Basolateral amino acid transport systems in the perfused exocrine pancreas: sodium-dependency and kinetic interactions between influx and efflux mechanisms

Basolateral amino acid transport systems have been characterized in the perfused exocrine pancreas using a high-resolution paired-tracer dilution technique. Significant epithelial uptakes were measured for L-alanine, L-serine, alpha-methylaminoisobutyric acid, glycine, methionine, leucine, phenylalanine, tyrosine and L-arginine, whereas L-tryptophan and L-aspartate had low uptakes. alpha-Methylaminoisobutyric acid transport was highly sodium dependent (81 +/- 3%), while uptake of L-serine, L-leucine and L-phenylalanine was relatively insensitive to perfusion with a sodium-free solution. Cross-inhibition experiments of L-alanine and L-phenylalanine transport by twelve unlabelled amino acids indicated overlapping specificities. Unidirectional L-phenylalanine transport was saturable (Kt = 16 +/- 1 mM, Vmax = 12.3 +/- 0.4 mumol/min per g), and weighted non-linear regression analysis indicated that influx was best described by a single Michaelis-Menten equation. The Vmax/Kt ratio (0.75) for L-phenylalanine remained unchanged in the presence of 10 mM L-serine. Although extremely difficult to fit, L-serine transport appeared to be mediated by two saturable carriers (Kt1 = 5.2 mM, Vmax1 = 7.56 mumol/min per g; Kt2 = 32.8 mM, Vmax2 = 22.9 mumol/min per g). In the presence of 10 mM L-phenylalanine the Vmax/Kt ratio for the two L-serine carriers was reduced, respectively, by 79% and 50%. Efflux of transported L-[3H]phenylalanine or L-[3H]serine was accelerated by increasing perfusate concentrations of, respectively, L-phenylalanine and L-serine, and trans-stimulated by other amino acids. In the pancreas neutral amino acid transport appears to be mediated by Na+-dependent Systems A and ASC, the classical Na+-independent System L and another Na+-independent System asc recently identified in erythrocytes. The interactions in amino acid influx and efflux may provide one of the mechanisms by which the supply of extracellular amino acids for pancreatic protein synthesis is regulated.     

Autoradiographic localization of transported neutral amino acids in epithelia of cat submandibular and sublingual salivary glands

Summary Light-microscopic autoradiography was used to localize the cellular sites for neutral amino acid uptake in submandibular and sublingual salivary gland epithelia. The vasculature of isolated glands was perfused for 3–5 min with either L-(3-³H)serine or L-(4-³H)phenylalanine and then fixed by perfusion with buffered glutaraldehyde. In the submandibular gland the small neutral amino acid L-serine and the aromatic amino acid L-phenylalanine were localized to central acinar cells, demilunar cells and ductal cells. In the sublingual gland silver grains associated with each of these tritiated amino acids were localized to central acinar and ductal cells. Perfusion of both submandibular and sublingual glands with unlabelled L-serine (25 mM) or L-phenylalanine (30 mM) resulted in a significant decrease in the silver grain density associated with each labelled amino acid. The absence of silver grains in the lumina of acinar and ductal cells and the presence of tight junctions near the apical surface of the epithelium strongly suggest that the initial uptake of these amino acids was mediated by basolateral plasma membrane carriers.     

Unidirectional flux of phenylalanine into Vero cells. Measurement using paired tracers in perfused cultures

The uptake of phenylalanine by Vero cells in perfused culture was measured using a double-label technique. Cells were anchored in microcarrier beads and maintained in a column perfused at a constant rate. The extracellular tracer [14C]mannitol and the test tracer [3H]phenylalanine were injected as a bolus, and the column effluent was sampled at 10-s intervals. The proportion of the test tracer retained by the cells was calculated by analysis of time-dilution curves of test and reference tracers. Uptake measurements were specific and highly reproducible. Uptake of [3H]phenylalanine was inhibited by unlabelled phenylalanine and by other amino acids that utilize transport system L. This new approach proved useful for rapid measurement of unidirectional uptake, and for determination of kinetics parameters of uptake under steady state conditions. This rapid technique obviates some of the limitations associated with uptake measurements in whole organs and with measurements in conventional cell cultures.     

Hypoxia inhibits amino acid uptake in human lung fibroblasts.

Hypoxia and amino acid deprivation downregulate expression of extracellular matrix genes in lung fibroblasts. We examined the effect of hypoxia on amino acid uptake and protein formation in human lung fibroblasts. Low O(2) tension (0% O(2)) suppressed incorporation of [(3)H]proline into type I collagen without affecting [(35)S]methionine labeling of other proteins. Initial decreases in intracellular [(3)H]proline incorporation occurred after 2 h of exposure to 0% O(2), with maximal suppression of intracellular [(3)H]proline levels at 6 h of treatment. Hypoxia significantly inhibited the uptake of radiolabeled proline, 2-aminoisobutyric acid (AIB), and 2-(methylamino)isobutyric acid (methyl-AIB) while inducing minor decreases in leucine transport. Neither cycloheximide nor indomethacin abrogated hypoxia-related suppression of methyl-AIB uptake. Efflux studies demonstrated that hypoxia inhibited methyl-AIB transport in a bidirectional fashion. The downregulation of amino acid transport was not due to a toxic effect; function recovered on return to standard O(2) conditions. Kinetic analysis of AIB transport revealed a 10-fold increase in K(m) accompanied by a small increase in maximal transport velocity among cells exposed to 0% O(2). These data indicate that low O(2) tension regulates the system A transporter by decreasing transporter substrate affinity.     

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.     

Inhibition by a nonmetabolized analogue of L-leucine of protein biosynthesis in tumoral pancreatic islet cells

The effect of L-leucine, its deaminated metabolite 2-ketoisocaproate and its nonmetabolized analogue b(+/-)2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) upon protein labelling was examined in tumoral islet cells (RINm5F line) exposed to L-[4-3H]phenylalanine or L-[3-3H]serine. The interpretation of the results, in terms of changes in biosynthetic activity, was obscured by a possible interference of the tested nutrients with the uptake and further metabolism of the tracer tritiated amino acids. Nevertheless, when the cells were preincubated with the nutrient secretagogues and then incubated in the sole presence of L-[3-3H]serine, BCH, but not L-leucine or 2-ketoisocaproate, still inhibited protein labelling, this coinciding with a decrease in the ratio between TCA-precipitable and total radioactivity in the RINm5F cells. The inhibitory action of BCH was antagonized, to a limited extent, by D-glucose. It is proposed that BCH could be used as a tool to interfere with the function and growth of insulinoma cells.     

In vivo and in vitro evidence for ACh-stimulated L-arginine uptake

Whereas L-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y(+) transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), the effects of ACh (9.25 and 37 microg/min), Iso (25-50 and 200 microg/min), and sodium nitroprusside (SNP) (1-2 and 8 microg/min) on forearm plasma flow (FPF), L-[(3)H]arginine uptake, and L-[(3)H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on L-[(3)H]arginine uptake were studied in BAEC. L-Arginine uptake was inversely related to FPF (r = -0.50; P < 0.005). At a similar FPF (ACh 56.82 +/- 9.25, Iso 58.49 +/- 5.56, SNP 57.92 +/- 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of L-[(3)H]arginine (54,655 +/- 8,018 dpm/min), compared with that observed with either Iso (40,517.23 +/- 6,841 dpm/min; P = 0.01) or SNP (36,816 +/- 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced L-[(3)H]citrulline release compared with Iso and SNP (P = 0.046). Similarly, in BAEC, ACh significantly increased L-[(3)H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 x 10(7) +/- 1.83 x 10(7) vs. control 6.67 x 10(7) +/- 1.16 x 10(7) vs. Iso 7.35 x 10(7) +/- 1.63 x 10(7) vs. SNP 6.01 x 10(7) +/- 1.11 x 10(7) fmol.min(-1).mg(-1) at 300 micromol/l L-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.     

Influx and incorporation into protein of L-phenylalanine in the perfused rat pancreas: effects of amino acid deprivation and carbachol.

The rate of protein synthesis in the isolated perfused rat pancreas was measured from the rate of incorporation of L-[3H]phenylalanine into total protein, and was compared with the transport of this amino acid into the epithelium. Unidirectional (15 s) and net (15-30 min) uptake of L-[3H]phenylalanine was measured relative to D-[14C]mannitol (extracellular marker) using a cell loading technique. The fractional rate of protein synthesis in the pancreas was also measured in vivo using a flooding dose technique and found to be 118 +/- 10% day-1 (corresponding to an absolute rate of incorporation of L-Phe into protein of 36.1 +/- 3 nmol min-1 g-1) in overnight fasted rats. Compared with the in vivo rate, the perfused pancreas exhibited a markedly lower rate of protein synthesis which increased significantly when amino acids were added to the perfusate (15.6 +/- 1.9 vs. 22.5 +/- 0.9% day-1 or 4.7 +/- 0.6 vs. 6.9 +/- 0.3 nmol L-Phe min-1 g-1). Carbachol (3 x 10(-7) M) stimulated protein synthesis provided amino acids were also supplied in the perfusate. Protein synthesis rates measured under all conditions in vivo and in vitro were at least an order of magnitude lower than the unidirectional influx (121 +/- 14 nmol min-1 g-1) of L-phenylalanine into the pancreatic epithelium. These results demonstrate that amino acid transport across the basolateral membrane of the epithelium is not rate-limiting for pancreatic protein synthesis.     

Novel localization of OCTN1, an organic cation/carnitine transporter, to mammalian mitochondria

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. We report novel localization of the organic cation/carnitine transporter, OCTN1, to mitochondria. We made GFP- and RFP-human OCTN1 cDNA constructs and showed expression of hOCTN1 in several transfected mammalian cell lines. Immunostaining of GFP-hOCTN1 transfected cells with different intracellular markers and confocal fluorescent microscopy demonstrated mitochondrial expression of OCTN1. There was striking co-localization of an RFP-hOCTN1 fusion protein and a mitochondrial-GFP marker construct in transfected MEF-3T3 and no co-localization of GFP-hOCTN1 in transfected human skin fibroblasts with other intracellular markers. L-[(3)H]Carnitine uptake in freshly isolated mitochondria of GFP-hOCTN1 transfected HepG2 demonstrated a K(m) of 422 microM and Western blot with an anti-GFP antibody identified the expected GFP-hOCTN1 fusion protein (90 kDa). We showed endogenous expression of native OCTN1 in HepG2 mitochondria with anti-GST-hOCTN1 antibody. Further, we definitively confirmed intact L-[(3)H]carnitine uptake (K(m) 1324 microM), solely attributable to OCTN1, in isolated mitochondria of mutant human skin fibroblasts having <1% of carnitine acylcarnitine translocase activity (alternate mitochondrial carnitine transporter). This mitochondrial localization was confirmed by TEM of murine heart incubated with highly specific rabbit anti-GST-hOCTN1 antibody and immunogold labeled goat anti-rabbit antibody. This suggests an important yet different role for OCTN1 from other OCTN family members in intracellular carnitine homeostasis.     

The transport of alanine, serine, and cysteine in cultured human fibroblasts

The transport of L-alanine, L-serine, and L-cysteine has been studied in skin-derived diploid human fibroblasts in culture. Competition analysis, mathematical discrimination by nonlinear regression, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of these amino acids. All the adopted criteria yielded results showing that L-alanine, L-serine, and L-cysteine enter the cell by two Na+-dependent systems, System A and System ASC, and by a Na+-independent route, whose major component has been identified as System L. The apparent affinity of L-alanine, L-serine, and L-cysteine for the putative carrier was higher for System ASC than for System A. The transport Vmax for System A increased in response to cell starvation; after 12 h, its values were similar or higher than those exhibited by System ASC. At amino acid concentrations approaching those present in human plasma, System ASC appeared to be the primary mediation for the inward transport of L-alanine, L-serine, and L-cysteine in human fibroblasts. The contribution of System A was negligible in nonstarved cells and became appreciable under conditions of cell starvation. The Na+-independent System L made no substantial contribution to the uptake of L-alanine and L-serine and accounted for approximately one-fourth of the total uptake of L-cysteine.     

Membrane protein changes in an L1210 leukemia cell line with a translocation defect in the methotrexate-tetrahydrofolate cofactor transport carrier

We report on membrane protein changes in an L1210 leukemia cell line with a highly specific defect in the function of the methotrexate (MTX)-tetrahydrofolate cofactor transport carrier. This clonal line, MTXrA, made 100-fold resistant to MTX, was derived in a single step and exhibited stable resistance over 120 generations in the absence of drug. The transport defect was associated with a 10-fold decrease in influx Vmax without a change in influx Km. There was no difference between the MTXrA and parent lines in the levels or affinities of specific cell surface binders for MTX nor in the labeling of the 44-kDa membrane protein upon treatment with the specific affinity label, N-hydroxysuccinimide ester of tritiated MTX. Consistent with impaired carrier function was the observation that trans-stimulation of MTX influx by intracellular 5-formyltetrahydrofolate observed in the parent line was not demonstrated in the MTXrA line. The transport defect was highly specific for the MTX-tetrahydrofolate cofactor transport carrier. Initial uptake rates for 5-fluoro-2'-deoxyuridine and 2-deoxyglucose were unchanged and influx and net transport of alpha-aminoisobutyric acid were, in fact, increased. There was no cross-resistance of this line to phenylalanine mustard or cytosine arabinoside, agents that utilize specific amino acid and nucleoside transport carriers, respectively. SDS-polyacrylamide gel electrophoresis of purified plasma membrane preparations stained with Coomassie Blue revealed several protein differences between the parental and MTXrA lines. Most prominent is a band at approximately 190 kDa which ran with slightly greater mobility than a lesser staining band in the parent line. [3H]Borohydride labeling of cells also identified a distinct protein peak in the MTXrA line at approximately 190 kDa eliminated by prior treatment of cells with neuraminidase. Absence of expression of protein or mRNA related to the multidrug resistance gene as well as lack of cross-resistance to daunorubicin or trimetrexate indicate that this mechanism of resistance to MTX is completely unrelated to the multidrug resistance phenomenon observed with high molecular weight heterocyclic compounds. These data represent the first demonstration of membrane protein differences in a highly resistant L1210 murine leukemia cell line with a marked unique defect in MTX transport which appears to be related to impaired mobility of the tetrahydrofolate-cofactor carrier. Further studies are now required to elucidate the possible role of one or more of these proteins in the transport defect.     

Normal amino acid uptake by cultured human fibroblasts does not require gamma-glutamyl transpeptidase

The uptake kinetics for four amino acids (cystine, glutamine, methionine, and alanine) which are among the best gamma-glutamyl acceptors have been determined for normal human fibroblasts and for a cell line containing undetectable quantities (< 0.5% normal mean) of gamma-glutamyl transpeptidase activity. Apparent Km and V(max) for uptake for each of the four amino acids were normal in the mutant fibroblasts. Insulin increased the uptake of alpha-aminoisobutyrate as in control cells. levels of 16 amino acids were also normal in this cell strain; the intracellular concentrations of phenylalanine, cystine, and cysteine were increased. In human fibroblasts, amino acid transport appears to proceed normally in the absence of active gamma-glutamyl transpeptidase.     

Transport and intracellular localization of cortisol-asialotranscortin complexes in rat liver

The influence of desialylation of human transcortin on transport of the transcortin-cortisol complex into the liver cells and its intracellular distribution was investigated in perfused rat liver. Under experimental conditions used the half-time of cortisol in perfusion medium was decreased more than 200 times in presence of asialotranscortin compared to that of native transcortin. Experiments with [3H]cortisol and [131I]asialotranscortin demonstrated a simultaneous uptake of cortisol and asialotranscortin by the hepatocytes. Distribution of [3H]cortisol and [131I]asialotranscortin in subcellular fractions showed the following pathway of cortisol-asialotranscortin complex: cell membrane, lysosomes, cytoplasm. The complex dissociates in lysosomes.     

Treatment of glutathione synthetase deficient fibroblasts by inhibiting gamma-glutamyl transpeptidase activity with serine and borate.

Glutathione synthetase deficiency results in decreased cellular glutathione content and consequent overproduction of 5-oxoproline. L-serine in borate buffer inhibits γ-glutamyl transpeptidase, the major catabolic enzyme for glutathione. Treatment of glutathione synthetase deficient fibroblasts with 40mM serine and borate for 24 hours produced more than a 2-fold increase in cellular glutathione content. L-serine alone led to a smaller increase in glutathione level, and borate alone was without effect. On exposure to serine and borate, 5-oxoproline formation from L-glutamate was decreased to normal levels in glutathione synthetase deficient fibroblasts, presumably secondary to feedback inhibition of γ-glutamylcysteine synthetase by the increased intracellular glutathione concentration. Cellular free amino acid content was generally unaffected by such exposure although increases were observed in serine and phosphoserine. This model system suggests that γ-glutamyl transpeptidase inhibition may be a rational approach to alleviating the effects of glutathione synthetase deficiency.     

D-Serine inhibits serine palmitoyltransferase, the enzyme catalyzing the initial step of sphingolipid biosynthesis

Serine palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L-serine to produce 3-ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [(3)H]KDS from L-[(3)H]serine was performed using purified SPT. D-Serine inhibited [(3)H]KDS production as effectively as non-radioactive L-serine, whereas neither D-alanine nor D-threonine showed any significant effect. Incubation of purified SPT with [palmitoyl 1-(14)C]palmitoyl coenzyme A and D-serine did not produce [(14)C]KDS, while the control incubation with L-serine did. These results suggest that D-serine competes with L-serine for the amino acid recognition site of SPT, but that D-serine is not utilized by this enzyme to produce KDS.     

Comparison of the Properties of γ-Aminobutyric Acid and L-Glutamate Uptake into Synaptic Vesicles Isolated from Rat Brain

Rat brain synaptic vesicles exhibit ATP-dependent uptake of gamma-[3H]amino-n-butyric acid ([3H]GABA) and L-[3H]glutamate. After hypotonic shock, the highest specific activities of uptake of both L-glutamate and GABA were recovered in the 0.4 M fraction of a sucrose gradient. The uptakes of L-glutamate and GABA were inhibited by similar, but not identical, concentrations of the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone and the ionophores nigericin and gramicidin, but they were not inhibited by the K+ carrier valinomycin. N,N'-Dicyclohexyl-carbodiimide and N-ethylmaleimide, Mg2+-ATPase inhibitors, inhibited the GABA and L-glutamate uptakes similarly. Low concentrations of Cl- stimulated the vesicular uptake of L-glutamate but not that of GABA. The uptakes of both L-glutamate and GABA were inhibited by high concentrations of Cl-. These results indicate that the vesicular GABA and L-glutamate uptakes are driven by an electrochemical proton gradient generated by a similar Mg2+-ATPase. The vesicular uptake mechanisms are discussed in relation to other vesicle uptake systems.     

Functional relevance of carnitine transporter OCTN2 to brain distribution of l-carnitine and acetyl-l-carnitine across the blood–brain barrier

Transport of L-[3H]carnitine and acetyl-L-[3H]carnitine at the blood-brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl-L-[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl-L-carnitine and in the absence of sodium ions. Similar transport properties for L-[3H]carnitine and/or acetyl-L-[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of L-[3H]carnitine and acetyl-L-[3H]carnitine by RBEC1 were sodium ion-dependent, saturable with K(m) values of 33.1 +/- 11.4 microM and 31.3 +/- 11.6 microM, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT-PCR method. Furthermore, the uptake of acetyl-L-[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of L-[3H]carnitine and acetyl-L-[3H]carnitine in jvs mice were slightly lower than those of wild-type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of L-carnitine and acetyl-L-carnitine from the circulating blood to the brain across the BBB.     

Hexose transport by brain slices: Further studies on energy dependence

We studied the uptake of [3H]2-deoxyglucose [( 3H]2DG) by slices of rat cerebral cortex in vitro as a model of glucose transport by brain. Slices were incubated with [3H]2DG, or with L-[3H]glucose as a marker for diffusion; the difference between [3H]2DG uptake and L-[3H]glucose uptake was defined as net [3H]2DG transport. Net [3H]2DG transport was a function of incubation temperature, with an estimated temperature coefficient of 1.87 from 15 degrees C to 25 degrees C. The net uptake of [3H]2DG was not inhibited by phlorizin or phloretin in concentrations well above the reported Ki of these inhibitors for hexose uptake in other systems. To examine the hypothesis that [3H]2DG transport by brain slices is dependent on mitochondrial energy, we studied net [3H]2DG uptake by slices which had been preincubated in media designed to alter intracellular ATP stores. The transport process was very sensitive to inhibition by DNP, but the correlation between [3H]2DG transport and ATP levels was unclear. In contrast to our published hypothesis that the transport process required mitochondrial energy, these data indicate that dependence on energy is not absolute.     

An antibody for macrophage migration inhibitory factor suppresses tumour growth and inhibits tumour-associated angiogenesis

To verify the role of macrophage migration inhibitory factor (MIF) in tumourigenesis, we examined the effect of an anti-MIF antibody on tumour growth and angiogenesis. We inoculated murine colon adenocarcinoma cell line colon 26 cells subcutaneously into the flank in BALB/c mice. After nine days, we treated tumour-bearing mice with an anti-rat MIF antibody by intraperitoneal injection on days 9, 11, 13, 15, 17, 19 and 21. We found significant inhibition of tumour growth by this treatment from day 15 to day 22. Next, we implanted a chamber filled with colon 26 cells, which only passes soluble factors, in the subcutaneous fascia of the flank, and treated mice with the anti-rat MIF antibody at days 1, 3 and 5. By histological examination at day 6, angiogenesis within the subcutaneous fascia in contact with the chamber was markedly suppressed. In vitro, we added an anti-human MIF antibody to human umbilical vein endothelial cells (HUVEC) to evaluate its effect on cell growth by measurement of [3H]thymidine incorporation. We observed that the anti-MIF antibody significantly suppressed [3H]thymidine uptake by HUVEC. These results suggest the possibility that MIF is involved in tumourigenesis via promotion of angiogenesis.     

Ascorbate Transport and Intracellular Concentration in Cerebral Astrocytes

Abstract: Regulation of the initial rate of uptake and steady-state concentration of ascorbate (reduced vitamin C) was investigated in rat cerebral astrocytes. Although these cells did not synthesize vitamin C, they accumulated millimolar concentrations of ascorbate when incubated with medium containing the vitamin at a level (200 µ M ) typical of brain extracellular fluid. Initial rate of [¹⁴C]-ascorbate uptake and intracellular ascorbate concentration were dependent on extracellular Na⁺ and sensitive to the anion transport inhibitor sulfinpyrazone. Comparison of the efflux profiles of ascorbate and 2',7'-bis(carboxyethyl)-5 (or -6)-carboxyfluorescein from astrocytes permeabilized with digitonin localized most intracellular ascorbate to the cytosol. Pretreatment of astrocytes with dibutyryl cyclic AMP (dBcAMP) doubled their initial rate of sulfinpyrazone-sensitive [¹⁴C]ascorbate uptake compared with cells treated with either n -butyric acid or vehicle. dBcAMP also increased steady-state intracellular ascorbate concentration by 39%. The relatively small size of the change in astrocytic ascorbate concentration was explained by the finding that dBcAMP increased the rate of efflux of the vitamin from ascorbate-loaded cells. These results indicate that uptake and efflux pathways are stimulated by cyclic AMP-dependent mechanisms and that they regulate the cytosolic concentration of ascorbate in astrocytes.