Biomedical subjects

Y Sai

Publications and source records attributed to Y Sai.

At least 55 records · Page 3Linked to original sources

PubMed1998

Inhibitory monoclonal antibodies to human cytochrome P450 1A2: analysis of phenacetin O-deethylation in human liver.

Human cytochrome P450 1A2 metabolizes a large number of common drugs and engages in carcinogen metabolism and activation. Baculovirus-expressed 1A2 was used to immunize mice producing hybridomas yielding monoclonal antibodies (MAbs). Three of 2050 clones assayed yielded the MAbs, MAb 26-7-5, MAb 951-5-1, MAb 1812-2-4, which were specific for 1A2 as assessed by enzyme-linked immunosorbent assay and immunoblots. The three MAbs inhibited 1A2-catalysed metabolism of phenacetin, 7-ethoxycoumarin, chlorzoxazone and phenanthrene by more than 85%. The MAbs were highly specific to 1A2 and did not inhibit 11 other human P450s. The phenancetin O-deethylation activity varied from 0.44-2.49 nmol/min/nmol P450 in eight human liver microsomes samples. MAb 26-7-5 inhibited 1A2-dependent phenacetin O-deethylation in these samples by 64-84% indicating the amount of 1A2 contribution to this reaction and in addition a role for other P450s in the O-deethylation. Independent analysis of recombinant human P450s showed that 1A1, 1A2, 2A6 and 2C19 exhibited phenacetin O-deethylation activity, with 1A1 and 1A2 being the most active followed by 2C19 and 2A6. Eight other P450s were inactive towards phenacetin O-deethylation. The role of different P450 in eight liver samples was analysed with specific individual inhibitory MAbs. Inhibitory antibodies to 1A2, 2C8/9/18/19, 2A6, 2D6, 2E1, and 1A1 were combinatorially added to the microsomes. The O-deethylation activity was inhibited by antibodies to 1A2 (64-84%), to 2C19 (4.6-20%) and to 2A6 (0-8.8%). The total activity inhibited by antibodies to P450 2E1, 2D6 and 1A1 was less than 4.5%, indicating a minor role for these P450s in phenancetin metabolism in human liver microsomes. Thus, 1A2, 2C 9 and 2A6 are the dominant P450s for phenacetin O-deethylation. These studies demonstrate the use of inhibitory MAbs to P450s for a simple and precise assessment of the quantitative role of each P450 in the metabolism of substrates, including drugs, carcinogens, mutagens, environmental chemicals and endobiotics.

Antibodies, Monoclonal↗

PubMed1998

Adsorptive-mediated endocytosis of a basic peptide in enterocyte-like Caco-2 cells.

The internalization of a basic peptide, 001-C8 [H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2], into enterocyte-like Caco-2 cells was evaluated. Internalization of 125I-labeled 001-C8 (125I-001-C8) increased time dependently and reached steady state at 60 min. The steady-state internalization of 125I-001-C8 (7.24 +/- 0. 41 microl/mg protein) was temperature and concentration dependent and was significantly decreased by dansylcadaverine (500 microM), protamine (1 mM), poly-L-lysine (1 mM), E-2078 (1 mM), and ebiratide (1 mM), whereas poly-L-glutamic acid (1 mM), tyrosine (1 mM), and glycylglycine (25 mM) were not inhibitory. Predigestion of acid mucopolysaccharides by heparinase I, heparitinase, and chondroitinase ABC also decreased the internalization. The maximal internalization, the half-saturation constant, and the nonsaturable internalization of 125I-001-C8 were 1.13 +/- 0.23 pmol/mg protein, 0. 47 +/- 0.43 microM, and 3.13 +/- 0.19 microl/mg protein, respectively. Confocal microscopy also indicated the internalization of fluorescence-derived 001-C8 [001-C8-4-nitrobenz-2-oxa-1,3-diazole (001-C8-NBD)]. Granular staining seen within the cell, excluding nuclei, indicated the sequestration of 001-C8-NBD within endocytotic vesicles. Dansylcadaverine and protamine strongly decreased the granular distribution of 001-C8-NBD within the cell. These results demonstrate that 001-C8 is taken up by Caco-2 cells via adsorptive-mediated endocytosis.

Caco-2 Cells↗

PubMed1998

Hepatobiliary transport kinetics of HSR-903, a new quinolone antibacterial agent.

HSR-903 is a newly synthesized quinolone antibacterial agent with low toxicity. The biliary and urinary excretion of unchanged HSR-903, its R-isomer, and their glucuronides was determined after iv bolus administration (5 mg/kg) to normal Sprague-Dawley rats (SDR) and Eisai hyperbilirubinemic mutant rats (EHBR). The values for the biliary excretion clearance of HSR-903 and its glucuronide in EHBR were decreased to approximately 40 and 2% of those in SDR, respectively, whereas the values for the urinary excretion clearance of HSR-903 and its glucuronide were comparable in SDR and EHBR. The biliary excretion clearance values for the R-isomer and its glucuronide were approximately 3 times greater than those for HSR-903. These results demonstrated that the enantiomers of HSR-903 and their conjugates were excreted into bile in a stereospecific manner. The hepatic uptake of [14C]HSR-903 in vivo was evaluated by means of integration plot analysis. The results indicated that the hepatic uptake of [14C]HSR-903 was very fast and was blood flow-limited. To clarify the mechanism of excretion of HSR-903 into bile, the uptake and efflux of [14C]HSR-903 were studied using isolated hepatocytes from SDR and EHBR. The initial uptake of HSR-903 by hepatocytes was temperature-dependent, saturable, and stereospecific. Unlabeled HSR-903 (S-isomer), the R-isomer, grepafloxacin, and sparfloxacin significantly inhibited the uptake of [14C]HSR-903. The efflux of [14C]HSR-903 from hepatocytes from EHBR was significantly slower than that from hepatocytes from SDR. The addition of sodium azide or bromosulfophthalein reduced the efflux of [14C]HSR-903. These results demonstrate that HSR-903 is actively excreted into bile via the canalicular multispecific organic anion transporter, which is deficient in EHBR.

Animals↗

PubMed1997

Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1.

cDNA for a novel proton/organic cation transporter, OCTN1, was cloned from human fetal liver and its transport activity was investigated. OCTN1 encodes a 551-amino acid protein with 11 transmembrane domains and one nucleotide binding site motif. It is strongly expressed in kidney, trachea, bone marrow and fetal liver and in several human cancer cell lines, but not in adult liver. When expressed in HEK293 cells, OCTN1 exhibited saturable and pH-dependent [3H]tetraethyl ammonium uptake with higher activity at neutral and alkaline pH than at acidic pH. Furthermore, treatment with metabolic inhibitors reduced the uptake, which is consistent with the presence of the nucleotide binding site sequence motif. Although its subcellular localization and detailed functional characteristics are not clear at present, OCTN1 appears to be a novel proton antiporter that functions for active secretion of cationic compounds across the renal epithelial brush-border membrane. It may play a role in the renal excretion of xenobiotics and their metabolites.

Adenosine Triphosphate↗

PubMed1997

Carrier-mediated transport of oligopeptides in the human fibrosarcoma cell line HT1080.

To explore the feasibility of targeting human tumor cells via their transport systems, dipeptide uptake was studied in the human fibrosarcoma cell line HT1080 and the human fibroblast cell line IMR-90 by the use of hydrolysis-resistant glycylsarcosine (Gly-Sar). The uptake of [14C]Gly-Sar into HT1080 was time dependent. Kinetic analysis of the concentration dependence of the initial rate of [14C]Gly-Sar uptake showed that a carrier-mediated transport system with a K(m) of 11.4 +/- 3.3 mM and V(max) of 26.8 +/- 4.0 (nmol/15 min/mg protein) and a nonsaturable component (k(d) of 0.80 microl/15 min/mg protein) were responsible for the dipeptide uptake by HT1080 cells. The optimal pH for the maximal uptake was around 6.0. [14C]Gly-Sar uptake was inhibited by various di- and tripeptides and peptide-mimetic drugs, such as bestatin and cefadroxil. [14C]Gly-Sar uptake was not affected by the presence of amino acids or tetra- or pentapeptides. The uptake of cefadroxil was reduced significantly by unlabeled Gly-Sar. Moreover, Gly-Gly and Gly-Leu produced an increase in the apparent K(m) of the uptake of Gly-Sar without altering V(max). On the other hand, dipeptide uptake by IMR-90, which is a normal diploid cell line (not malignant), showed no saturable transport. These results suggest that HT1080 cells take up dipeptides via a pH-dependent transporter. This is the first report showing that a dipeptide transport system, which is similar but not identical to the well-characterized oligopeptide transporters PepT1 and PepT2, exists in fibroblast-derived tumor cells but not in normal fibroblasts. The present finding could be the basis of a novel strategy for the specific delivery of oligopeptide-mimetic anticancer drugs into tumor cells.

Biological Transport↗

PubMed1997

Brain-to-blood active transport of beta-alanine across the blood-brain barrier.

A high-affinity antiluminal uptake system for beta-alanine was demonstrated in primary cultured bovine brain capillary endothelial cells (BCEC) for which K(t) is 66.9 microM. beta alanine uptake was energy-, sodium- and chloride ion-dependent. beta-amino acids strongly inhibited the uptake, while alpha- and gamma-amino acids had a little or no inhibitory effect. In ATP-depleted cells, the uptake was stimulated by preloading beta-alanine or taurine but not by L-leucine. These results suggest that beta-alanine is actively transported across the antiluminal membrane of BCECs that is common to beta-amino acids. The system may function for the efflux from the brain to blood.

Amino Acids↗

PubMed1997

Recognition of novel artifacts produced during the microsomal incubation of secondary alicyclic amines in the presence of cyanide.

1. During the in vitro microsomal metabolism of the secondary alicyclic amines, nornicotine, anabasine or 4-benzylpiperidine in the presence of cyanide, a new product was formed. 2. Comparison of these new products with authentic compounds by hplc and ms showed they were the corresponding 1'-N-cyanomethyl compounds. 3. The source of the methylene group was shown to be formaldehyde, arising during incubation, from either microsomal lipid and/or glycerol used to store microsomes for long periods at low temperature. 4. The formation of the 1'-N-cyanomethyl compounds was enhanced in the presence of substrates producing formaldehyde during their metabolism, in direct relationship to the amount of formaldehyde generated. 5. The results indicate that care should be taken in interpreting data obtained from incubates containing secondary amines and cyanide, especially in the presence of substrates capable of donating formaldehyde.

Amines↗

PubMed1997

The predominant contribution of oligopeptide transporter PepT1 to intestinal absorption of beta-lactam antibiotics in the rat small intestine.

Although recent evidence suggests that certain beta-lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of beta-lactam antibiotics was evaluated. For evaluation of transport activity complementary RNA (cRNA) of rat PepT1 was synthesized in-vitro and expressed in Xenopus laevis oocytes. cRNA induced uptake of several beta-lactam antibiotics and the dipeptide [14C]glycylsarcosine; this was specifically inhibited by various dipeptides and tripeptides but not by their constituent amino acids or by tetra- or pentapeptides. The transport activity of PepT1 for beta-lactam antibiotics correlated well with their in-vivo intestinal transport and absorption. Furthermore, mutual inhibitory effects on uptake were observed between glyclsarcosine and beta-lactam antibiotics. Hybrid depletion of the functional expression of rat PepT1 in oocytes injected with rat intestinal epithelial total mRNA was studied using an antisense oligonucleotide corresponding to the 5'-coding region of PepT1. In oocytes injected with rat mRNA pre-hybridized with the antisense oligonucleotide against rat PepT1, the uptake of [14C]glycylsarcosine was almost completely abolished, whereas its uptake was not influenced by a sense oligonucleotide for the same region of PepT1. Similarly, the uptake of beta-lactam antibiotics was also reduced by the antisense oligonucleotide against rat PepT1. These results demonstrate that the intestinal proton-coupled oligopeptide transporter PepT1 plays a predominant role in the carrier-mediated intestinal absorption of beta-lactam antibiotics and native oligopeptides in the rat.

Animals↗

PubMed1997

Intestinal brush-border membrane transport of monocarboxylic acids mediated by proton-coupled transport and anion antiport mechanisms.

Intestinal brush-border membrane transport of monocarboxylic acids was investigated by using rabbit intestinal brush-border membrane vesicles (BBMVs) and isolated intestinal tissues mounted on Ussing-type chambers. [3H]Mevalonic acid uptake by BBMVs showed an overshoot phenomenon in the presence of an inwardly directed proton gradient, but not in the presence of an inwardly directed sodium gradient or an outwardly directed HCO3- or chloride gradient. Initial uptake of mevalonic acid was saturable in the presence of a proton gradient. Uptake of [3H]mevalonic acid was inhibited by various monocarboxylic acids, including acetic acid, benzoic acid, lactic acid, nicotinic acid, pravastatin, salicylic acid and valproic acid, but not by dicarboxylic acid or amino acids. Acetic acid, which is transported by both anion antiport and proton-coupled transport systems, induced serosal bicarbonate-dependent alkalinization in the mucosal-side bathing solution of rabbit jejunal tissues, when examined in Ussing-type chambers. Pravastatin, which is a structural analogue of mevalonic acid and is absorbed via proton-coupled transport like mevalonic acid, did not. The result demonstrates that acetic acid is transported by the bicarbonate-dependent anion antiport system, whereas pravastatin is not. So, it is suggested that monocarboxylic acids are transported by at least two independent transporters, namely, a proton-coupled transporter for most monocarboxylic acids, including mevalonic acid, pravastatin and acetic acid, and an anion antiporter for acetic acid, but not for mevalonic acid or pravastatin. Activation of anion antiporter can induce HCO3- secretion in intact intestine.

Acetic Acid↗

PubMed1997

Structure-internalization relationship for adsorptive-mediated endocytosis of basic peptides at the blood-brain barrier.

For the purpose of the brain delivery of peptides, the structural specificity of adsorptive-mediated endocytosis at the blood-brain barrier was studied by measuring transport of a newly synthesized basic peptide 001-C8, H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)aNH2, using primary cultured bovine brain capillary endothelial cells. The apparent uptake of [125I]001-C8 increased time-dependently and reached a steady-state at 60 min. The steady-state uptake of [125I]001-C8 was temperature and concentration dependent and was significantly decreased in the presence of dansylcadaverine, protamine or poly-L-lysine. Uptakes of peptides modified by 1,8-octanediamine, 1,5-pentanediamine, 1,2-ethanediamine or ethylamide and peptides with a free carboxyl terminal were significantly higher than, and similar to, that of [3H]PEG900, respectively. The half-saturation constants and the maximal uptake capacities of these peptides were in the ranges of 0.2 to 134 microM and 1.1 to 408 pmol/mg protein, respectively. These values were correlated with the basicity of the molecules. In conclusion, not the number of constituent amino acids of peptides, but rather the C-terminal structure and the basicity of the molecules are the most important determinants for the uptake by the adsorptive-mediated endocytosis system at the blood brain barrier.

Adsorption↗

PubMed1997

Nonlinear intestinal absorption of 5-hydroxytryptamine receptor antagonist caused by absorptive and secretory transporters.

The mechanism of the nonlinear concentration dependence of intestinal absorption of the 5-hydroxytryptamine receptor antagonist azasetron was studied by use of rat in situ intestinal perfusion, as well as an in vitro Ussing-type chamber method mounted with rat intestinal tissue and cultured monolayers of human adenocarcinoma Caco-2 cells. The intestinal absorption rate constant of azasetron evaluated by the Doluisio method increased significantly with increasing concentration of azasetron up to 10 mM in a nonlinear fashion and tended to decrease at higher concentrations. Mucosal-to-serosal directed permeation of [14C]azasetron across rat ileal sheets evaluated by the in vitro Ussing-type chamber method also increased in a nonlinear fashion in a low concentration range, followed by a decrease as the concentration was further increased, whereas serosal-to-mucosal directed permeation decreased in a concentration-dependent manner. Vectorial transport of [14C]azasetron across a Caco-2 cell monolayer was observed, with higher transport in the basolateral-to-apical direction at a trace concentration of azasetron. When the initial uptake rate of azasetron by Caco-2 cells was measured, it was saturable with an apparent half-saturation concentration of 15 mM and was reduced in the presence of several cationic compounds. These observations suggest that azasetron is taken up by a carrier-mediated transport mechanism across the intestinal epithelial cells. When the steady-state uptake of [14C]azasetron was measured, it was increased in the presence of unlabeled azasetron and ondansetron. In addition, the steady-state uptake was enhanced in the presence of a P-glycoprotein inhibitor, cyclosporin A, and by ATP-depletion of the cells, although these treatments had no effect on the initial uptake of [14C]azasetron. Furthermore, the multidrug-resistant cancer cell line K562/ADM that overexpresses P-glycoprotein accumulated azasetron less extensively than did the parental drug-sensitive K562 cells. These results strongly suggest that azasetron is secreted into the intestinal lumen predominantly by P-glycoprotein. We conclude that intestinal transport of azasetron involves specialized transporters in both the absorptive and secretory directions, and the complex nonlinear intestinal absorption characteristics can be ascribed to the participation of multiple transport mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Immunolocalization and pharmacological relevance of oligopeptide transporter PepT1 in intestinal absorption of beta-lactam antibiotics.

A polyclonal antibody (anti-PepT1/C) was raised against the rabbit intestinal H(+)-coupled oligopeptide transporter, PepT1. Anti-PepT1/C detected 70-80-kDa protein in crude membranes obtained from rabbit duodenum, jejunum and ileum. PepT1 was localized in the brush-border of the absorptive epithelial cells by subcellular fractionation of membranes on a sucrose density gradient and by immunohistochemistry using light and electron microscopy. Transport activity for cephalosporins and dipeptide expressed in Xenopus laevis oocytes injected with total mRNA obtained from rabbit small intestine was eliminated completely by prehybridization of the mRNA with antisense oligonucleotide against the 5'-coding region of rabbit PepT1 cDNA.

Amino Acid Sequence↗

PubMed1996

Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter.

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.

Age Factors↗

PubMed1996

Contribution of lysosomes to the subcellular distribution of basic drugs in the rat liver.

PURPOSE: We examined the subcellular distribution of the basic drugs, chlorpromazine (CPZ), imipramine (IMP) and biperiden (BP), in rat liver, and evaluated the contribution of lysosome (Lys) to their intracellular distribution in comparison with that of mitochondria (Mit). METHODS: In an in vivo distribution, the concentrations of CPZ, IMP and BP in the liver subcellular fractions were determined. In an in vitro study, uptake of [3H]IMP into Lys and Mit fractions was determined in the presence or absence of several agents. RESULTS: The distribution of these drugs 10 min after administration was quite similar. However, the relative specific contents (the drug concentration per protein of each fraction divided by that of the total homogenate) in Lys were 7.3, 9.6 and 4.2, respectively for CPZ, IMP and BP, whereas those in the other organella were only 0.4 approximately 1.7. In an in vitro uptake study, the dose response of IMP uptake into Lys was biphasic, while that into Mit fractions was monophasic. The binding of IMP to the high affinity sites of Lys was pH dependent and disappeared in 50 mM NH4Cl or 50 microM CPZ, both of which increased the intralysosomal pH, the low affinity sites were not affected by these drugs. CONCLUSIONS: The results indicated that Lys has the highest affinity for the basic drugs in the liver and that its contribution to their subcellular distribution depends on the intralysosomal pH, which is also affected by these drugs. The importance of these effects may become significant in combination therapy using various basic drugs.

Animals↗

PubMed1996

Stereoselective and carrier-mediated transport of monocarboxylic acids across Caco-2 cells.

PURPOSE: To characterize the transport mechanism of monocarboxylic acids across intestinal epithelial cells by examining the stereoselectivity of the transcellular transport of several chiral monocarboxylic acids. METHODS: The transport of monocarboxylic acids was examined using monolayers of human adenocarcinoma cell line, Caco-2 cells. RESULTS: The permeability of L-[14C]lactic acid at a tracer concentration (1 microM) exhibited pH- and concentration-dependencies and was significantly greater than that of the D-isomer. The permeabilities of both L-/D-[14C]lactic acids involve saturable and nonsaturable processes; the saturable process showed a higher affinity and a lower capacity for L-lactic acid compared with the D-isomer, while no difference between the isomers was seen for the nonsaturable process. The transport of L-lactic acid was inhibited by chiral monocarboxylic acids such as (R)/ (S)-mandelic acids and (R)/(S)-ibuprofen in a stereoselective manner. Mutually competitive inhibition was observed between L-lactic acid and (S)-mandelic acid. CONCLUSIONS: Some chiral monocarboxylic acids are transported across the intestinal epithelial cells in a stereoselective manner by the specific carrier-mediated transport mechanism.

Biological Transport↗

PubMed1996

Sodium and chloride ion-dependent transport of beta-alanine across the blood-brain barrier.

The characteristics of beta-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the beta-[3H]alanine transport indicated that the transporter for beta-alanine functions with Kt of 25.3 +/- 2.5 microM and Jmax of 6.90 +/- 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. Beta-[3H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN3) and low temperature reduced the uptake significantly. Furthermore, the uptake of beta-[3H]alanine required Na+ and Cl- in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one beta-alanine molecule. The Na+ and Cl--dependent uptake of beta-[3H]alanine was stimulated by a valinomycin-induced inside-negative K+-diffusion potential. beta-Amino acids (beta-alanine, taurine, and hypotaurine) inhibited strongly the uptake of beta-[3H]-alanine, whereas alpha- and gamma-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of beta-[3H]alanine was stimulated by preloading of beta-alanine or taurine but not L-leucine. These results show that beta-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to beta-amino acids.

Amino Acids↗

PubMed1996

Blood-brain-barrier transport of lipid microspheres containing clinprost, a prostaglandin I2 analogue.

Because the permeability of the blood-brain barrier to lipid microspheres (LMs) has not hitherto been demonstrated, blood-brain-barrier permeability to LM containing the prostaglandin I2 analogue clinprost has been evaluated for an in-vitro system of primary cultured monolayers of bovine brain capillary endothelial cells (BCECs), by a capillary depletion study in rats and by an in-situ brain perfusion study in normal and 4-vessel-occluded fore brain ischaemic rats. Although energy-dependency was not observed in [3H]clinprost uptake by BCECs, in accordance with results for simple diffusional transport, uptake of [3H]clinprost contained in lipid microspheres (denoted [3H]clinprost(LM)) was significantly inhibited by the endocytosis inhibitor, dansylcadaverine. The transport of LM into BCECs by endocytosis was also confirmed by fluorescence microscopy and flow-cytometric analysis using LM labelled with a fluorescent probe, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). The absolute uptake of Dil(LM) by BCECs, measured by HPLC, was, however, almost 1/10 that of [3H]clinprost(LM), results which suggest the superiority of simple diffusion of clinprost over endocytosis of its LM form in the uptake of clinprost(LM) by BCECs. In the capillary-depletion study with rat-brain-perfused [3H]clinprost(LM) from the internal carotid artery, the parenchyma apparent distribution volume was about 45 times larger than that of the capillary, showing that [3H]clinprost(LM) was transported through the blood-brain barrier into the brain. The permeability coefficients of [3H]clinprost and [3H]clinprost(LM) determined by insitu brain perfusion in normal rats were considerably higher than those of the active metabolite [3H]isocarbacyclin and its LM form. In addition, the Blood-brain-barrier permeabilities to [3H]clinprost, [3H]isocarbacyclin and their LM forms in ischaemic rats were almost identical to those in normal rats. It was concluded that clinprost(LM) was transported through the blood-brain barrier by endocytosis of LM, simple diffusion of clinprost released from LM, and transport of isocarbacyclin generated by hydrolysis of clinprost. The blood-brain-barrier permeability of clinprost(LM) is not reduced in ischaemic conditions, because the simple diffusion of clinprost released from LM contributed mainly to clinprost(LM) transport.

Animals↗

PubMed1996

Active secretion of drugs from the small intestinal epithelium in rats by P-glycoprotein functioning as an absorption barrier.

Because the significance of P-glycoprotein in the in-vivo secretion of beta-blockers in intestinal epithelial cells is unclear, the secretory mechanism for beta-blockers and other drugs has been evaluated. Uptake of the beta-blockers acebutolol, celiprolol, nadolol and timolol, and the antiarrhythmic agent, quinidine by the multidrug-resistant leukaemic cell line variant K562/ADM was significantly lower than that by drug-sensitive K562 cells, suggesting that these beta-blockers are transported by P-glycoprotein out of cells. The reduced uptake of acebutolol by the drug-resistant K562/ADM cells was reversed by treating the cells with anti-P-glycoprotein monoclonal antibody, MRK16, whereas no such alteration in uptake was observed for drug-sensitive K562 cells. Acebutolol uptake by K562/ADM cells was, moreover, markedly enhanced, in a concentration-dependent manner, in the presence of the specific P-glycoprotein inhibitors, MS-209 and cyclosporin. Caco-2 cells were used for evaluation of the role of P-glycoprotein in intestinal permeability to drugs in-vitro. Basolateral-to-apical transport of acebutolol was twice that in the reverse direction. A similar polarized flux was also observed in the transport of vinblastine, but not in that of acetamide or mannitol. When in-vivo intestinal absorption was evaluated by the rat jejunal loop method, with simultaneous intravenous administration of a P-glycoprotein inhibitor, cyclosporin, intestinal absorption of both acebutolol and vinblastine increased 2.6- and 2.2-fold, respectively, but no such enhancement was observed in the absorption of acetamide. The effect of cyclosporin on the intestinal absorption of several drugs was further examined, and the extent of the contribution of P-glycoprotein as an absorption barrier to those drugs was evaluated. ATP depletion by occlusion of the superior mesenteric artery resulted in a clear increase in epithelial permeability to vinblastine, but not to 3-O-methylglucose or acetamide, indicating that vinblastine is secreted by ATP-dependent P-glycoprotein into the lumen. These findings demonstrate that P-glycoprotein plays a role as an absorption barrier by transporting several drugs from intestinal cells into the lumen.

ATP Binding Cassette Transporter, Subfamily B, Mem↗