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Stereoselective transport of histidine in rat lung microvascular endothelial cells

Eiichi Sakurai1, Tomoya Sakurada1, Yoshinori Ochiai1, Jun Yamakami2, and Yorihisa Tanaka1

1 Department of Pharmaceutics I, Tohoku Pharmaceutical University, Aoba-ku, Sendai 981-8558; and 2 Department of Pharmacy, Sapporo National Hospital, Sapporo 003-0804, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The transport characteristics of L- and D-histidine through the blood-lung barrier were studied in cultured rat lung microvascular endothelial cells (LMECs). L-Histidine uptake was a saturable process. The addition of metabolic inhibitors [2,4-dinitrophenol (DNP) and rotenone] reduced the uptake rate of L-histidine. Ouabain, an inhibitor of Na+-K+-ATPase, also reduced uptake of L-histidine. Moreover, the initial L-histidine uptake rate was reduced by the substitution of Na+ with choline chloride and choline bicarbonate in the incubation buffer. The system N substrate, L-glutamic acid gamma -monohydroxamate, also inhibited uptake of L-histidine. However, system N-mediated transport was not pH sensitive. These results demonstrated that L-histidine is actively taken up by a system N transport mechanism into rat LMECs, with energy supplied by Na+. Moreover, the Na+-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH), had an inhibitory effect on L-histidine uptake in Na+ removal, indicating facilitated diffusion by a Na+-independent system L transport into the rat LMECs. These results provide evidence for there being at least two pathways for L-histidine uptake into rat LMECs, a Na+-dependent system N and Na+-independent system L process. On the other hand, the uptake of D-histidine into rat LMECs was not reduced by the addition of DNP, rotenone, or ouabain, or by Na+ replacement. Although the uptake of D-histidine was reduced in the presence of BCH, the addition of L-glutamic acid gamma -monohydroxamate did not significantly decrease uptake of D-histidine. These results suggest that the uptake of D-histidine by rat LMECs has different characteristics compared with its isomer, L-histidine, indicating that system N transport did not involve D-histidine uptake.

sodium-dependent system N transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE LUNG has a vast endothelial surface area available for the exchange of solutes and liquid (5, 32). Two different transendothelial pathways can be utilized for the passage of solutes: 1) diffusive nonvesicular flux pathways that are likely present at intercellular junctions; and 2) vesicle-mediated pathways activated by receptor-ligand binding or by fluid-phase uptake of solutes into caveolae on the endothelial cell membrane. Transport of macromolecules across the endothelium may be dependent on both paracellular and transcellular routes (18, 26). However, the plasma membrane of endothelial cells has been shown to be the site of several carrier-mediated transport systems (25, 29), including those for glucose, monocarboxylic acid, and amino acids. System N amino acid transport was first described by Kilberg et al. (11) as mediating L-glutamine uptake in hepatocytes. This transport system has since been identified in skeletal muscle, placenta, lymphocytes, astrocytes, and neurons (8, 10, 14, 20, 30). Three amino acids, L-glutamine, L-histidine, and L-asparagine, all of which have a nitrogen in their side group, are the natural substrates for this transporter (8, 11). In particular, L-histidine, an essential amino acid, is a precursor of histamine. Histamine initiates transitory increases in endothelial permeability in situ and in vitro (19, 33). Under in situ conditions, increased permeability is associated with the development of small gaps between adjacent endothelial cells, and restored barrier function is associated with the reapposition of adjacent cells (15, 33). Therefore, it is of interest to investigate the transport mechanism of L-histidine from blood to lung and metabolism of L-histidine in the microvascular endothelial cells. Xiang et al. (34) reported that uptake of L-histidine into the rat choroids plexus is mediated not only via the system L amino acid transporter described by Segal et al. (28) and Davson et al. (3) but also via system N amino acid transporters similar to that described in liver and skeletal muscle (8, 11). Recently, we (35) also found evidence for at least two pathways for L-histidine uptake into cultured rat brain microvascular endothelial cells, which are major structural and functional components of the blood-brain barrier (BBB), a Na+-independent and a Na+-dependent process. The former was inhibited by 2-amino-2-norbornanecarboxylic acid (BCH), indicating system L transport, while the latter appears to be a system N transporter because uptake was inhibited by glutamine, asparagine, and L-glutamic acid gamma -monohydroxamate. In the present study, we examine the uptake system of L-histidine in cultured rat lung microvascular endothelial cells (LMECs).

Current evidence also indicates the presence of some D-amino acids in rodent, bovine, and human peripheral and central nervous system tissues (4, 21). Oldendorf et al. (22) reported that 12 common amino acids have stereospecific BBB permeability as determined using rapid amino acid injection into the rat common carotid artery followed by decapitation 15 s later. Our previous study demonstrated the enantiomer-specific pharmacokinetics of histidine after bolus intravenous administration of each enantiomer (27), suggesting the possible existence of stereoselective transport and elimination systems for histidine enantiomers at the endothelial cells. To analyze the correlation with these in vivo experiments, the uptake of D-histidine into rat LMECs was compared with that of L-histidine.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Materials. S-alpha -fluoromethylhistidine (FMH) was supplied by Dr. J. Kollonitsch (Merck, Sharp, and Dohme Research Laboratories, Rahway, NJ). L- and D-histidine were obtained from the Peptide Institute (Osaka, Japan). Medium 199 (M199), DMEM/nutrient mixture F-12 (DMEM/F-12), heparin, HEPES, dispase, epidermal growth factor (EGF), fetal bovine serum (FBS), and donor horse serum (HS) were obtained from GIBCO-BRL Life Technologies (Rockville, MD). Percoll was purchased from Pharmacia (Uppsala, Sweden). Collagenase P was obtained from Boehringer Mannheim (Mannheim, Germany). Gentamicin sulfate and amphotericin B were purchased from Sigma (St. Louis, MO). Acetylated density lipoprotein labeled with a fluorescent probe, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-Ac-LDL), was obtained from Biomedical Technologies (Stoghton, MA). 2,4-Dinitrophenol (DNP) and ouabain were purchased from Wako (Osaka, Japan). All other chemicals were of reagent grade and were obtained commercially.

Isolation and culture of rat lung microvascular endothelial cells. Three-week-old male Wistar rats purchased from Japan SLC (Hamamatsu, Japan) were housed at constant temperature (23 ± 1°C) and constant humidity (55 ± 5%) with automatically controlled lighting. Rat LMECs were isolated using a modification of the technique described previously by Magee et al. (17). Twenty rats were killed by decapitation. The lungs were removed and placed in a beaker containing M199 with 0.005% antibiotic solution. The visceral pleura were first stripped from each lobe, and the outer 3-5 mm of the peripheral lung tissue was dissected free of the remaining tissue. The pooled pieces of lung periphery were finely minced and washed with M199, and the fragments were collected on 40-µm nylon mesh. With constant gentle mechanical agitation, the tissue was digested with 0.6% collagenase in M199 at 37°C for 20 min and then incubated with 2.1% dispase in M199 at 37°C for 30 min. The suspension was mixed in M199 with 5% FBS. After centrifugation at 600 g for 10 min, the resulting tissue pellet was resuspended in M199 and filtered through 100-µm mesh. The microvessels were collected by centrifugation at 600 g for 10 min and resuspended in M199. The suspension was layered on a Percoll gradient formed by centrifugation of 50% Percoll at 26,000 g at 4°C for 60 min and was then centrifuged at 600 g for 10 min. After Percoll gradient centrifugation, three layers were observed. The endothelial cell aggregates formed a band around the middle third of the gradient, and the entire middle layer was collected from gradients. The cells were resuspended in M199 and collected by centrifugation at 600 g for 10 min. The cell suspensions were seeded onto collagen-coated 225-cm2 tissue culture flasks (Iwaki Glass, Funabashi, Japan). Cells were allowed to attach and grow to monolayers at 37°C in a humidified atmosphere of 5% CO2-95% air. The culture medium (DMEM/F-12 containing 14 mM sodium bicarbonate, 20 ng/ml of EGF, 50 µg/ml of gentamicin-amphotericin B solution, 10 U/ml of heparin, 5% FBS, and 5% HS) was changed every 3 days. Subculture was performed when the cells reached confluence (after ~6-7 days). Cells were trypsinized at a 1:3 ratio after reaching confluence using 0.025% trypsin in Hanks' balanced salt solution containing 0.02% EDTA. Secondary subcultured cells were grown on collagen-coated 25-cm2 tissue culture flasks. Uptake experiments were performed when cells reached confluence, in ~4-5 days. Identification of cells possessing factor VIII antigen was done using immunofluorescence with goat anti-human von Willebrand factor antiserum as the primary antibody. Cellular uptake of DiI-Ac-LDL was assessed by the method of Voyta et al. (31) (data not shown). The culture also became progressively homogenous by the third passage, and all cells had the morphology (17). Furthermore, it is expected that monolayers of LMECs remain polarized because several membrane functions have been reported to localize in a manner that is consistent with the lung (5, 23, 24) and brain (1, 9, 13) microvascular endothelial cells in vivo. These features of the cultured cells enable us to study the polarized transport characteristics at luminal or antiluminal membrane.

L- or D-histidine uptake. The uptake of each enantiomer of histidine by rat LMECs attached to the culture flask (25 cm2) was measured as described by Hughes and Lantos (7) with some modifications. After removal of the culture medium, each flask was washed three times with 2 ml of incubation solution [Krebs buffer (in mM: 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 CaCl2, 1.2 MgSO4, 11.1 glucose, pH 7.4)]. Then, 2 ml of Krebs buffer containing L- or D-histidine was added to each flask, and the cells were incubated for a specified period. At the end of the incubation period, the medium was removed immediately by suction, and the flask was rinsed rapidly three times with 2 ml of ice-cold Krebs buffer. For measurement of histidine enantiomers in the cells, the cells were scraped with a rubber policeman into 2 ml of ice-cold Krebs buffer and homogenized for 15 s in a sonicator (Sonifier 450) in an ice bath in 150 µl of ice-cold 0.4 M perchloric acid. The sonicated samples were centrifuged at 9,000 g for 10 min at 4°C, and the supernatant obtained was filtered through an Ultra-free C3GC filter (Millipore, Tokyo, Japan) and stored at -80°C until assayed. The concentrations of L- or D-histidine in clear samples were analyzed by ion-exchange chromatography as described previously (27). The concentrations of L- and D-histidine in the rat LMECs were determined by subtracting the concentrations of endogenous L-histidine. The perchloric acid-insoluble residue was solubilized with 0.1 N NaOH for determination of protein content. Protein content of cultured cells per flask was determined by the method of Lowry et al. (16), using bovine serum albumin as a standard.

To estimate the kinetic parameters of L- or D-histidine uptake in cultured monolayers of rat LMECs, the uptake rate (V; nmol · mg of protein-1 · min-1) was fitted to the following equation, consisting of two saturable terms, using the nonlinear least-squares regression analysis program MULTI (36)
V=V<SUB>max1</SUB>S<IT>/</IT>(<IT>K</IT><SUB>m1</SUB> + S)<IT>+V</IT><SUB>max2</SUB><IT>/</IT>(<IT>K</IT><SUB>m2</SUB> + S) (1)
where Vmax is maximum uptake rate for the carrier-mediated process, S is concentration of substrate, and Km is the half-saturation concentration (Michaelis-Menten constant); subscript integers indicate the saturable processes (1, high affinity; 2, low affinity).

Effects of FMH on L-histidine uptake. Cultured cells were preincubated for 15 min at 37°C in Krebs buffer in the absence or presence of FMH, a specific L-histidine decarboxylase inhibitor (100 µM). The uptake of L- or D-histidine was determined by the method described above.

Effects of metabolic inhibitors and ion replacement in Krebs buffer on the uptake of histidine. Cultured cells were preincubated for 15 min 37°C in Krebs buffer in the absence or presence of metabolic inhibitors (250 µM DNP or 25 µM rotenone) and Na+-K+-ATPase inhibitor (100 µM ouabain). In the sodium ion-replacement studies, incubation solution free from Na+ was prepared by substitution of choline chloride for NaCl and choline bicarbonate for NaHCO3. The uptake of L- or D-histidine was determined by the method described above.

Effects of BCH and various amino acids on the uptake of histidine. Cultured cells were incubated for 2 min at 37°C in Krebs buffer in the absence or presence of the Na+-independent system L substrate BCH (1 mM) with L- or D-histidine (100 µM). Similarly, rates of histidine uptake were measured when NaCl and NaHCO3 in the Krebs buffer was substituted with an equimolar concentration of choline chloride or choline bicarbonate, respectively. Moreover, cultured cells were incubated for 2 min at 37°C in Krebs buffer in the absence or presence of the Na+-dependent system N substrate L-glutamic acid gamma -monohydroxamate (1 mM) with L- or D-histidine (100 µM). Similar measurements for histidine uptake were made in the incubation buffer substituted with choline chloride and choline bicarbonate. BCH (1 mM) was added to all buffer solutions to inhibit system L-mediated uptake.

Effects of pH on L- or D-histidine uptake. In the uptake experiment at various pH values, 1 N HCl was used to adjust the Krebs buffer to pH 5.0, 6.0, 7.0, or 7.4. The uptake of L- or D-histidine (100 µM) was determined in the absence or presence of BCH (1 mM) by the method described above.

Statistics. The uptake data are presented as means ± SE of means for n experiments. Comparisons of data among groups were carried out using analysis of variance and Dunnett's post hoc multiple-comparisons test. Differences were considered significant at P < 0.05 (two-tailed).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FMH is a specific inhibitor of L-histidine decarboxylase, a histamine-forming enzyme, and strongly inhibits histamine formation in vitro and in vivo (12). In this experiment, there were no significant differences in uptake of L-histidine into cultured monolayers of rat LMECs by FMH (Table 1). Metabolism of L-histidine in the cells was not observed within 2 min of incubation, and accumulation of L-histidine was due only to intact L-histidine. Therefore, the uptake experiments of L-histidine were carried out without an inhibitor of L-histidine decarboxylase. D-Histidine, however, may not be metabolized in rat tissues because most of the enzymes responsible for metabolism of histidine are specific to the L-enantiomer.

                              
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Table 1.   Effects of FMH on the uptake of L-histidine by cultured monolayers of rat LMECs

Figure 1 shows the time courses of the uptake of L- and D-histidine into cultured monolayers of rat LMECs. The normal L-histidine concentration in rat plasma was 80 µM. When 100 µM L- and D-histidine was added to Krebs buffer, both L- and D-histidine uptake rates were linear over a 3-min incubation period. Based on these results, all subsequent uptake experiments were conducted for 2 min. At all time points measured at 37°C, the uptake of L-histidine was significantly greater than that of D-histidine.


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Fig. 1.   Time courses of L- () and D- (black-triangle) histidine uptake by cultured monolayers of rat lung microvascular endothelial cells (LMECs). The rat LMECs were incubated at 37°C in 2 ml of Krebs buffer (pH 7.4) containing 0.1 mM L- or D-histidine for the indicated times. Each point represents the mean ± SE of 4 determinations.

Figure 2 shows the relationship between the initial uptake rate and the concentration of L- or D-histidine (0.01-5 mM). Eadie-Hofstee plots suggested that uptake is a saturable process. Nonlinear least-squares regression analysis of these data based on Eq. 1 yielded the following kinetic parameters: Km1 = 0.058 mM, Vmax1 = 0.841 nmol · mg of protein-1 · min-1 for the high-affinity process and Km2 = 1.627 mM, Vmax2 = 3.905 nmol · mg of protein-1 · min-1 for the low-affinity process for L-histidine; and Km1 = 0.061 mM, Vmax1 = 0.342 nmol · mg of protein-1 · min-1 for the high-affinity process and Km2 = 1.590 mM and Vmax2 = 1.137 nmol · mg of protein-1 · min-1 for the low-affinity process for D-histidine.


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Fig. 2.   Concentration dependence of L- () and D- (black-triangle) histidine uptake by cultured monolayers of rat LMECs. L- or D-histidine uptake for 2 min at concentrations between 0.01 and 3 mM was determined at 37°C. Inset: the Eadie-Hofstee plot. Each point represents the mean ± SE of 4 determinations. V, histidine uptake rate (nmol · mg protein-1 · min-1); S, histidine concentration (mM).

The effects of metabolic inhibitors and ion replacement in the incubation solution on the uptake of L- or D-histidine are summarized in Table 2. Addition of 250 µM DNP, an uncoupler of oxidative phosphorylation, or 25 µM rotenone, a respiratory chain inhibitor, significantly reduced the uptake of L-histidine at 37°C. Moreover, 100 µM ouabain, an inhibitor of Na+-K+-ATPase, significantly reduced the uptake of L-histidine. The substitution of Na+ with choline and choline bicarbonate in the incubation buffer also decreased the initial L-histidine uptake rate. However, L-histidine uptake was not completely inhibited by these treatments. On the other hand, the uptake of D-histidine into rat LMECs was not reduced by the addition of DNP (100 µM), rotenone (25 µM), or ouabain (100 µM) or by sodium ion replacement.

                              
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Table 2.   Effects of metabolic inhibitors and ion replacement in Krebs buffer on the uptake of histidine

The uptake of L-histidine was reduced by 24% in the presence of an Na+-independent system L substrate, BCH (1 mM), and it was also reduced by 66% when NaCl and NaHCO3 in the Krebs buffer were substituted with equimolar concentrations of choline chloride and choline bicarbonate (Table 2 and Fig. 3). Moreover, the effects of BCH (1 mM) and Na+ removal were additive; the combination resulted in an ~88% decline in L-histidine uptake. In the presence of BCH (1 mM), a system N substrate, L-glutamic acid gamma -monohydroxamate (1 mM), significantly inhibited uptake of L-histidine. This inhibition was due to a reduction in Na+-dependent uptake. On the other hand, although the uptake of D-histidine was reduced in the presence of BCH (1 mM), the addition of L-glutamic acid gamma -monohydroxamate (1 mM) had no significant decrease in uptake of D-histidine in the presence of 143 mM Na+ plus 1 mM BCH.


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Fig. 3.   Rates of uptake of L- (left) or D- (right) histidine into rat LMECs. Measurements were made in 143 mM Na+ (control), Na+ free, Na+ free + 1 mM 2-amino-2-norbornanecarboxylic acid (BCH), 143 mM Na+ + 1 mM BCH, and 1 mM BCH + 1 mM L-glutamic acid gamma -monohydroxamate (L-Glu-gamma -MH) to inhibit uptake via the Na+-dependent system N in the presence of 143 mM Na+. Each point represents the mean ± SE of 4 determinations. * P < 0.05 and ** P < 0.01, significant difference from control. dagger  P < 0.05, significant difference between Na+ free and Na+ free + 1 mM BCH. Dagger  P < 0.01, significant difference between 1 mM BCH and 1 mM L-Glu-gamma -MH + 1 mM BCH.

No significant differences were observed in the uptake of L- or D-histidine in the absence or presence of BCH (1 mM) in the pH range from 5 to 7.4 (Fig. 4).


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Fig. 4.   Effects of pH on L- (, ) and D- (black-triangle, triangle ) histidine uptake by cultured monolayers of rat LMECs. L- or D-histidine uptake for 2 min at pH between 5.0 and 7.4 was determined in the absence (closed symbols) or presence (open symbols) of BCH (1 mM) at 37°C. Each point represents the mean ± SE of 4 determinations.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

L-Histidine, an essential amino acid, is a precursor of histamine, an inflammatory mediator in the lung. Histamine activates local target cells and tissues, such as endothelial cells, fibroblasts, and smooth muscle (6). Therefore, it is important to elucidate the uptake and metabolic mechanisms involving L-histidine in rat LMECs. In 1994, a method to isolate and culture LMECs from rats was developed by Magee et al. (17). In this study, we initially investigated the mechanism of uptake of L-histidine into rat LMECs using the protocol described by Magee et al. (17) with some modifications. Moreover, as small amounts of D-amino acids that exist in certain plants and bacteria and appear in blood may be restricted from entry into the lung by the stereospecificity of the carrier systems, D-histidine was studied in rat LMECs.

L-Histidine uptake was a saturable process. Furthermore, addition of DNP or rotenone reduced the uptake rate of L-histidine, demonstrating that L-histidine uptake is metabolic energy dependent. Ouabain, an inhibitor of Na+-K+-ATPase, which is localized in the antiluminal membrane (2), also inhibited uptake of L-histidine, possibly by reducing the sodium gradient and membrane potential. The effect of ouabain suggested that one of the driving forces for L-histidine transport is Na+, since choline did not substitute for Na+. These results suggested that L-histidine is actively taken up by a carrier-mediated mechanism with energy supplied by Na+. However, L-histidine uptake was reduced in the presence of the Na+-independent system L substrate, BCH, suggesting the coexistence of facilitated diffusion (Na+-independent process) by a carrier-mediated mechanism into the LMECs and an active transport system consuming Na+.

The Na+-dependent process for L-histidine uptake appears to involve the system N transporter, as uptake was inhibited by glutamine, asparagine, and L-glutamic acid gamma -monohydroxamate, system N substrates but not substrates for system A and system ASC transporters (11, 34). In this experiment, in the presence of 143 mM Na+ and 1 mM BCH, L-glutamic acid gamma -monohydroxamate (1 mM) resulted in a progressive decrease in L-histidine uptake. This result suggested that system N transport plays an important role in L-histidine uptake into rat LMECs. Oldendorf et al. (22) demonstrated pH dependence of histidine affinity for BBB carrier transport systems for neutral and cationic amino acids with the single-pass carotid injection technique. Reductions in pH markedly inhibited Na+-dependent but not Na+-independent transport, indicating that system N-mediated transport at the choroid plexus was also pH sensitive (34). Thus such pH sensitivity may contribute to the derangement of brain amino acid composition during cerebral acidosis. However, the uptake of L-histidine into the rat brain microvascular endothelial cells was reported previously not to be affected by pH (5.0-7.4) in the absence or presence of BCH (35). In this study, the system N-mediated transport system was also not pH sensitive in rat LMECs, suggesting that the transport mechanism of L-histidine is very similar in both the lung and brain microvascular endothelial cells. Moreover, as shown in Fig. 2, Eadie-Hofstee plots demonstrated at least two types of transport processes. The (Vmax/Km) value of the high-affinity process was ~5.4-fold greater than that of the low-affinity process. Further investigations would be necessary to confirm whether the high-affinity process dominates Na+-dependent or Na+-independent uptake of L-histidine in rat LMECs.

On the other hand, the uptake of D-histidine into rat LMECs was not reduced by the addition of DNP, rotenone, or ouabain, or by sodium ion replacement. Although the uptake of D-histidine was reduced in the presence of BCH, the addition of L-glutamic acid gamma -monohydroxamate did not significantly decrease uptake of D-histidine in 143 mM Na+ plus 1 mM BCH. These results suggest that the uptake of D-histidine by rat LMECs has different characteristics compared with its isomer L-histidine, indicating that the system N transporter does not play a role in D-histidine uptake. However, because two processes are involved in uptake of D-histidine, at least as suggested by Eadie-Hofstee plots, another process may be involved in addition to system L transport. Therefore, further investigations of the mechanism of D-histidine transport into rat LMECs are necessary.


    FOOTNOTES

Address for reprint requests and other correspondence: E. Sakurai, Dept. of Pharmaceutics I, Tohoku Pharmaceutical Univ., Aoba-ku, Sendai 981-8558 (E-mail: sakuraie{at}tohoku-pharm.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00405.2001

Received 17 October 2001; accepted in final form 10 January 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 282(6):L1192-L1197
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