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Biomedical subjects

A Tsuji

Publications and source records attributed to A Tsuji.

At least 343 records · Page 19Linked to original sources
PubMed1990

Specific binding and clearance of [3H]dynorphin (1-13) in the perfused rat lung: an application of the multiple-indicator dilution method.

The clearance and binding of a kappa-selective opioid peptide, dynorphin (1-13), in the perfused rat lung has been examined, using the multiple indicator dilution method. More than 50% of [3H]dynorphin (1-13) entering the pulmonary circulation was eliminated by the lung during a single passage of a tracer dose. By contrast, when a high dose (100 microM) of dynorphin (1-13) was concomitantly injected, [3H]dynorphin (1-13) behaved like [14C]sucrose, an extracellular marker. The kinetic analyses of the pulmonary venous outflow curves of [3H]dynorphin (1-13) and [14C]sucrose at low and high doses of dynorphin (1-13) indicated that the initial uptake rate constant, extraction ratio and distribution volume of [3H]dynorphin (1-13) decreased significantly in the presence of a high concentration of unlabelled dynorphin (1-13). These results suggest that [3H]dynorphin (1-13) is eliminated by a saturable process and binds to a specific binding site in the perfused lung, which may be the kappa-type binding site. The multiple indicator dilution technique, in combination with a moment analysis, was successfully applied to demonstrate the specific binding and clearance of dynorphin (1-13) in the perfused lung.

Analgesics↗
PubMed1990

Dysfunction of choline transport system through blood-brain barrier in stroke-prone spontaneously hypertensive rats.

The blood-brain barrier (BBB) transport of choline was compared between stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar KY rats (WKY). The permeability surface area product (PS) of [3H]choline through the BBB in SHRSP (3.03 X 10(-3) +/- 1.09 X 10(-3) ml/min/g brain) was significantly lower than that in WKY (7.23 X 10(-3) +/- 0.97 X 10(-3) ml/min/g brain) in the presence of respective rat sera. No significant difference in the brain vascular space was indicated from the apparent uptake of [3H]sucrose between SHRSP and SKY. There was no significant difference for the Michaelis constant of choline transport between SHRSP (262 +/- 97 microM) and WKY (180 +/- 32 microM). However, the maximum velocity in SHRSP (3.41 +/- 1.19 nmol/min/g brain) was 37% lower than in WKY (5.40 +/- 0.38 nmol/min/g brain). Brain microdialysis technique was employed to collect the brain interstitial fluid in the rat hippocampus. The concentration of free choline in the brain dialysate in SHRSP was about half of that in WKY, while no significant difference was observed for the plasma concentration of free choline between SHRSP and WKY. In contrast, no significant difference was observed for the transport of D-[3H]glucose, 3-methyl-[3H]D-glucose and [3H]-phenylalanine through the BBB between SHRSP and WKY. Accordingly, the decreased choline concentration in the brain interstitial fluid ascribed to the specific dysfunction of the BBB choline transport has been demonstrated in SHRSP.

3-O-Methylglucose↗
PubMed1990

Acidic drug transport in vivo through the blood-brain barrier. A role of the transport carrier for monocarboxylic acids.

The relationship of the transports between acidic drugs and monocarboxylic acids through the blood-brain barrier (BBB) was examined using the carotid artery injection technique in rats. The BBB uptakes of [3H]acetic acid and [14C]salicylic acid were significantly reduced by the presence of the respective unlabeled compounds, valproic acid, lactic acid, benzoic acid, nicotinic acid or beta-lactam antibiotics (benzylpenicillin, propicillin and cefazolin), but was not reduced by choline, phenylalanine and a basic drug, eperisone. A remarkable pH dependency was observed for the BBB uptake of [14C]salicylic acid at the pH region of 4.0 to 7.4. Interestingly, 10 mM of salicylic acid diminished significantly the pH dependent BBB uptake of [14C]salicylic acid. Similar results were obtained in the BBB uptake of [14C]nicotinic acid. No significant difference was observed in the transport of monocarboxylic acids through the BBB between normotensive Wistar KY rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). From these observations, acidic drugs could be transported by a carrier-mediated system for monocarboxylic acids at the BBB and the transport system was not changed by the disease state.

Acetates↗
PubMed1990

Sodium and pH dependent carrier-mediated transport of antibiotic, fosfomycin, in the rat intestinal brush-border membrane.

The mechanism of intestinal absorption of an antimicrobial agent, fosfomycin (FOM), was investigated in rats using small intestinal brush-border membrane vesicles (BBMV). The uptake of [3H]FOM by BBMV was osmolarity- and temperature-sensitive and showed apparently saturable uptake kinetics consistent with the Michaelis-Menten equation, having Kt = 15.3 mM and Jmax = 7.78 nmol/30 s/mg protein at 37 degrees C. An overshoot uptake of FOM was observed in the presence of an inwardly direct Na+ gradient. The replacement of extravesicular Na+ with choline or mannitol significantly reduced the uptake. An addition of a protonophore, FCCP, significantly decreased the initial uptake of FOM in the absence of Na+ gradient but in the presence of a H+ gradient (pHin = 7.5, pHout = 6.0), whereas in the absence of a H+ gradient no significant difference was observed between the uptakes at an acidic pH (pHin = pHout = 6.0) and a neutral pH (pHin = pHout = 7.5). An inside negative potassium diffusion potential induced by valinomycin enhanced significantly the uptake of FOM. The uptake of FOM in the presence of both Na(+)- and H(+)-gradients was significantly inhibited by phosphate, arsenate and phosphonoformic acid (PFA), which are specific inhibitors of phosphate transport, but not by D-glucose. Based on these results, it is concluded that FOM transport in the small intestine is partially shared with the Na(+)-phosphate cotransport system and in part occurs via a H(+)-gradient dependent carrier-mediated system.

Animals↗
PubMed1990

Carrier-mediated uptake of nicotinic acid by rat intestinal brush-border membrane vesicles and relation to monocarboxylic acid transport.

The intestinal transport of [14C]nicotinic acid was investigated at 27 degrees C by using brush-border membrane vesicles (BBMV) isolated from the rat small intestine. The osmolarity sensitive uptake by BBMV showed a remarkable overshoot phenomenon in the presence of an inward-directed H+ gradient (pHin = 7.5, pHout = 6.0). In contrast, the imposition of a Na+ gradient ([Na+]in = 0 mM, [Na+]out = 100 mM) had no stimulatory effect on the uptake of [14C]nicotinic acid. The remarkable pH-dependence of the initial uptake showing an increase of the uptake rate with decreasing the extravesicular pH disappeared completely in the presence of a structural analogue, isonicotinic acid, at pH below 6.5. In the presence of a H+ gradient, the initial uptake of [14C]nicotinic acid was saturable with the apparent Kt of 4.43 mM and Jmax of 2.55 nmol/mg protein/15 s. The uptake was increased by the imposition of an inside-positive membrane potential and was significantly inhibited by monocarboxylic acids such as benzoic acid, salicylic acid, acetic acid, propionic acid, valproic acid and L-lactic acids as well as two isomers (isonicotinic acid and picolinic acid). The uptake was not inhibited by nicotinamide, nicotinyl alcohol, D-glucose, p-aminohippuric acid, glycyl-L-proline, succinic acid and an exchange transport inhibitor. From these results it was concluded that nicotinic acid is transported through the intestinal brush-border membrane by a carrier-mediated system and the system can recognize some acidic drugs with a monocarboxylic group. The pH dependent intestinal uptake of nicotinic acid can be ascribed to the proton-coupled and active carrier-mediated transport mechanism rather than a simple diffusion of the undissociated nicotinic acid to follow a pH-partition hypothesis.

Animals↗
PubMed1990

In vivo and in vitro evidence for a common carrier mediated transport of choline and basic drugs through the blood-brain barrier.

The blood-brain barrier (BBB) transport system for choline and basic drugs was characterized by the in vivo carotid artery injection technique (Oldendorf, Brain Res., 24, 372-376, 1970) and in vitro uptake into isolated bovine brain capillaries (Pardridge et al., J. Neurochem., 44, 1178-1184, 1985). Basic drugs such as eperisone, thiamine and scopolamine significantly inhibited choline uptake by the BBB with the half inhibitory concentration, IC50 value of 1.45, 2.06, and 0.47 mM, respectively. On the contrary, the uptake of choline was not inhibited by amino acids (L-phenylalanine and L-arginine) and acidic drugs (nicotinic acid, salicylic acid and valproic acid). Choline was taken up by the isolated brain capillaries in concentration and temperature dependent manners. The uptake of choline by the isolated bovine brain capillaries was significantly inhibited by eperisone, scopolamine and thiamine in consistent with the in vivo results. Furthermore, eperisone inhibited competitively the uptake of choline with the inhibition constant, Ki value of 455 microM. According to these results it was suggested that in the BBB choline and basic drugs would share a common carrier-mediated transport system.

Animals↗
PubMed1990

Specific binding of beta-endorphin to the isolated renal basolateral membranes in vitro.

Binding of human beta-endorphin (beta-EP) to rat renal basolateral membranes was characterized using [125I]Tyr27-beta-EP ([125I]beta-EP) as a primary ligand. Ten millimolar of ethylenediaminetetra acetic acid (EDTA) completely inhibited the degradation of [125I]beta-EP in the incubation mixture at 4 degrees C, thus making it possible to quantitatively examine the [125I]beta-EP binding. The specific binding of [125I]beta-EP to the basolateral membranes was reversible and saturable, and a nonlinear least-squares regression analysis of a saturation isotherm revealed two different classes of specific binding sites. One class had an apparent dissociation constant (Kd) of 0.68 nM and a lower number of binding sites (33 fmol/mg protein), whereas the other class had a lower affinity (apparent Kd of 210 nM) and a higher number of binding sites (7.3 pmol/mg protein). Inhibition of the [125I]beta-EP binding by naloxone (10 microM) was approximately only 20%, and that by D-Ala2-D-Leu5-enkephalin (10 microM) was null, suggesting the major role of a non-opioid binding component in specific [125I]beta-EP binding to basolateral membranes. Moreover, a 50% inhibition by 10 microM of dynorphin(1-13) suggests that a certain region of the primary structure of beta-EP, excluding at least the NH2-terminal enkephalin sequence, is of particular importance for the [125I]beta-EP binding. These lines of evidence suggest the existence of two different classes of specific binding sites for beta-EP on the renal basolateral membranes, and the high-and low-affinity bindings may be attributed to opioid and non-opioid receptors, respectively, as judged by known characteristics of opioid and non-opioid receptors in other peripheral tissues.

Animals↗
PubMed1990

Application of HPLC in disposition study of A14-125I-labeled insulin in mice.

To describe quantitatively the in vivo distribution and elimination of insulin, high-performance liquid chromatography (HPLC) separation was applied to the pharmacokinetic study of human insulin labeled with 125I at tyrosine A14 (A14-125I-insulin) as a tracer. Intact A14-125I-insulin levels were determined by HPLC and trichloroacetic acid (TCA) precipitation in plasma and various tissues after its intravenous bolus injection into mice. TCA precipitation consistently overestimated the intactness of A14-125I-insulin compared with HPLC, possibly due to the presence of both a TCA-precipitable intermediate degradation product of labeled insulin found in HPLC elution profiles and reported high-molecular-weight forms of labeled insulin in plasma. Thus, TCA precipitation gave a considerably lower total plasma clearance (Cltot) value than HPLC. The half-life of A14-125I-insulin was prolonged by a simultaneous injection of 8 U/kg unlabeled insulin, and labeled insulin behaved similarly to [14C]inulin (an extracellular fluid marker). The concentration time profiles of HPLC-separated labeled insulin in plasma were analyzed by a noncompartmental moment method, and both Cltot and steady-state apparent volume distribution (VDss) of A14-125I-insulin were considerably decreased by unlabeled insulin coadministration. In particular, VDss of labeled insulin decreased by 79%, similar to that of inulin (181 ml/kg), suggesting that the nonspecific binding of labeled insulin to tissues was so small that VDss of labeled insulin was reduced to the extracellular fluid volume (approximately 20% of the body weight) when its receptor binding was blocked effectively by unlabeled insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

[The effect of leukotriene B4 inhalation on airway responsiveness in dogs].

We studied the effect of leukotriene B4 (LTB4) inhalation on airway responsiveness in 12 dogs. LTB4 (10 micrograms/ml) was delivered as an aerosol, generated from a Devilbiss 646 nebulizer for ten minutes. Airway responsiveness to inhaled methacholine was determined by modified Astograph (7 Hz oscillation method) 1 hr (n = 6) and 6 hr (n = 6) after LTB4 inhalation. After measurement of airway responsiveness, total cell counts, differential cell counts, thromboxane B2 (TxB2) and 6-keto-prostaglandin (PG)F1 alpha levels in bronchoalveolar lavage fluid (BALF) were measured. The total cell counts in BALF increased after LTB4 inhalation (p less than 0.05), and the neutrophil counts in BALF increased significantly 1 hr (p less than 0.05) and 6 hr after (p less than 0.01) LTB4 inhalation. Airway responsiveness to inhaled methacholine decreased significantly 1 hr (p less than 0.05) and 6 hr (p less than 0.01) after LTB4 inhalation. There were no significant changes in the levels of TxB2 or 6-keto-PGF1 alpha in BALF 1 hr and 6 hr after LTB4 inhalation. These results suggest that inhaled LTB4 causes neutrophil recruitment into the airway but does not increase airway responsiveness to inhaled methacholine, and the possibility that LTB4 reduces airway responsiveness to inhaled methacholine exists in dogs.

Administration, Inhalation↗
PubMed1990

In vivo evidence for carrier-mediated uptake of beta-lactam antibiotics through organic anion transport systems in rat kidney and liver.

The transport mechanisms of beta-lactam antibiotics in the rat kidney and liver were studied with an in vivo tissue-sampling single-injection technique using [3H]benzylpenicillin [( 3H]PCG) as a substrate. Concentration-dependent uptake of [3H]PCG was observed in the kidney, and the in vivo kinetic parameters were estimated as follows: the maximum uptake rate (Jmax) was 6.88 mumol/min/g of kidney, MIchaelis constant (Kt) was 1.39 mM and nonsaturable first-order rate constant (kd) was 0.414 ml/min/g of kidney. The uptake of [3H]PCG was inhibited by organic anions but not by organic cations. Several beta-lactam antibiotics also reduced the uptake of [3H]PCG. Furthermore, the organic anion, probenecid, and beta-lactam antibiotic, cefpiramide, showed a dose-dependent inhibitory effect. These results suggest participation of an organic anion transport system in uptake of beta-lactam antibiotics across the renal plasma membrane. Saturable uptake of [3H]PCG was also observed in the liver and Jmax, Kt and Kd were estimated to be 3.62 mumol/min/g of liver, 3.59 mM and 0.223 ml/min/g of liver, respectively. The in vivo influx rate calculated from Jmax/Kt in the liver was 1.01 ml/min/g of liver and was close to the in vitro value, 1.54 ml/min/g of liver, estimated previously from isolated hepatocytes. Although dipeptides and organic cations showed no effect on the hepatic uptake of [3H]PCG, probenecid significantly reduced its uptake. Several beta-lactam antibiotics also reduced the uptake of [3H]PCG by the liver. These features of the hepatic uptake of beta-lactam antibiotics through an organic anion transport system are in agreement with the previous results obtained in isolated hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Membrane potential-dependent and carrier-mediated transport of cefpiramide, a cephalosporin antibiotic, in canalicular rat liver plasma membrane vesicles.

The biliary excretion mechanism of cefpiramide, a typical biliary excretion type cephalosporin antibiotic, was investigated by measuring the uptake by purified rat liver bile canalicular membrane vesicles. The initial uptake of cefpiramide showed significant temperature and concentration dependencies; the kinetic parameters were estimated to be 1.73 mM, 2.64 nmol/40 sec/mg protein and 0.17 nmol/40 sec/mg protein/mM for Michaelis constant, maximum uptake rate and nonsaturable uptake rate constant, respectively. An intravesicular positive membrane potential induced by valinomycin significantly enhanced the initial uptake of cefpiramide. Furthermore, by inducing an inside negative membrane potential, the initial efflux was significantly enhanced. Organic anions such as bile acids, bromosulfophthalein, bilirubin, probenecid, furosemide and 4,4'-diisothiocyanostilben-2,2'-disulfonic acid significantly reduced the uptake of cefpiramide, whereas amino acids, dipeptides, organic neutral compounds and cationic compounds had no effect. The inhibitory effects of taurocholic acid and bromosulfopthalein were especially competitive. Most of the beta-lactam antibiotics tested significantly reduced the uptake of cefpiramide in a competitive manner. Furthermore, benzylpenicillin showed a countertransport effect on the cefpiramide uptake. From these results it is suggested that neither organic cation nor dipeptide carrier systems participate in the uptake of beta-lactam antibiotics, and most of the derivatives are transported by recognition as organic anions in the bile canalicular membrane.

Animals↗
PubMed1989

Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors.

We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.

Amino Acids↗
PubMed1989

Chemiluminescent assay of various enzyme activities and its application to enzyme immunoassays.

A highly sensitive chemiluminescent assay for NAD(P)H have been developed. The principle of the method is as follows; NAD(P)H reduces molecular oxygen to superoxide anion (O2-) and hydrogen peroxide (H2O2) in the presence of 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS) as electron mediator. The produced O2- and H2O2 can be measured by chemiluminescent reaction using isoluminol (IL) and microperoxidase (m-POD). A linear relationship between chemiluminescence intensity and NAD(P)H concentration (log/log) was obtained ranged from 10(-9) mol/l to 10(-5) mol/l. This chemiluminescent reaction has been coupled to the assay of glucose-6-phosphate dehydrogenase (G6PDH), beta-D-galactosidase (beta-Gal) and alkaline phosphatase (ALP). The detection limits of G6PDH, beta-Gal and ALP were 10(-18) mol, 10(-20) mol and 10(-18) mol per assay, respectively. The chemiluminescent assay of these enzymes applied to chemiluminescent enzyme immunoassay for 17 alpha-hydroxy-progesterone and DNA hybridization assay using these enzymes as label.

Alkaline Phosphatase↗
PubMed1989

Chemiluminescent assay of co-factors.

Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperoxidase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 X 10(-14) to 5 X 10(-12) mol/assay. The detection limit of NADH was 30 fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.

Adenosine Triphosphate↗
PubMed1989

Two-dimensional electrophoresis aided by personal computer analysis for screening of mutant proteins in inherited diseases.

A rapid and reproducible method of two-dimensional electrophoresis was developed for screening of abnormal proteins expressed in fibroblasts from patients with inherited diseases. After silver staining, the electrophoresis gel was subjected to semiautomatic digitizer-personal computer analysis: scanning with an image sensor video camera connected to a digitizer, followed by quantitative determination and statistical analysis with a personal computer. The protein spots analyzed by this method showed quantitative variations of various degrees, particularly in 2 of 247 spots examined. Seven spots were not always detected in control and pathological cells in this study. Slight variations in molecular weight were observed in 3 different spots.

Adolescent↗
PubMed1989

Degradation kinetics of (+/-)-4'-ethyl-2-methyl-3-(1-pyrrolidinyl)propiophenone hydrochloride (HY-770) and structure-stability relationship among its analogues in aqueous solution.

The kinetics and pathways for degradation of (+/-)-4'-ethyl-2-methyl-3-(1-pyrrolidinyl)propiophenone hydrochloride (HY-770; 1), a newly developed muscle-relaxing agent, and its analogues were studied in aqueous solution at 50 degrees C, ionic strength 0.5 M, and pH 8.0-12.0. Compound 1 and its four analogues followed pseudo-first-order degradation kinetics at constant pH and temperature. From the analysis of the pH degradation-rate profiles, it is evident that specific hydroxide ion-catalyzed degradations of ionized and un-ionized species occur for 1 and its structural analogue, 3'-fluoro-2-methyl-3-(1-pyrrolidinyl)propiophenone hydrochloride (HN-961; 5). The hydroxide ion-catalyzed degradation of the ionized species was found to be 100 times faster than that of the un-ionized species and to be the major process at pH less than 9.0. On the contrary, 1 was extremely stable in 0.5 M HCl at 50 degrees C, suggesting that the hydronium ion-catalyzed degradation and the spontaneous degradation of the ionized species is negligible. The Arrhenius plot for the degradation of 1 at 35-50 degrees C and pH 9.0 showed that the apparent energy of activation was 22.0 kcal/mol. The degradation rates of the five structural analogues were significantly dependent on the electron withdrawing effect of the benzene substituents of the molecule.

Boric Acids↗