Methotrexate transport in the human intestine. Evidence for heterogeneity.

The transport of methotrexate (MTX) was investigated in organ-cultured endoscopic biopsy specimens of intestinal mucosa from normal subjects. In biopsy specimens from the proximal small intestine incubated with [3H]MTX (0.1 microM) for 2 hr at pH 5.5, [3H]MTX accumulated in the intracellular fluid to a concentration 3.5-fold higher than that of the medium, but at pH 6.5 and 7.5, the concentration was the same as that of the medium. In biopsy specimens from the cecum incubated under similar conditions, no accumulation against a concentration gradient was found. However, the accumulation of MTX was significantly higher at pH 5.5 than at 7.5. At [MTX] = 0.1 microM, the initial rate of MTX transport in the small intestine was significantly affected by medium pH and was optimal at pH 5.5. The relationship between the initial rate of uptake and medium [H+] was hyperbolic, suggestive of saturability with respect to [H+] with a Km of 132.2 nm [H+], corresponding to a medium pH of 6.88. At medium [MTX] = 10 microM, this effect was abolished. At pH 5.5, the relationship between the initial rate of uptake and medium [MTX] was sigmoidal, suggestive of a positive cooperativity, with napp of 1.8. [MTX] at 0.5 Vmax was 20.37 microM. Folic acid inhibited 37% of MTX flux. At pH 7.5, the relationship between the initial rate of uptake of MTX and medium [MTX] was linear. These data indicate the presence of a proton-dependent active transport of MTX in the human proximal small intestine, which is partially shared with folic acid.     

Binding and transport of methotrexate and its derivative, MX-68, across the brush-border membrane in rat kidney

Binding and transport properties of methotrexate (MTX) and its novel derivative, MX-68, were examined in brush-border membrane vesicles (BBMVs) isolated from rat kidneys. The uptake of MTX, MX-68 and folic acid by BBMVs was stimulated by an inwardly-directed H(+) gradient. Such H(+)-dependent uptake of folic acid is compatible with a previous report (Bhandari et al., Biochim Biophys Acta 1988; 937: 211). The MTX uptake exhibits saturation with a K(m) of 0.834 microM. Although the uptake of these three compounds at optimal pH depended on the osmolarity of the medium, a substantial portion of the uptake was osmolarity-insensitive. By changing the medium osmolarity, the uptake by BBMVs could be separately discriminated as osmolarity-sensitive and insensitive portions, representing transport into the intravesicular space and binding to the surface of BBMVs, respectively. For all three compounds, the binding increased in a time-dependent manner, while the amount transported reached a maximum after a relatively short incubation period. The transport of folic acid, but not its binding, exhibited an overshoot phenomenon under conditions of an inward H(+) gradient. The present results suggest that reabsorption of MTX and MX-68 in the kidney is governed by both their binding and transport mechanisms, with a similar kinetic profile to that of folic acid. The involvement of a transport system seems to make a relatively small contribution to the reabsorption of MTX assessed in BBMVs compared with MX-68 and folic acid.     

Carrier-mediated transport of folate compounds in L1210 cells. Initial rate kinetics and extent of duality of entry routes for folic acid and diastereomers of 5-methyltetrahydrohomofolate in the presence of physiological anions

Comparison of the kinetic parameters for influx of highly purified [3H]folic acid versus [3H]methotrexate in L1210 cells under anionic buffer conditions showed a marked discordancy. In addition, the kinetics for influx of [3H]folic acid were unchanged in variant L1210 cells defective in [3H]methotrexate transport. In these variant cells, the Vmax for methotrexate was reduced 17-fold and the Km was increased 3-fold. The results show that [3H]folic acid influx is mediated by a system which has a low affinity, but a 20-fold higher capacity, for folate compounds than the classical high-affinity system mediating [3H]methotrexate influx. Since the latter system also exhibits very low affinity for [3H]folic acid, it would not be expected to contribute significantly to the total influx of [3H]folic acid. The high-capacity system for [3H]folic acid influx is different from that believed to mediate pterin influx in L1210 cells since it was not inhibited by adenine, a potent inhibitor of pterin influx. However, exposure of cells to [3H]folic acid in a nonanionic buffer resulted in marked stimulation of initial influx, and a fraction of influx under these conditions was inhibited by methotrexate. These results suggest that anions modulate the extent of multiplicity of [3H]folic acid influx by their known effects on the high-affinity, reduced folate/methotrexate system. The diastereomers, at carbon 6, of [14C]5-methyltetrahydrohomofolate shared both transport systems. The influx Km for the natural diastereomer was one-half that of the unnatural form for both transport systems. Both diastereomers showed a much greater differential in affinity between the two transport systems than did [3H]folic acid. Our results suggest that an analog which could be effectively transported by the low-affinity/high-capacity route may be useful in the treatment of tumors resistant to methotrexate due to a defective high-affinity/low capacity influx system. We also found that incubation of L1210 cells with [3H]folic acid or the natural diastereomer [14C]5-methyltetrahydrohomofolate for 10 min resulted in the formation of a nonexchangeable fraction of radioactivity amounting to 20-40% of the total accumulation. This non-exchangeable fraction may be explained by the accumulation of metabolites other than polyglutamates. Preloading of cells with methotrexate prior to incubation with [3H]folic acid prevented the accumulation of radioactivity as a nonexchangeable fraction.     

Blood-Brain Barrier Transport of Reduced Folic Acid

Purpose. The brain is relatively resistant to folic acid deficiency, indicating specialized transport systems may exist for this vitamin localized within the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. The present studies quantify the BBB transport of [³H]-methyltetrahydrofolic acid (MTFA) in vivo and in isolated human brain capillaries in vitro. Methods. BBB transport of [³H]-MTFA was compared to that of [¹⁴C]-sucrose, a plasma volume marker, following either intravenous injection or intracarotid perfusion in anesthetized rats. Competition by 10 M MTFA or 10 M folic acid was examined to determine whether folic acid is also transported by the MTFA uptake system. Results. The BBB permeability-surface area (PS) product of [³H]-MTFA, 1.1± 0.3 L/min/g, was 6-fold greater than that of [¹⁴C]-sucrose following intravenous injection. The BBB PS product determined by intracarotid arterial perfusion was not significantly different from the BBB PS product calculated following intravenous injection. A time- and temperature- dependent uptake of [³H]-MTFA in human brain capillaries was observed. The uptake of [³H]-MTFA by either rat brain in vivo or by human brain capillaries in vitro was equally inhibited by 10 M concentrations of either unlabeled MTFA or unlabeled folic acid. Conclusions. (1) A saturable transport system exists at the BBB for folic acid derivatives and since this transport is equally inhibited by either folic acid or MTFA, it is inferred that this transport system is the folic acid receptor, and not the reduced folic acid carrier. (2) The presence of a folate transport system at the BBB may offer an endogenous transport system for brain drug delivery of conjugates of folates and drugs that do not normally cross the BBB in vivo.     

Interaction of sulfonylureas with the transport of bile acids into hepatocytes.

The sulfonylurea compounds glisoxepide and glibenclamide inhibit the uptake of bile acids into isolated rat hepatocytes. The Ki values for the inhibition of cholate uptake was 9 microM with glibenclamide and 200 microM with glisoxepide. The inhibition of cholate uptake by both sulfonylureas was noncompetitive. Uptake of the conjugated bile acid taurocholate was inhibited by glibenclamide, Ki = 75 microM. Again the inhibition was noncompetitive. Glisoxepide inhibited taurocholate uptake only in the absence of sodium ions. Under sodium-free conditions glisoxepide also strongly inhibited cholate uptake. The inhibition was competitive, Ki = 42 microM. Both bile acids interfered with the hepatocellular uptake of [3H]glisoxepide, with IC50 values of 375 and 467 microM for cholate and taurocholate, respectively. The uptake of [3H]glibenclamide was inhibited by cholate, IC50 = 328 microM, but not by taurocholate. Glisoxepide uptake was further inhibited by blockers of the hepatocellular monocarboxylate transporter, by the loop diuretic bumetanide, by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and by sulfate. Glibenclamide uptake was weakly inhibited by DIDS and by anthracene-9-carboxylic acid (A-9-C) but not by bumetanide and sulfate. Neither bromosulfophthalein nor the fatty acid oleate inhibited glisoxepide or glibenclamide uptake. These results are consistent with the transport of glisoxepide via the transport system for the unconjugated bile acid cholate. Glibenclamide uptake is mediated by a still unknown hepatocellular transport system.     

丹酚酸A抑制核苷转运并增强化疗药物的抗肿瘤作用丹酚酸A抑制核苷转运并增强化疗药物的抗肿瘤作用

目的观察丹酚酸A(SAA)的抑制核苷转运活性及其抗肿瘤作用。方法用3H-TdR和3H-UR转运测定法,克隆生成测定法以及小鼠移植性肉瘤180模型。结果SAA抑制艾氏腹水癌细胞的胸苷和尿苷的转运,其IC₅₀分别为18.1和17.1 μmol·L^-1。SAA能明显增强5-FU、丝裂霉素C、MTX对KB细胞、肝癌BEL-7402细胞的细胞毒性。体内试验,SAA 200 mg·kg^-1和5-FU 10 mg·kg^-1单独使用的抑瘤率分别为41%和27%;SAA和5-FU联合使用的抑瘤率为63%(CDI=0.86)。结论SAA有抑制肿瘤细胞核苷转运的活性,可增强5-氟尿嘧啶等药物的抗肿瘤作用,有可能用于肿瘤联合化疗。     

Hepatic transport of PKI166, an epidermal growth factor receptor kinase inhibitor of the pyrrolo-pyrimidine class, and its main metabolite, ACU154.

PKI166, a specific inhibitor of the tyrosine kinase activity of two epidermal growth factor receptors, was under development for the treatment of cancer. In preclinical studies PKI166 was mainly cleared by metabolism, and its metabolites were eliminated by biliary excretion, emphasizing the role of liver transport processes for its disposition. Here the transport properties of [14C]PKI166 and its main metabolite [14C]ACU154, an O-glucuronide, were analyzed using 1) Madin-Darby canine kidney II (MDCKII) cells stably transfected with human multidrug resistance-associated protein 2 (MRP2) and/or human organic anion-transporting peptide 2 (OATP2) and 2) liver canalicular membrane vesicles (CMVs) prepared from Wistar and mrp2-deficient TR- rats. Analysis of transport through MDCKII cells revealed that [14C]ACU154 was a substrate of MRP2 and OATP2. Rat mrp2 was shown to transport [14C]ACU154 with a Km of approximately 1 microM. [14C]PKI166 efficiently crossed MDCKII cells, particularly toward the apical side, but expression of MRP2 and/or OATP2 did not increase the flux. The effect of PKI166 and ACU154 on transport of [3H]estradiol-17beta-d-glucuronide (EG; via mrp2/MRP2 and OATP2) or [3H]taurocholic acid (TCA; via bile salt export pump (bsep) was analyzed. PKI166 inhibited the transport of [3H]EG by OATP2. ACU154 did strongly inhibit [3H]TCA uptake into CMVs from Wistar but not from TR- rats, demonstrating a dependence of bsep inhibition on mrp2 activity. ATP-dependent uptake of [3H]EG into CMVs from Wistar rats was inhibited by ACU154 but up to 4-fold increased by PKI166. In conclusion, OATP2 and MRP2/mrp2 were identified as transporters involved in ACU154 transport into bile. Both PKI166 and its O-glucuronide ACU154 affected mrp2/MRP2-, OATP2-, and/or bsep-mediated transport processes.     

Inhibitory effects of prostaglandin A1 on membrane transport of folates mediated by both the reduced folate carrier and ATP-driven exporters.

Studies are reported that describe the multifaceted inhibitory effects of prostaglandin A1 (PGA1) on processes that govern the transport of folates across the plasma membrane of Chinese hamster ovary (CHO) cells: the reduced folate carrier, RFC1, and ATP-dependent exporters. PGA1 was a noncompetitive inhibitor of MTX influx mediated by RFC1 with a Ki of approximately 21 microM. The onset of inhibition was virtually instantaneous, not reversible, and appeared to require the incorporation of PGA1 into the lipid membrane; surface adsorption alone was insufficient for inhibition of RFC1 transport activity. In contrast, the effect of PGA1 on folic acid transport was small (approximately 20% inhibition of total influx), consistent with the observation that the major portion of folic acid transport in CHO cells is mediated by a low pH mechanism distinct from RFC1. PGA1 was also a potent inhibitor of the ATP-driven efflux of both MTX and folic acid. At a concentration of 7 microM PGA1, the efflux rate constants for these folates were depressed by approximately 70 and approximately 50%, respectively. The net effects of PGA1 on the bidirectional folate fluxes translated into marked alterations in net transport. The addition of 7 microM PGA1 to cells at steady state with 1 microM MTX produced a rapid onset of net uptake and the achievement of an approximately 3-fold increase in the steady-state free MTX level as compared with untreated CHO cells. The addition of 7 microM PGA1 to cells at steady state with 1 microM folic acid produced an approximately 5-fold increase in the free folate level. These studies establish PGA1 as a potent inhibitor of both the reduced folate carrier and ATP-driven folate exporter(s). The noncompetitive nature of the inhibition of RFC1 is unique among anionic compounds, which are usually competitive inhibitors of the carrier.     

Characterization of prostaglandin E1 transport by rat renal cortical slices

The purpose of this study was to examine prostaglandin E1 (PGE1) transport in rat kidney. [3H]PGE1 administered intravenously was accumulated most abundantly in the renal cortex. Infusion of probenecid and bromcresol green (BCG) decreased [3H]PGE1 accumulation in the renal cortex after injection of [3H]PGE1. To further investigate PGE1 transport in the kidney, [3H]PGE1 uptake by renal cortical slices was examined. Probenecid and BCG inhibited [3H]PGE1 uptake by the slices. Unlabeled PGE1 decreased [3H]PGE1 uptake by renal cortical slices in a concentration-dependent manner. The inhibitory effect of various dicarboxylates with different carbon atoms on [3H]PGE1 uptake was maximal at 6 carbon atoms. Preloading cortical slices with glutarate significantly increased [3H]PGE1 uptake. [3H]PGE1 uptake was inhibited by various eicosanoids and compounds with other structures (p-aminohippurate, benzylpenicillin, estrone-3-sulfate, etc.). These findings suggest that PGE1 uptake by renal cortical slices may be mediated by the members of the organic anion transporter family.     

Uptake mechanism of valproic acid in human placental choriocarcinoma cell line (BeWo)

Valproic acid is an anticonvulsant widely used for the treatment of epilepsy. However, valproic acid is known to show fetal toxicity, including teratogenicity. In the present study, to elucidate the mechanisms of valproic acid transport across the blood-placental barrier, we carried out transcellular transport and uptake experiments with human placental choriocarcinoma epithelial cells (BeWo cells) in culture. The permeability coefficient of [3H]valproic acid in BeWo cells for the apical-to-basolateral flux was greater than that for the opposite flux, suggesting a higher unidirectional transport in the fetal direction. The uptake of [3H]valproic acid from the apical side was temperature-dependent and enhanced under acidic pH. In the presence of 50 microM carbonyl cyanide p-trifluoromethoxylhydrazone, the uptake of [3H]valproic acid was significantly reduced. A metabolic inhibitor, 10 mM sodium azide, also significantly reduced the uptake of [3H]valproic acid. Therefore, valproic acid is actively transported in a pH-dependent manner on the brush-border membrane of BeWo cells. Kinetic analysis of valproic acid uptake revealed the involvement of a non-saturable component and a saturable component. The Michaelis constant for the saturable transport (K(t)) was smaller under acidic pH, suggesting a proton-linked active transport mechanism for valproic acid in BeWo cells. In the inhibitory experiments, some short-chain fatty acids, such as acetic acid, lactic acid, propanoic acid and butyric acid, and medium-chain fatty acids, such as hexanoic acid and octanoic acid, inhibited the uptake of [3H]valproic acid. The uptake of [3H]valproic acid was also significantly decreased in the presence of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, salicylic acid and furosemide, which are well-known inhibitors of the anion exchange system. Moreover, p-aminohippuric acid significantly reduced the uptake of [3H]valproic acid. These results suggest that an active transport mechanism for valproic acid exists on the brush-border membrane of placental trophoblast cells and operates in a proton-linked manner.     

Blood-brain barrier and neuronal membrane transport of 6-[18F]fluoro-L-DOPA.

The transport of 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]FDOPA) across the blood-brain barrier (BBB) and neuronal membranes was compared with that of L-3,4-dihydroxyphenylalanine (L-DOPA) in rats. The carotid injection method was used as a direct measurement of [18F]FDOPA, 1-[14C]-L-DOPA, and 3-[14C]-L-DOPA transport across the BBB, while isolated nerve terminals were used to examine neuronal membrane transport of [3H]-L-DOPA. [18F]FDOPA appeared to use the same large neutral amino acid carrier for BBB transport as L-DOPA and L-phenylalanine. In addition, carbidopa [L-alpha-hydrazino-alpha-methyl-beta-(3,4-dihydroxyphenyl)propionic acid] was found not to have direct interference with the transport carrier on the BBB, but indirectly inhibited aromatic L-amino acid decarboxylase (AAAD) activity in brain endothelium by depletion of pyridoxal phosphate, a necessary cofactor of the enzyme. In striatal and cortical synaptosomes, [3H]-L-DOPA uptake was inhibited by non-radioactive L-DOPA, FDOPA, and 6-fluoro-L-meta-tyrosine (6-FMT). The inhibition was significantly greater in terminals isolated from the striatum than in those from the cerebral cortex. FDOPA, 6-FMT, and L-DOPA equally inhibited the neuronal transport of [3H]-L-DOPA. This suggests that FDOPA and 6-FMT compete with L-DOPA at similar transport sites at the neuronal membrane.     

Characterization of the transport of the organic cation [3H]MPP+ in human intestinal epithelial (Caco-2) cells

The aim of this study was to characterize the transport of organic cations at the intestinal level, by studying the characteristics of the transport of 1-methyl-4-phenylpyridinium (MPP+) in Caco-2 cells. Transepithelial flux as well as cellular accumulation of [3H]MPP+ were quantitatively similar when substrate was applied from the basolateral or apical cell membrane. Verapamil (100 microM) and rhodamine123 (10 microM) significantly reduced [3H]MPP+ transepithelial flux in the apical-to-basolateral direction. When cells were grown on plastic supports, [3H]MPP+ was rapidly accumulated in the cells, both by saturable and nonsaturable mechanisms. The kinetic parameters of the saturable component were: Km: 449 microM and Vmax: 2,249 pmol per mg protein and 5 min. Uptake of [3H]MPP+ was metabolic energy-dependent and Na+-, pH- and potential-independent. It was inhibited by several organic cations (verapamil, rhodamine123, daunomycin, vinblastine, tetrabutylammonium and vecuronium) but not by others (tetraethylammonium and N-methylnicotinamide). Decynium22 and corticosterone inhibited [3H]MPP+ uptake into the cells. The P-glycoprotein antibody UIC2 (20 microg/ml) had no effect. In conclusion, [3H]MPP+ is efficiently transported by Caco-2 cells in both basolateral-to-apical (secretion) and apical-to-basolateral (absorption) directions. Absorption of [3H]MPP+ at the apical membrane seems to occur through a carrier-mediated mechanism belonging to the Amphiphilic Solute Facilitator (ASF) family of transporters, but distinct from the known members of this family.     

Influx and efflux transport of H1-antagonist epinastine across the blood-brain barrier.

We investigated influx and efflux transporters involved in blood-brain barrier transport of the nonsedative H1-antagonist epinastine. The basal-to-apical transport of [14C]epinastine was markedly higher than that in the opposite direction in LLC-GA5-COL150 cells stably transfected with human multidrug resistance (MDR)1 gene. The brain-to-plasma concentration ratio of [14C]epinastine in mdr1a/b(-/-) mice was 3.2 times higher than that in wild-type mice. The uptake of both [3H]mepyramine and [14C]epinastine into immortalized rat brain capillary endothelial cells (RBEC)1 showed temperature and concentration dependence. The kinetic parameters, K(m), V(max), and uptake clearance (V(max)/K(m)), of the initial uptake of [3H]mepyramine and [14C]epinastine by RBEC1 were 150 microM, 41.8 nmol/min/mg protein, and 279 microl/min/mg protein for mepyramine and 10.0 mM, 339 nmol/min/mg protein, and 33.9 microl/min/mg protein for epinastine, respectively. The uptake of [3H]mepyramine and [14C]epinastine by RBEC1 was inhibited by organic cations such as quinidine, amantadine, and verapamil, but not by other organic cations, tetraethyl ammonium, guanidine, and carnitine. Organic anions such as benzoic acid, estrone-3-sulfate, taurocholate, and neutral digoxin were not inhibitory. Furthermore, some cationic H1 antagonists (chlorpheniramine, cyproheptadine, ketotifen, and desloratadine) inhibited the [3H]mepyramine and [14C]epinastine uptake into RBEC1. In conclusion, the present study demonstrated that the combination of efficient efflux transport by P-glycoprotein and poor uptake by the influx transporter, which is identical with that responsible for the uptake of mepyramine, account for the low brain distribution of epinastine.     

In-vivo and in-vitro evidence of a carrier-mediated efflux transport system for oestrone-3-sulphate across the blood-cerebrospinal fluid barrier

The efflux transport of oestrone-3-sulphate, a steroid hormone sulphate, across the blood-cerebrospinal fluid barrier has been examined following its intracerebroventricular administration. [3H]Oestrone-3-sulphate was eliminated from cerebrospinal fluid (CSF) with an apparent efflux clearance of 205 microL min(-1) per rat. There was 25% of unmetabolized [3H]oestrone-3-sulphate in the plasma 5 min after intracerebroventricular administration, indicating that at least a part of [3H]oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier. This efflux transport was inhibited by co-administration of excess oestrone-3-sulphate (25 mM 10 microL = 0.25 micromol) into rat cerebral ventricle. To characterize the oestrone-3-sulphate transport process, an in-vitro uptake experiment was performed using isolated rat choroid plexus. Oestrone-3-sulphate uptake by isolated rat choroid plexus was found to be a saturable process with a Michaelis-Menten constant (Km) of 18.1 +/- 6.3 microM, and a maximum uptake rate (Vmax) of 48.0 +/- 15.1 pmol min(-1) microL(-1) of tissue. The oestrone-3-sulphate transport process was temperature dependent and was inhibited by metabolic inhibitors such as 2,4-dinitrophenol and rotenone, suggesting an energy dependence. This uptake process was also inhibited by steroid hormone sulphates (1 mM dehydroepiandrosterone sulphate and 1 mM oestrone sulphate), bile acids (1 mM taurocholic acid and 1 mM cholic acid) and organic anions (1 mM sulphobromophthalein and 1 mM phenolsulphonphthalein), whereas 1 mM p-aminohippuric acid, 1 mM p-nitrophenol sulphate, 0.1 mM methotrexate and the cardiac glycoside, 2.5 microM digoxin, had little effect. In conclusion, these results provide evidence that oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier via a carrier-mediated efflux transport system.     

Effect of P-glycoprotein modulators on the human extraneuronal monoamine transporter.

The aim of this work was to investigate the effect of P-glycoprotein modulators on human extraneuronal monoamine transporter (EMT)-mediated transport. The experiments were performed using a cell line from human embryonic kidney (HEK293 cells) stably transfected with pcDNA3hEMT (293(hEMT)), or with pcDNA3 alone (293(control)). Of the P-glycoprotein modulators tested, rhodamine123, verapamil and daunomycin concentration-dependently inhibited EMT-mediated uptake of [3H]1-methyl-4-phenylpyridinium ([3H]MPP(+)). The corresponding IC(50)'s were found to be 3.6, 37 and 130 microM, respectively. By contrast, vinblastine, digitoxin and cyclosporine A were devoid of effect. The endogenous organic cation tyramine, but not choline, inhibited EMT-mediated transport (IC(50) of 468 microM). Moreover, L-arginine and L-histidine (up to 1 mM) did not affect [3H]MPP(+) uptake. Finally, MPP(+) and tyramine trans-stimulated [3H]MPP(+) uptake, but rhodamine123 had no effect, and verapamil and daunomycin trans-inhibited [3H]MPP(+) uptake. In conclusion, this study shows that several cationic modulators of P-glycoprotein inhibit EMT-mediated transport. As a consequence, the interaction of P-glycoprotein modulators with EMT must be taken into account, and the consequences of this interaction must not be forgotten when using such drugs in vivo.     

In rat alveolar macrophages lipopolysaccharides exert divergent effects on the transport of the cationic amino acids l-arginine and l-ornithine

In rat alveolar macrophages (AMphi) it was tested whether induction of iNOS by lipopolysaccharides (LPS) is accompanied by changes in L-arginine transport and whether L-ornithine, the product of arginase released from AMphi, could, via inhibition of L-arginine uptake, act as a paracrine inhibitor of NO synthesis. Rat AMphi (cultured for 20 h in the absence or presence of 1 microg/ml LPS) were incubated in Krebs-HEPES solution containing [3H]-L-arginine (0.1 microM for 2 min or 100 microM for 5 min) and the cellular radioactivity was determined as a measure of L-arginine uptake. In parallel, cells were incubated for 6 h in Krebs-HEPES solution containing 0-1 mM L-arginine and nitrite accumulation was determined. [3H]-L-arginine uptake (0.1 microM or 100 microM) occurred independently of sodium ions and was inhibited by L-ornithine (EC50: 117 and 562 microM, respectively) and with similar potencies by L-lysine. In LPS-treated AMphi the concentration inhibition curve of L-ornithine was shifted to the right by about a factor of 4, whereas that of L-lysine was only marginally shifted to the right. L-Leucine (0.1 and 1 mM) inhibited [3H]-L-arginine (0.1 microM) by 43 and 58%, respectively, and the effect of 0.1 mM L-leucine was partially sodium dependent. In LPS-treated AMphi, 0.1 mM L-leucine no longer inhibited [3H]-L-arginine and the effect of 1 mM L-leucine was attenuated. Kinetic analysis of the transport of [3H]-L-arginine and [14C]-L-ornithine revealed two components for each amino acid with Km values of 21 and 114 microM (L-arginine) and 39 and 1050 microM (L-ornithine), respectively. After LPS treatment Km2 of L-arginine transport was reduced to 63 microM and Vmax of both components was increased, whereas Km2 of L-ornithine transport was enhanced to 1392 microM and Vmax1 reduced. LPS-stimulated AMphi, incubated in amino acid-free Krebs-HEPES solution, produced about 4 nmol nitrite/10(6) cells per 6 h, and L-arginine enhanced nitrite accumulation maximally about threefold (EC50: 30 microM). L-ornithine, up to 3 mM, failed to affect significantly nitrite accumulation observed in the presence of 30 or 100 microM L-arginine. Rat AMphi express mRNA for two cationic amino acid transporters (CAT-1 and CAT-2B), and LPS markedly up-regulated mRNA for CAT-2B in parallel with mRNA for iNOS, but had no effect on that for CAT-1. In conclusion, in rat AMphi LPS up-regulates L-arginine transport and induces changes in the characteristics of the cationic amino acid transport resulting in preferential transport of L-arginine. These effects may be regarded as cellular measures to ensure a high L-arginine supply for iNOS.     

Pharmacological characteristics of choline transport system in mouse cerebral cortical neurons in primary culture

The characteristics of [3H]choline transport with high affinity were investigated using primary cultured neurons obtained from the mouse cerebral cortex. [3H]Choline uptake was saturable as a function of extracellular [3H]choline concentration. Analysis by Lineweaver-Burk plot revealed that [3H]choline was transported into neurons by a high affinity transport system with a Km value of 19.8 +/- 0.8 microM and Vmax value of 0.334 +/- 0.022 nmol/mg protein/min. This high affinity transport of [3H]choline was significantly inhibited by the withdrawal of sodium from the incubation medium, incubation at low temperature (4 degrees C) and addition of metabolic inhibitors such as monoiodoacetate. These results indicate that the high affinity [3H]choline uptake in primary cultured neurons is sodium- and energy-dependent. Hemicholinium-3 also showed a competitive inhibition on the [3H]choline transport. Depolarization by high K+ induced an enhancement of the [3H]choline uptake in the presence of Ca2+. The crude synaptosomal fraction obtained from primary cultured neurons possessed approximately forty-fold higher synthesizing activity of [3H]acetylcholine from [3H]choline than that found in the homogenate preparation of cultured neurons. The present results strongly suggest that the primary cultured neurons used in this study possess a sodium- and energy-dependent high-affinity choline uptake system as well as a synthesizing system for acetylcholine. Possible usefulness of these neurons for investigating neuronal uptake of choline and its functional role in the biosynthesis of acetylcholine are also suggested.     

Carrier-Mediated Transport of H1-Antagonist at the Blood-Brain Barrier: A Common Transport System of H1-Antagonists and Lipophilic Basic Drugs

The blood-brain barrier (BBB) transport system for H₁ antagonists was studied using primary cultured bovine brain capillary endothelial cells (BCEC). The uptake of [³H]mepyramine was inhibited by various H₁-antagonists. Ketotifen competitively inhibited [³H]mepyramine uptake with an inhibition constant (K_i ) of 46.8 µM. Lipophilic basic drugs such as propranolol, lidocaine and imipramine significantly inhibited [³H]mepyramine uptake. In particular, propranolol inhibited [³H]mepyramine uptake competitively at an inhibition constant (K_i) of 51.1 µM. Moreover, in ATP-depleted BCEC, [³H]mepyramine uptake was stimulated by preloading with H₁- antagonists and lipophilic basic drugs. These results indicated that H₁-antagonists are transported across the BBB via a carrier-mediated transport system common to lipophilic basic drugs.     

Influence of neuroglial transport on the action of gamma-aminobutyric acid on mammalian ganglion cells.

1 The effect of inhibiting the transport of gamma-aminobutyric acid (GABA) by neuroglial cells on the depolarizing action of exogenous amino acids on isolated superior cervical ganglia of the rat was studied. 1 Transport (measured by uptake of [3H]-GABA) was inhibited by (a) reducing external [na+] from 143 to 2mM and (b) administering alternative carrier-sbustrates, 3-amino-n-butyric acid (beta-amino-butyric acid, BABA) and (+/-)-nipecotic acid at a concentration of 1 mM. 3 All three procedures enhanced the depolarization produced by low concentrations of GABA (less than or equal to 10 muM) but did not alter the maximum response, nor the response to 3-aminopropanesulphonic acid (a gabamimetic with low affinity for the neuroglial carrier). 4 It is concluded that the neuroglial uptake process can limit the action of exogenous GABA upon neurones, by reducing the interstitial GABA concentration.