenitors were assayed weekly with cultures initiated with 1 or 5000 cells. While the fold expansion of nucleated cells was greater in cultures initiated with one cell per well (> 5000 compared to 791-fold expansion for 5000 cells), the fold expansion of progenitors was greater than 5000 cells were used to initiate cultures. Under optimal conditions, there was, respectively, a 160-, 164-, and 57-fold output of high proliferative potential colony-forming cells, granulocyte-macrophage colony-forming units, and erythroid burst-forming units/granulocyte erythroid macrophage megakaryocyte colony-forming units within 1-3 weeks for cultures initiated with 5000 CD34 cells compared with respective fold increases of 29, 16, and 1, for single-initiated cultures. These results demonstrate the expansion capacity of single CD34 cord blood cells and demonstrate that factors in addition to SLF, IL-1 alpha, and IL-3 are necessary for optimal expansion of progenitors from single isolated CD34 cells.

Antigens, CD↗
PubMed1994

Regulation of CFTR channel gating.

Findings outlined here support a complex model for the regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel gating that incorporates incremental protein kinase A (PKA) phosphorylation of CFTR at multiple sites which, in turn, differentially control the activity of CFTR's two nucleotide-binding domains (NBDs). The NBDs are functionally distinct: only one can respond to the non-hydrolyzable ATP analogue AMP-PNP, and then only after ATP has acted at the other. Moreover, the nature of the responses to AMP-PNP, and to the inorganic phosphate analogue orthovanadate, argues that ATP hydrolysis normally occurs at both NBDs, at one to initiate channel opening and at the other to initiate closing.

Adenosine Triphosphate↗
PubMed1994

Additional similarity of cardiac cAMP-activated Cl- channels to CFTR Cl- channels.

Isoproterenol, forskolin, or cAMP activated a time-independent Cl- current (ICl.cAMP), which is known to be activated by A-kinase-mediated phosphorylation, in guinea pig ventricular myocytes. External glibenclamide inhibited ICl.cAMP in a concentration-dependent manner with IC50 of around 30 microM. Thus, it is concluded that glibenclamide inhibits cardiac cAMP-activated Cl- channels and that the Cl- channel is, in this respect, similar to epithelial CFTR Cl- channel. Northern analysis actually indicated that CFTR messenger RNA is expressed in the guinea pig ventricle.

Animals↗
PubMed1994

Inhibitory pathway of cardiac PKA-dependent Cl- conductance via pertussis toxin-sensitive G proteins.

The inhibitory pathway of cardiac cAMP-dependent protein kinase regulated Cl- conductance was investigated using the whole-cell configuration of patch-clamp techniques in single guinea pig ventricular myocytes. Pertussis toxin-sensitive G proteins (Gi), mediating the signal transductions between muscarinic receptors and adenylate cyclase, have a substantial tonic activity even in the absence of muscarinic receptor modulators. Muscarinic agonists or antagonists (like atropine) either increase or decrease this basal activity of Gi by altering the proportion of active and inactive forms of the receptors. Similar to L-type Ca-channel currents, the Cl- conductance showed a transient over-recovery upon cessation of brief muscarinic receptor stimulation by carbachol (CCh) (rebound). Atropine alone enhanced the Cl- conductance elicited by low concentrations of Iso (reverse agonist). After washout of atropine, the over-suppression of the conductance was observed as a mirror image of CCh-induced rebound (reverse rebound). Both types of rebound became prominent when cell dialysis with pipette solutions containing 100 microM GTP was minimized with high-resistance pipettes. Endogenous GTP is therefore an intracellular modulator, and not simply a mediator, of Gi-dependent signal transduction.

Adenylate Cyclase Toxin↗
PubMed1994

The effects of endothelin-1 on the PKA-dependent Cl- current in the heart.

The effects of endothelin-1 (ET-1) on whole-cell cardiac PKA-dependent Cl- currents (ICl) were investigated using patch clamp techniques. ET-1 inhibited the isoproterenol-induced ICl with a half-maximally effective concentration of approximately 1 nM. ET-1 also inhibited the forskolin-induced current in a similar concentration range. The effects of ET-1 were abolished by pre-treatment of the cells with pertussis toxin. Since ET-1 was ineffective at inhibiting the ICl induced by internal dialysis with cyclic AMP, it is unlikely that the Gi-protein had a direct effect on channel gating or phosphorylation of the channel by PKA. It is concluded that ET-1 inhibited the cardiac PKA-dependent ICl by attenuating activation of adenylate cyclase and that this effect was mediated by a pertussis toxin-sensitive G-protein, presumably Gi.

Adenylate Cyclase Toxin↗
PubMed1994

[Measurement and fluctuation of urinary glucaric acid in pregnant women].

It is known that urinary excretion of glucaric acid (GA) is an indirect index of hepatic P-450 microenzyme induction. We measured and analyzed urinary excretion of GA and plasma lipids in non-pregnant women, pregnant women and postpartum women. GA was measured by a new method for the inhibition of beta-glucuronidase activity and plasma lipids were measured by routine laboratory methods and we obtained the following results. 1. The concentration of urinary GA was correlated with that of urinary creatinine in pregnant women. 2. The urinary GA and plasma HDL-cholesterol did not change during the first of gestation, but steeply increased in the middle of gestation, and postpartum values were lower than at term. 3. Plasma total lipids, triglycerides, and total cholesterol continuously increased throughout gestation. 4. Plasma free fatty acids and lipid peroxide steeply increased in late in gestation. These results and several reports suggested that the change in GA in pregnant women reflected their own metabolism not fetal or placental metabolism. It seems that grasping and understanding their metabolism can make their disease clear.

Cholesterol↗
PubMed1994

[Lipids metabolism evaluated of by vitamin E to total lipid ratio in maternal and cord sera in normal pregnancy].

Changes in plasma levels of lipids peroxide (LPO) and vitamin E(VE) are observed in pregnant women. We measured levels of VE and lipids in 80 maternal and cord sera in normal delivery and obtained the following results. 1. For each lipid and VE, the concentration gradients were preserved in maternal sera to cord sera through placentas. 2. The concentration of VE correlated with that of total lipids (TL) in both sera. 3. The VE to TL ratio (VE/TL ratio) inversely correlated with the concentration of LPO in both sera. 4. The concentration of VE correlated with the VE/TL ratio and there was a more significant correlation of the VE and VE/TL ratio than that of VE and TL in both sera. 5. The VE/TL ratio in maternal serum correlated with VE/TL ratio and the concentration of VE in cord serum. These results suggested that lipids in serum was a regulatory factor in VE and that VE and lipids maintained the gradation of the concentration according to their own placental transport systems. It seems that the fetus has a unique VE which is comparatively lowered. This may be due to the effects of the accelerated lipid metabolism in the fetus.

Female↗
PubMed1994

[Studies on hypogeusia in hyperemesis gravidarum].

We measured changes in gustatory sensitivities and gustation threshold levels and those of some trace elements, vitamin E and lipids in the serum of 38 patients suffering from hyperemesis gravidarum, 12 normal pregnant women in the first trimester and 22 healthy non-pregnant women by means of filter discs and an electrogusto meter (EGM) and we obtained the following results. 1. Gustatory sensitivity to sweetness was found to be significantly lowered in the patients with hyperemesis gravidarum when tested with filter discs (p < 0.05). 2. Gustation threshold levels were significantly lowered in the patients with hyperemesis gravidarum and the normal pregnant women tested with the EGM (p < 0.01). 3. There was considerable heterogeusia of women in the patients with hyperemesis gravidarum (p < 0.05). 4. Zn and Mg in serum decreased (p < 0.01), but Cu increased significantly in the patients with hyperemesis gravidarum and the normal pregnant women (p < 0.01). 5. Total cholesterol and phospholipid in serum significantly decreased, but lipid peroxide in serum significantly increased in the patients with hyperemesis gravidarum (p < 0.01). These results suggested that the gustatory test was helpful in investigating hyperemesis gravidarum and that biological defense systems (scavenger systems) to free radicals were damaged in patients with hyperemesis gravidarum.

Adult↗
PubMed1993

Detection of micrometastatic prostate cancer cells in lymph nodes by reverse transcriptase-polymerase chain reaction.

We have developed a highly sensitive method for detecting prostate cancer cells using reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for prostate-specific antigen gene. Forty-four lymph nodes obtained from 22 patients with prostate cancers were analyzed by RT-PCR to detect metastatic prostate cancer cells. RT-PCR could detect prostate-specific antigen mRNA in five lymph nodes with histologically and/or immunohistochemically identifiable metastases and in four lymph nodes with negative histological and immunohistochemical analyses for metastases. RT-PCR was a more sensitive method than histology and immunohistochemistry in detecting metastatic prostate cancer cells and could be applied for diagnosing micrometastases of prostate cancer to lymph nodes. This highly sensitive RT-PCR will be a relevant tool to allow a more accurate clinical assessment of lymph node metastases of prostate cancer and to understand lymphatic dissemination of prostate cancer biologically.

Base Sequence↗
PubMed1993

Effects of NB-598, a potent squalene epoxidase inhibitor, on the apical membrane uptake of cholesterol and basolateral membrane secretion of lipids in Caco-2 cells.

Caco-2 cells grown on membrane filters were used as a model to study the effects of NB-598, an inhibitor of squalene epoxidase, on cholesterol absorption from the intestinal epithelia. NB-598 (10 microM) inhibited the synthesis of sterol and sterol ester from [14C]acetate without affecting the synthesis of other lipids such as phospholipids (PL), free fatty acids (FFA) and triacylglycerol (TG). When labeled lipid was apically loaded as a micellar lipid solution into Caco-2 cell cultures, NB-598 reduced basolaterally secreted radioactivity in cholesterol, cholesterol ester, PL and TG. Furthermore, NB-598 suppressed the basolateral secretion of apolipoprotein (apo) B. When microsomes prepared from control Caco-2 cells were incubated with 10 microM NB-598, acyl CoA:cholesterol acyltransferase (ACAT) activity was inhibited slightly. After incubating Caco-2 cells with 10 microM NB-598, a slight reduction in cellular ACAT activity was also observed. These results suggest that suppression of the secretion of particles containing apo B and reduction of cellular ACAT activity in the intestinal epithelia are part of the mechanism of the cholesterol-lowering effect of NB-598.

Anticholesteremic Agents↗