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Biomedical subjects

I Tamai

Publications and source records attributed to I Tamai.

At least 109 records · Page 6Linked to original sources
PubMed1993

Transport mechanism of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors at the blood-brain barrier.

The transport mechanism of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors through the blood-brain barrier was studied in vitro by using primary cultures of bovine brain capillary endothelial cells (BCEC). The uptake of HMG-CoA reductase inhibitors with the lactone form, [14C]lovastatin and [14C]simvastatin, was slightly decreased to 65% of the control uptake (37 degrees C) at low temperature (4 degrees C) and was not affected by pretreatment of the BCEC with metabolic inhibitors (2,4-dinitrophenol and rotenone). [14C]Simvastatin acid (the lactone ring-opened form) was taken up in a markedly temperature- and concentration-dependent fashion, whereas the uptake of [14C] pravastatin was negligible. At pH below 7.4, the uptake rate of [14C]simvastatin acid by the BCEC increased markedly with decreasing medium pH, whereas almost pH-independent uptake was observed in the presence of 1 mM simvastatin acid. Additional studies using an in situ rat brain perfusion method showed that the in vivo cerebrovascular permeation of [14C]simvastatin acid in rats was significantly inhibited in the presence of 1 mM simvastatin acid, demonstrating that the transport system for the acid forms of HMG-CoA reductase inhibitors functions under in vivo conditions. Several monocarboxylic acids significantly inhibited the uptake of [14C]simvastatin acid by the BCEC, whereas dicarboxylic acids did not. The uptake of [14C]simvastatin acid by the BCEC was competitively inhibited by 15 mM acetic acid. Accordingly, we concluded that HMG-CoA reductase inhibitors in lactone form are transported via simple diffusion, whereas those having an acid form are transported across the blood-brain barrier via a carrier-mediated transport mechanism for monocarboxylic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1992

P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells.

The expression of a functional P-glycoprotein (P-gp) which pumps drugs out of brain capillary endothelial cells (BCEC) into blood was studied by evaluating the steady-state uptake and efflux of vincristine (VCR) by primary cultured bovine BCEC. The steady-state uptake of VCR was increased in the presence of metabolic inhibitors, and an anti-P-gp monoclonal antibody, MRK16, as well as verapamil and steroid hormones which are known to reverse multidrug resistance in tumor cells. Furthermore, efflux of VCR from BCEC was inhibited by verapamil. By immunohistochemistry, P-gp was localized at the luminal side of the capillary endothelial cells in both gray matter of bovine brain and primary cultured BCEC. These data suggest that P-gp functions as a drug efflux pump at the luminal side of BCEC and regulates the transfer of certain lipophilic drugs from the blood into the brain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Mechanisms of intestinal absorption of the antibiotic, fosfomycin, in brush-border membrane vesicles in rabbits and humans.

In order to clarify the mechanism of intestinal absorption of an antibiotic, fosfomycin (FOM), the uptakes of FOM by rabbit and human small intestinal brush-border membrane vesicles (BBMV) were studied. The initial uptake of FOM by BBMV at 15 s was saturable at a higher concentration of FOM. The kinetic parameters at 37 degrees C of the saturable uptake expressed by the Michaelis-Menten equation were Kt = 5.17 mM and Jmax = 3.88 nmol/15 s/mg protein for rabbits, and Kt = 4.03 mM and Jmax = 1.90 nmol/15 s/mg protein for humans. The most efficient uptake was observed in the presence of both inward-directed Na(+)- and H(+)-gradients in both mammals. The uptake of FOM was inhibited by inorganic phosphate, FOM glycol which is a degradation product of FOM in the gastric juice and specific inhibitors of phosphate transport such as arsenate and phosphonoacetic acid. These findings confirmed that FOM absorption from rabbit and human small intestines is associated with the phosphate transport system. These transport phenomena of FOM are in close agreement with those obtained previously in rat BBMV studies. Judging from the results obtained for three mammalian species, rat, rabbit and human, it was concluded that carrier-mediated transport via the phosphate transport system is a very important pathway of intestinal absorption of FOM.

Animals↗
PubMed1991

Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug resistant cells.

It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells. In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins. The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL). Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine. P-gp in these cells was the only protein specifically bound to [3H]azidopine in photoaffinity experiments. The specific photoaffinity labeling of P-gp by [3H]azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively. The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein. Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Transport of the new quinolone antibacterial agents of lomefloxacin and ofloxacin by rat erythrocytes, and its relation to the nicotinic acid transport system.

Transport mechanism of quinolonecarboxylic acid derivatives (quinolones), lomefloxacin and ofloxacin, in rat erythrocytes was investigated. The uptake of lomefloxacin by erythrocytes was apparently nonsaturable over the concentration range of 0.1-1.0 mM at 27 degrees C and pH 7.4. In the case of ofloxacin, however, the erythrocytes took up ofloxacin in a concentration dependent manner at 20 degrees C and pH 7.4, with a maximum rate (Jmax) of 28.8 +/- 0.98 pmol/10(6) cells/s and a Michaelis constant (Kt) of 0.92 +/- 0.15 mM. An increase in the medium osmolarity caused a decrease in the [14C]lomefloxacin uptake, indicating that the drug is transported into the intracellular space of erythrocytes. The uptake of [14C]lomefloxacin by the erythrocytes was independent of pH but increased above pH 6.0, and, moreover, was dependent on temperature with an activation energy of 16.6 kcal/mol. The uptake of [14C]lomefloxacin was competitively inhibited by a water-soluble vitamin, nicotinic acid, with an inhibition constant (Ki) of 16.1 mM, but not inhibited by 10 mM of L-alanine, glucose, p-aminohippuric acid (PAH), N-methyl-nicotinamide (NMN) or tetraethylammonium chloride (TEA). These lines of evidence suggest that the transport system of lomefloxacin and ofloxacin in rat erythrocytes is common with that of a water-soluble vitamin, nicotinic acid, to which quinolones are structurally analogous.

Animals↗
PubMed1991

Participation of monocarboxylic anion and bicarbonate exchange system for the transport of acetic acid and monocarboxylic acid drugs in the small intestinal brush-border membrane vesicles.

A participation of bicarbonate exchange system for the transport of acetic acid and the related monocarboxylic acid drugs in the intestinal brush-border membrane vesicles (BBMVs) was investigated. The uptake of [3H]acetic acid at 37 degrees C by BBMVs was markedly stimulated and showed a clear overshoot phenomenon in the presence of outward-directed bicarbonate gradient (pHin = 7.5, [KHCO3]in or [NaHCO3]in = 100 mM; pHout = 7.5, [K-gluconate]out or [Na-gluconate]out = 100 mM). This uptake process was saturable (Kt = 50.4 +/- 4.96 mM and Jmax = 11.6 +/- 0.61 nmol/mg protein/10 s) and was inhibited by DIDS (4,4-diisothiocyano-2,2'-disulfonic acid stilbene disodium salt) and furosemide, anion exchange inhibitors, and by many monocarboxylates. The initial uptake of [3H]acetic acid was competitively inhibited by salicylic acid, suggesting the common transport between acetic acid and salicylic acid. At lower extravesicular pHs and in the presence of outward-directed bicarbonate gradient (pHin = 7.5 [KHCO3]in = 100 mM; pHout = 6.0 or 5.0, [K-gluconate]out = 100 mM) where membrane potential was clamped to zero by K(+)-valinomycin, the uptake of [3H]acetic acid showed an overshoot phenomenon, whereas the uptake was significantly decreased in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a protonophore. It was concluded, therefore, that there are one or two mechanisms for the carrier-mediated transport of acetic acid and monocarboxylates related to bicarbonate exchange systems in rabbit intestinal BBMVs: 1) proton gradient independent and bicarbonate exchange system; 2) proton gradient dependent and bicarbonate exchange system.

Acetates↗
PubMed1990

Competitive interaction of cyclosporins with the Vinca alkaloid-binding site of P-glycoprotein in multidrug-resistant cells.

The mechanism of reversal of resistance to Vinca alkaloids by cyclosporins is unclear. We investigated the molecular mechanism of reversal of Vinca alkaloid resistance by cyclosporin A (CsA) and its nonimmunosuppressive analog O-acetyl C9(1) CsA (SDZ 33-243) in multidrug resistant DC-3F/VCRd-5L Chinese hamster cells. CsA at 3 microM increased vincristine (VCR) sensitivity and almost totally reversed VCR resistance. SDZ 33-243 at 1 microM reduced the IC50 for VCR in resistant cells from 62.0 to 0.00062 microM. CsA and SDZ 33-243 at 10 microM increased [3H]vinblastine (VBL) accumulation in DC-3F/VCRd-5L cells by 27- and 22-fold, respectively. At 10 microM, these compounds also increased [3H]VCR accumulation by 3.5- and 4.0-fold, respectively. [3H]VCR uptake by membrane vesicles from DC-3F/VCRd-5L cells showed high and low affinity components with Michaelis-Menten kinetics, and apparent Km values were 0.140 +/- 0.0523 and 24.8 +/- 6.67 microM, respectively. Kinetic analysis of [3H]VCR uptake in membrane vesicles in the presence of 0.2 microM CsA revealed that CsA competitively inhibited the high affinity [3H]VCR uptake with an apparent inhibition constant (Ki) of 0.126 +/- 0.0173 microM. In addition, CsA and SDZ 33-243 inhibited VBL photoaffinity labeling of P-glycoprotein in a dose-dependent manner, with half-maximum inhibition at 0.5 and 0.4 microM, respectively, compared with that of VBL at 0.6 microM. These data confirm that cyclosporins modulate Vinca alkaloid resistance at least partially through interaction with P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

The alpha 1-adrenergic photoaffinity probe [125I]arylazidoprazosin binds to a specific peptide of P-glycoprotein in multidrug-resistant cells.

Much evidence suggests that P-glycoprotein (P-gp) confers multidrug-resistance (MDR) in tumor cells by energy-dependent efflux of hydrophobic cytotoxic agents. In this study, we have used the alpha 1-adrenergic photoaffinity probe, [125I]arylazidoprazosin ([125I]AAP), and identified P-gp as a specific acceptor for prazosin. Drugs to which MDR cells are resistant, including vincristine, vinblastine, doxorubicin, actinomycin D and colchicine as well as agents reversing MDR, including verapamil, nicardipine, prenylamine, diltiazem, trifluoperazine, dibucaine, reserpine, monensin, and progesterone, differentially reduced [125I]AAP photolabeling of P-gp. We also analyzed the influence of alpha 2-adrenergic drugs and dopaminergic drugs on [125I]AAP photolabeling of P-gp. Limited proteolysis of [125I]AAP photolabeled P-gp with Staphylococcus aureus V8 protease revealed that prazosin binds to a single 8 kDa fragment of P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Molecular basis of preferential resistance to colchicine in multidrug-resistant human cells conferred by Gly-185----Val-185 substitution in P-glycoprotein.

Expression of P-glycoprotein, encoded by the human MDR1 gene, results in cross-resistance to many lipophilic cytotoxic drugs (multidrug resistance). P-glycoprotein is believed to function as an energy-dependent efflux pump that is responsible for decreased drug accumulation in multidrug-resistant cells. Previous work showed that preferential resistance to colchicine in a colchicine-selected multidrug-resistant cell line was caused by spontaneous mutations in the MDR1 gene that resulted in a Gly-185----Val-185 substitution in P-glycoprotein. We have now compared transfectant cell lines expressing either the wild-type Gly-185 or the mutant Val-185 P-glycoprotein with regard to their levels of resistance to and accumulation and binding of different drugs. In cells expressing the mutant protein, increased resistance to colchicine and decreased resistance to vinblastine correlated with a decreased accumulation of colchicine and increased accumulation of vinblastine. Expression of the mutant P-glycoprotein also resulted in significantly increased resistance to epipodophyllotoxin and decreased resistance to vincristine and actinomycin D; smaller changes in resistance were observed for several other drugs. Unexpectedly, the mutant P-glycoprotein showed increased binding of photoactive analogs of vinblastine and verapamil and the photoactive compound azidopine and decreased binding of a photoactive colchicine analog. These results suggest that the Gly-185----Val-185 substitution affects not the initial drug-binding site of P-glycoprotein but another site, associated with the release of P-glycoprotein-bound drugs to the outside of the cell.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
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 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

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↗
PubMed1989

Age-related change in tissue-to-plasma partition coefficient of cefazolin for noneliminating organs in the rat.

Age-related changes in tissue distribution characteristics of cefazolin, a cephalosporin antibiotic, were examined for noneliminating organs of rats. The in vivo tissue-to-plasma partition coefficients (Kp,vivo) varied markedly among different ages and organs. In particular, muscle and skin acted as reservoirs for cefazolin distribution. There were also marked differences in interstitial fluid space (IS), determined using [14C]inulin, among different ages and organs. For muscle and bone, the magnitude of the age-related changes in Kp,vivo of cefazolin and IS was in the order of 1-week-old greater than 7-week-old = 100-week-old greater than 50-week-old rats. This is well correlated with the age-related changes in the volume of distribution at the steady state of cefazolin per body weight (Vdss/BW) and the extracellular fluid volume per body weight (Vecw/BW) determined previously using [14C]inulin. The predicted Kp value (Kp,pred) was estimated by incorporating the serum protein binding parameters of cefazolin, the IS values, and an interstitial-to-plasma albumin concentration ratio (AR) into equations derived from an extracellular fluid model. The Kp,pred values exhibited a fairly good correspondence with the Kp,vivo values determined for various organs, except gut, in rats of all four ages. These results suggest that the determinant of the age-related change in Vdss/BW is the difference in the IS value of muscle and bone, while the age-related change in serum protein binding plays only a modest role.

Adolescent↗