Characterization of a human plasma membrane heme transporter in intestinal and hepatocyte cell lines

Heme is the most bioavailable form of dietary iron and a component of many cellular proteins. Controversy exists as to whether heme uptake occurs via specific transport mechanisms or passive diffusion. The aims of this study were to quantify cellular heme uptake with a fluorescent heme analog and to determine whether heme uptake is mediated by a heme transporter in intestinal and hepatic cell lines. A zinc-substituted porphyrin, zinc mesoporphyrin (ZnMP), was validated as a heme homolog in uptake studies of intestinal (Caco-2, I-407) and hepatic (HepG2) cell lines. Uptake experiments to determine time dependence, heme inhibition, concentration dependence, temperature dependence, and response to the heme synthesis inhibitor succinylacetone were performed. Fluorescence microscope images were used to quantify uptake and determine the cellular localization of ZnMP; ZnMP uptake was seen in intestinal and hepatic cell lines, with cytoplasmic uptake and nuclear sparing. Uptake was dose- and temperature dependent, inhibited by heme competition, and saturated over time. Preincubation with succinylacetone augmented uptake, with an increased initial uptake rate. These findings establish a new method for quantifying heme uptake in individual cells and provide strong evidence that this uptake is a regulated, carrier-mediated process.     

Evidence for carrier-mediated transport of L-leucine into isolated pea (Pisum sativum L.) chloroplasts

The uptake of leucine into isolated, intact, pea chloroplasts was investigated using the silicone oil centrifugation technique. The internal: external ratio of leucine exceeded unity at low external leucine concentrations. Uptake of leucine at different external concentrations showed passive diffusion and carrier-mediated transport components. Competition for uptake was shown between leucine and isoleucine but not between leucine and glycine. Rates of diffusion of leucine were found to be low compared with glycine, however, fast carrier-mediated transport of leucine assumed more importance at physiological concentrations.Abbreviations SIS Sucrose impermeable space - TWS Tritiated water space - SPS Sucrose permeable space - PGA 3-phosphoglyceric acid - TCA Trichloroacetic acid - TLC Thin layer chromatography     

The specificity of carrier-mediated auxin transport by suspension-cultured crown gall cells

1. The specificity of the auxin transport system of suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch- is examined with regard to 2,4-Dichlorophenoxyacetic acid (2,4 D), l-Naphthylacetic acid (NAA) and Benzoic acid (BA) as well as for indole-3-acetic acid (IAA). — 2. All four weak acids can be accumulated by the cells from a medium more acidic than the cytoplasm. This is by virtue of non-specific passive diffusion of their lipid-soluble protonated forms down a concentration gradient. The corresponding anionic species are much less permeant. The extent of the accumulation is dependent on the pH difference that is maintained by the cells between their cytoplasm and the incubation medium. Studies of the concentration dependence of BA and NAA net uptake at a series of external pHs suggest that an acidification of the cytoplasm can be eventually brought about by the entry of weak acid into the cells. — 3. The uptake of 2,4 D, as well as that of IAA, has a saturable carrier-mediated component in addition to the passive diffusion of the undissociated acid. These saturable components probably represent anion uptake and appear to be mediated by a common carrier. The kinetic studies provided no evidence for the participation of carriers in the transport of BA or NAA. — 4. It was shown that the efflux of 2,4 D also has a carrier-mediated component and it is suggested that both the influx and efflux of IAA and 2,4 D occur on a common carrier. — 5. The inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA), stimulates the net uptake of IAA by inhibiting carrier-mediated efflux of IAA from the cells. However, TIBA could not be demonstrated to have a significant effect on 2,4 D transport and any perturbation that occurs is very small in comparison with its effect on IAA movement. To account for this, the proposed common carrier could exhibit some difference in its internal binding characteristics betweend 2,4 D and IAA. An alternative explanation is that a second carrier is present, which mediates IAA efflux only, and which is inhibited by TIBA. — 6. TIBA has no significant effect on the transport of either BA or NAA, except to bring about an inhibition of net uptake, and a corresponding stimulation of efflux, when it is present at concentrations sufficient to acidify the cytoplasm. —7. The crown gall cells are compared to intact plant tissues capable of polar auxin transport with regard to the specificities exhibited for the transport of the auxins IAA, 2,4 D and NAA and the non-auxin BA.Abbreviations IAA indol-3-yl acetic acid - 2,4 D 2,4-Dichlorophenoxyacetic acid - NAA 1-Naphthylacetic acid - BA Benzoic acid - TIBA 2,3,5-triiodobenzoic acid     

Components of auxin transport in stem segments of Pisum sativum L.

1. The uptake of indol-3-yl acetic acid ([1-¹⁴C]IAA, 0–2.0 M) into light-grown pea stem segments was measured under various conditions to investigate the extent to which mechanisms of auxin transport in crown gall suspension culture cells (Rubery and Sheldrake, Planta 118, 101–121, 1974) are also found in a tissue capable of polar auxin transport. — 2. IAA uptake increased as the external pH was lowered. IAA uptake was less than that of benzoic acid (BA), naphthylacetic acid (NAA) or 2,4 dichlorophenoxyacetic acid (2,4D) under equivalent conditions. TIBA enhanced net IAA uptake through inhibition of efflux, and to a lesser extent, also increased uptake of NAA and 2,4D while it had no effect on BA uptake. — 3. Both DNP and, at higher concentrations, BA, reduced IAA uptake probably because of a reduction of cytoplasmic pH. However, low concentrations of both BA and DNP caused a slight enhancement of IAA net uptake, possibly through a reduction of carrier-mediated IAA efflux. In the presence of TIBA, the inhibitory effects of DNP and BA were more severe and there was no enhancement of uptake at low concentrations. — 4. Non-radioactive IAA (10 M) reduced uptake of labelled IAA but further increases in concentration up to 1.0 mM produced first an inhibition (0–10 min) of labelled IAA uptake, followed by a stimulation at later times. Non-radioactive 2,4 D decreased, but was not observed to stimulate, uptake of labelled IAA. In the presence of TIBA labelled IAA uptake was inhibited by non-radioactive IAA regardless of its concentration. — 5. Sulphydryl reagents PCMB and PCMBS promoted or inhibited IAA uptake depending, respectively, on whether they penetrated or were excluded from the cells. The penetrant PCMB also reduced the promotion of labelled IAA uptake by TIBA or by high concentrations of added non-labelled IAA. — 6. Our findings are interpreted as being consistent with the diffusive entry of unionised IAA into cells together with some carrier-mediated uptake. Auxin efflux from the cells also appears to have a carrier-mediated contribution, at least part of which is inhibited by TIBA, and which has a capacity at least as great as that of the uptake carrier. The data indicate that pea stem segments contain cells whose mechanisms of trans-membrane auxin transport fit the model of polar auxin transport proposed from experiments with crown gall suspension cells, although differences, particularly of carrier specificity, are apparent between the two systems.Abbreviations IAA indol-3-yl acetic acid - BA benzoic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - DNP 2,4-dinitrophenol - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzene sulphonic acid This work was performed in Cambridge during the tenure of a sabbatical leave by P.J.D. Supported by a grant for supplies from the American Philosophical Society to P.J.D.     

Evidence for Carrier-Mediated Transport of Monosaccharides in the Ehrlich Ascites Tumor Cell

Evidence for carrier-mediated transport of monosaccharides in the Ehrlich ascites tumor cells was provided through kinetic analysis of data obtained by: (a) studying sugar uptake by dilute cell suspensions with an optical densimetric apparatus, (b) studying sugar uptake by thicker cell suspensions by means of direct chemical analytical methods using packed cell plugs, (c) observing the effects of a competitive inhibitor upon sugar uptake with the chemical analytical method, and (d) measurement of tracer uptake of a high affinity sugar in thick cell suspensions in the absence of net movement. Quantitative application of the data obtained with the above experimental procedures to theoretical model systems derived for both carrier-mediated transport and simple passive diffusion indicated that the results were consonant with predictions for the carrier-mediated transport model, but could not be explained on the basis of uncomplicated diffusion.     

Uptake of the neutral amino acids glutamine, leucine, and serine by Pneumocystis carinii.

Experiments to elucidate the mechanism by which Pneumocystis carinii transports glutamine, leucine, and serine were performed in this study. Uptake of all three radiolabeled amino acids exhibited first-order, saturation kinetics as extracellular substrate concentrations were increased, thus ruling out simple diffusion and indicating carrier-mediated transport. Kinetic analyses of amino acid uptake and the results of competitive inhibition experiments suggested that leucine, serine, and glutamine were taken up via a common transporter system. The uptake of serine was examined in greater detail to characterize the nature of the carrier. Serine uptake was not affected by N, N'-dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl hydrazone, ouabain, gramicidin, valinomycin, sodium azide, salicylhydroxamine acid (SHAM), iodoacetate, iodoacetate plus SHAM, KCN, and azide. Thus serine uptake did not require sodium or energy from ATP, an electrochemical proton gradient or a membrane potential across the cell surface (i.e., proton-motive force). Serine uptake was dependent on glucose in the extracellular compartment. In the presence of glucose, serine uptake was inhibited by chloramphenicol but not cycloheximide. The results from these experiments are most consistent with facilitated diffusion as the mechanism. After 30 min of incubation, most of the radioactivity was in the cellular soluble fraction. In most cases, incorporation into the extractable total lipids and the remaining particulate cellular components were detectable after this incubation period.     

Uptake of L-carnitine, D-carnitine and acetyl-L-carnitine by isolated guinea-pig enterocytes

Uptake and metabolism of L-carnitine, D-carnitine and acetyl-L-carnitine were studied utilizing isolated guinea-pig enterocytes. Uptake of the D- and L-isomers of carnitine was temperature dependent. Uptake of L-[14C]carnitine by jejunal cells was sodium dependent since replacement by lithium, potassium or choline greatly reduced uptake. L- and D-carnitine developed intracellular to extracellular concentration gradients for total carnitine (free plus acetylated) of 2.7 and 1.4, respectively. However, acetylation of L-carnitine accounted almost entirely for the difference between uptake of L- and D-carnitine. About 60% of the intracellular label was acetyl-L-carnitine after 30 min, and the remainder was free L-carnitine. No other products were observed. D-Carnitine was not metabolized. Acetyl-L-carnitine was deacetylated during or immediately after uptake into intestinal cells and a portion of this newly formed intracellular free carnitine was apparently reacetylated. L-Carnitine and D-carnitine transport (after adjustment for metabolism and diffusion) were evaluated over a concentration range of 2-1000 microM. Km values of 6-7 microM and 5 microM, were estimated for L- and D-carnitine, respectively. Ileal-cell uptake was about half that found for jejunal cells, but the labeled intracellular acetylcarnitine-to-carnitine ratios were similar for both cell populations. Carnitine transport by guinea-pig enterocytes demonstrate characteristics of a carrier-mediated process since it was inhibited by D-carnitine and trimethylaminobutyrate, as well as being temperature and concentration dependent. The process appears to be facilitated diffusion rather than active transport since L-carnitine did not develop a significant concentration gradient, and was unaffected by ouabain or actinomycin A.     

The tfdK Gene Product Facilitates Uptake of 2,4-Dichlorophenoxyacetate by Ralstonia eutropha JMP134(pJP4)

Uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Ralstonia eutropha JMP134(pJP4) was studied and shown to be an energy-dependent process. The uptake system was inducible with 2,4-D and followed saturation kinetics in a concentration range of up to 60 μM, implying the involvement of a protein in the transport process. We identified an open reading frame on plasmid pJP4, which was designated tfdK, whose translation product TfdK was highly hydrophobic and showed resemblance to transport proteins of the major facilitator superfamily. An interruption of the tfdK gene on plasmid pJP4 decimated 2,4-D uptake rates, which implies a role for TfdK in uptake. A tfdA mutant, which was blocked in the first step of 2,4-D metabolism, still took up 2,4-D. A mathematical model describing TfdK as an active transporter at low micromolar concentrations fitted the observed uptake data best.     

Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid,naphthalene-1-acetic acid,and indole-3-acetic acid in suspension-cultured tobacco cells

Accumulation of radiolabelled naphthalene-1-acetic acid (1-NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-acetic acid (IAA) has been measured in suspension-cultured tobacco (Nicotiana tabacum) cells. In this paper is presented a simple methodology allowing activities of the auxin influx and efflux carriers to be monitored independently by measuring the cellular accumulation of [³H]NAA and [¹⁴C]2,4-D. We have shown that 1-NAA enters cells by passive diffusion and has its accumulation level controlled by the efflux carrier. By contrast, 2,4-D uptake is mostly ensured by the influx carrier and this auxin is not secreted by the efflux carrier. Both auxin carriers contribute to IAA accumulation. The kinetic parameters and specificity of each carrier have been determined and new information concerning interactions with naphthylphthalamic acid, pyrenoylbenzoic acid, and naphthalene-2-acetic acid are provided. The relative contributions of diffusion and carrier-mediated influx and efflux to the membrane transport of 2,4-D, 1-NAA, and IAA have been quantified, and the data indicate that plant cells are able to modulate over a large range their auxin content by modifying the activity of each carrier.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 1-NAA naphthalene-1-acetic acid - 2-NAA naphthalene-2-acetic acid - NPA N-1-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - V_m maximum transport capacity of the carrier In honour of Professor Dieter Klämbt's 65th birthdayThe authors thank Drs. A.E. Geissler and G.F. Katekar (CSIRO, Canberra City, Australia) for providing auxin efflux carrier inhibitors CPD, CPP, and PBA, and Dr. H. Barbier-Brygoo (Institut des Sciences Végétales, CNRS, Gif-sur-Yvette, France) for helpful discussions. This work was supported by funds from the Centre National de la Recherche Scientifique (UPR0040).     

The Kinetics of Fatty Acid Uptake By Paramecium Tetraurelia

ABSTRACT. The kinetics of radiolabeled fatty acid uptake by the ciliate Paramecium tetraurelia was examined on a homologous series of saturated, straight chain fatty acids of even carbon numbers. Uptake rates increased with chain length from acetate to palmitate. Saturation kinetics was demonstrated for most fatty acids examined, thus ruling out simple diffusion as the major mechanism for fatty acid transport and implicating carrier-mediated, facilitated transport as the major mechanism. Data from most competitive inhibition experiments were too scattered to determine the number of transporter systems present. Cholesterol uptake also exhibited saturation kinetics and hence other sterols, which can satisfy this nutritional requirement, may also be transported by a carrier-mediated mechanism. the uptake of the essential fatty acid oleate was faster than those observed for the saturated acids and could not be explained by only one transport mechanism. Therefore, fatty acid transport also occurs via other kinetically significant routes.     

Folate transport in the choroid plexus.

The uptake of 5-methyltetrahydrofolic acid (5-MTHF) by the isolated choroid plexus of hog was studied and shown to be both temperature and time dependent. Uptake of 5-MTHF by the isolated choroid plexus was a saturable process and exhibited a K_t of 0.9 × 10⁻⁶M and Vmax of 1.39 nmole/gm dry wt/min. The system did not require the presence of sodium ion nor was it ouabain sensitive. The presence of metabolic inhibitors, e.g., 2,4-dinitrophenol, did not suppress the uptake rate. Deprivation of oxygen also did not affect the rate of 5-MTHF transport. Addition of folic acid to the incubating medium led to countertransport of intracellular 5-MTHF. Efflux studies also indicated that the majority of the intracellular 5-MTHF was rapidly exchangeable and therefore probably present in the cell water in a free state. Chromatographic analyses confirmed that 5-MTHF was not metabolically altered during the transport process. It is suggested that 5-methyltetrahydrofolic acid is transported in the isolated choroid plexus via a carrier-mediated process.     

Monosaccharide uptake by erythrocytes of the embryonic and adult chicken

Rates of monosaccharide uptake by adult and 10-18 day old embryonic chicken erythrocytes were quantitated. The rate of carrier-mediated, stereospecific transport decreased 28% from day 10 to day 14 of incubation and was unchanged thereafter. At no time, however, did the rate of carrier-mediated transport by embryonic erythrocytes differ significantly from that of the adult cells. The rate of transfer by simple diffusion was 3-5 fold faster in embryonic than in adult erythrocytes. Uptake by simple diffusion decreased slightly as the embryo developed. Chronic hyperoxic incubation (70% O2) had little influence on total monosaccharide uptake by embryonic erythrocytes.     

Active Transport of Alcohol in Corynebacterium acetophilum

The transport of alcohols was studied in Corynebacterium acetophilum, which was isolated as a strain growing well on acetate and ethanol. The transport of ethanol was found to be inducible by ethanol, n-propanol, n-butanol, and acetate, whereas transport of methanol occurred by noninducible passive diffusion. The entry of ethanol into the cells occurred against a concentration gradient and showed saturation kinetics with two K(m) values of 2.4 x 10(-5) M and 6.0 x 10(-5) M. Uptake of ethanol was inhibited by sodium azide, sodium cyanide, 2,4-dinitrophenol, and p-chloromercuribenzoate. The transport of ethanol was competitively inhibited by normal alcohols, but not by iso- or tert-alcohols. From these studies, we concluded that an inducible active alcohol transport system mediates the entry of ethanol, n-propanol, or n-butanol into the cells of C. acetophilum.     

Carrier-mediated uptake of lactate in rat hepatocytes. Effects of pH and possible mechanisms for L-lactate transport

The rate of uptake and the distribution ratio between intra- and extracellular compartments of L- and D-lactate were studied in hepatocyte preparations from fed rats. L- and D-lactate uptake apparently depended on both passive diffusion and carrier-mediated components. The apparent Km of the high-affinity carrier for L-lactate was in the range of 1.8 mM. The reciprocal competitive inhibitions between isomers of lactate suggest that L- and D-lactate might be transported by distinct carriers. Lactate transport was inhibited by various anions; pyruvate was the most potent anion, whereas only high concentrations of ketone bodies were effective. Acidic extracellular pH enhanced lactate uptake, this effect being more pronounced for L-lactate. At low pH, L-lactate was concentrated into hepatocytes, but its affinity for the carrier appeared unchanged, suggesting the existence of a process gaining energy from the pH gradient across the cell membrane. In the hypothesis of a lactate/H+ symport, the affinity for H+ was not dependent on lactate concentration and the apparent Km for H+ corresponded to a pH of 7.34. No trans-stimulation of lactate uptake after prior loading of the cells with pyruvate or lactate was observed. The present data suggest that, at physiological concentrations, lactate uptake by the liver might be largely carrier-mediated and the rate of transport across the liver cell membrane may be of a magnitude relatively comparable to the rate of metabolism.     

Carrier-mediated uptake of cephalexin in human intestinal cells

A transport carrier for cephalexin, a cephalosporin antibiotic, was identified in a human intestinal cell line, HT-29. Uptake via the carrier was inhibited by dipeptides, phe-gly, gly-pro, carnosine, and cefaclor, a close drug analog. Uptake was unaffected by the presence of amino acids. The pH optimum for uptake was 6.2. Drug uptake was not dependent on the presence of sodium and was insensitive to metabolic inhibitors. The efflux of cephalexin was stimulated by extracellular carnosine, indicating counter-transport. Taken together, drug uptake is mediated by a dipeptide transport carrier and not by an amino acid transport carrier. This is the first demonstration of the carrier in an established cell line.     

Uptake of Benzoic Acid and Chloro-Substituted Benzoic Acids by Alcaligenes denitrificans BRI 3010 and BRI 6011

The mechanism of uptake of benzoic and 2,4-dichlorobenzoic acid (2,4-DCBA) by Alcaligenes denitrificans BRI 3010 and BRI 6011 and Pseudomonas sp. strain B13, three organisms capable of degrading various isomers of chlorinated benzoic acids, was investigated. In all three organisms, uptake of benzoic acid was inducible. For benzoic acid uptake into BRI 3010, monophasic saturation kinetics with apparent K(infm) and V(infmax) values of 1.4 (mu)M and 3.2 nmol/min/mg of cell dry weight, respectively, were obtained. For BRI 6011, biphasic saturation kinetics were observed, suggesting the presence of two uptake systems for benzoic acid with distinct K(infm) (0.72 and 5.3 (mu)M) and V(infmax) (3.3 and 4.6 nmol/min/mg of cell dry weight) values. BRI 3010 and BRI 6011 accumulated benzoic acid against a concentration gradient by a factor of 8 and 10, respectively. A wide range of structural analogs, at 50-fold excess concentrations, inhibited benzoic acid uptake by BRI 3010 and BRI 6011, whereas with B13, only 3-chlorobenzoic acid was an effective inhibitor. For BRI 3010 and BRI 6011, the inhibition by the structural analogs was not of a competitive nature. Uptake of benzoic acid by BRI 3010 and BRI 6011 was inhibited by KCN, by the protonophore 3,5,3(prm1), 4(prm1)-tetrachlorosalicylanilide (TCS), and, for BRI 6011, by anaerobiosis unless nitrate was present, thus indicating that energy was required for the uptake process. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation uptake kinetics were not observed. Uptake of 2,4-DCBA by BRI 6011 was inhibited by KCN, TCS, and anaerobiosis even if nitrate was present, but the compound was not accumulated intracellularly against a concentration gradient. Uptake of 2,4-DCBA by BRI 6011 appears to occur by passive diffusion into the cell down its concentration gradient, which is maintained by the intracellular metabolism of the compound. This process could play an important role in the degradation of xenobiotic compounds by microorganisms.     

Uptake Kinetics of 2,4-Dichlorophenoxyacetate by Delftia acidovorans MC1 and Derivative Strains: Complex Characteristics in Response to pH and Growth Substrate

The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20–100 μM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 μM 2,4-D at either pH, and amounted to 15–20 nmol/min*mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min*mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.     

Transport of aspartic acid, arginine, and tyrosine by the opportunistic protist Pneumocystis carinii

In order to improve culture media and to discover potential drug targets, uptake of an acidic, a basic, and an aromatic amino acid were investigated. Current culture systems, axenic or co-cultivation with mammalian cells, do not provide either the quantity or quality of cells needed for biochemical studies of this organism. Insight into nutrient acquisition can be expected to lead to improved culture media and improved culture growth. Aspartic acid uptake was directly related to substrate concentration, Q(10) was 1.10 at pH 7.4. Hence the organism acquired this acidic amino acid by simple diffusion. Uptake of the basic amino acid arginine and the aromatic amino acid tyrosine exhibited saturation kinetics consistent with carrier-mediated mechanisms. Kinetic parameters indicated two carriers (K(m)=22.8+/-2.5 microM and K(m)=3.6+/-0.3 mM) for arginine and a single carrier for tyrosine (K(m)=284+/-23 microM). The effects of other L-amino acids showed that the tyrosine carrier was distinct from the arginine carriers. Tyrosine and arginine transport were independent of sodium and potassium ions, and did not appear to require energy from ATP or a proton motive force. Thus facilitated diffusion was identified as the mechanism of uptake. After 30 min of incubation, these amino acids were incorporated into total lipids and the sedimentable material following lipid extraction; more than 90% was in the cellular soluble fraction.     

Transport of dehydroepiandrosterone and dehydroepiandrosterone sulphate into rat hepatocytes

The purpose of the present study was to characterize the transport of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) into hepatocytes at physiological and pharmacological concentrations. Hepatocytes were isolated from female Sprague-Dawley rats by collagenase perfusion. Uptake of [³H]DHEA and [³H]DHEAS at increasing concentrations (3.5 nM-100 μM) was measured by the rapid filtration technique at 30 s intervals up to 120 s. The uptake of DHEAS by hepatocytes was saturable (K_m = 17.0 μM; V_max = 3.7 nmol/min/mg cell protein). In contrast, a specific saturable transport system for DHEA could not be detected in rat hepatocytes. It is suggested that DHEA enters the cell by diffusion. The uptake of DHEAS could be inhibited by antimycin A, carbonylcyanide-m-chlorophenylhydrazone, and dinitrophenol (inhibitors of the mitochondrial respiratory chain), by dinitrofluorobenzene and p-hydroxymercuribenzoate (NH₂- and SH-blockers, respectively), and by monensin (Na⁺-specific ionophore). No inhibition was seen in the presence of ouabain (inhibitor of Na⁺-K⁺-ATPase) and phalloidin (inhibitor of cholate transport and actin-blocker). Interestingly, DHEAS uptake was inhibited by bile acids (cholate, taurocholate and glycocholate). Conversely, [³H]cholate uptake was strongly inhibited by DHEAS, which indicates a competition for the same carrier. Replacement of sodium ion with choline markedly decreased uptake velocity at pharmacological DHEAS concentrations. The results suggest that DHEAS uptake is a saturable, energy-dependent, carrier-mediated, partially Na⁺-dependent process, and that DHEAS may be taken up via the multispecific bile acid transport system.     

l-DOPA (l-3,4-dihydroxyphenylalanine) uptake by human red blood cells

The uptake of l-DOPA (l-3,4-dihydroxyphenylalanine) was studied in normal human red blood cells in vitro using l-[3-¹⁴C]DOPA. Uptake was slow, tending towards a distribution ratio close to unity with a half-time to equilibrium of one hour. Uptake was not Na⁺-dependent. Concentration dependence studies showed both saturable and non-saturable components of uptake, and inhibition studies using l-leucine and l-tryptophan suggest that the L and T systems of red cell amino acid uptake are involved. A powerful inhibitor of both systems, 3,4-dihydroxy-2-methylpropriophenone (U-0521), is described. It is concluded that uptake is by carrier-mediated facilitated diffusion via the L and T systems for which l-DOPA has low affinity.