Arg Uptake Research Articles

Cytokine treatment increases arginine metabolism and uptake in bovine pulmonary arterial endothelial cells.

L-Arginine (L-Arg) is metabolized to nitric oxide (NO) by NO synthase (NOS) or to urea by arginase (AR). L-Arg is transported into bovine pulmonary arterial endothelial cells (BPAECs) by cationic amino acid transporter-2 (CAT-2). We hypothesized that cytokine treatment would increase L-Arg metabolism and increase CAT-2 mRNA expression. BPAECs were incubated for 24 h in medium (control) or medium with lipopolysaccharide and tumor necrosis factor-alpha (L-T). L-T increased nitrite production (3.1 +/- 0.4 nmol/24 h vs. 1.8 +/- 0.1 nmol/24 h for control; P < 0.01) and urea production (83.5 +/- 29.5 nmol/24 h vs. 17.8 +/- 8.6 nmol/24 h for control; P < 0.05). L-T-treated BPAECs had greater endothelial and inducible NOS mRNA expression compared with control cells. Increasing the medium L-Arg concentration resulted in increased nitrite and urea production in both the control and the L-T-treated BPAECs. L-T treatment resulted in measurable CAT-2 mRNA. L-T increased L-[(3)H]Arg uptake (5.78 +/- 0.41 pmol vs. 4.45 +/- 0.10 pmol for control; P < 0.05). In summary, L-T treatment increased L-Arg metabolism to both NO and urea in BPAECs and resulted in increased levels of CAT-2 mRNA. This suggests that induction of NOS and/or AR is linked to induction of CAT-2 in BPAECs and may represent a mechanism for maintaining L-Arg availability to NOS and/or AR.

The uptake of amino acids, in particular leucine, by isolated perfused testes of rats.

The uptake of amino acids by the isolated rat testis perfused with Krebs-Ringer solution with albumin has been studied using the single-passage, multiple-tracer technique with [14C]-mannitol as the reference tracer. When the perfusate contained no added amino acids, the uptake of [3H]-Leu was between 60% and 80% of the uptake of mannitol at all times after injection of the bolus; there was a small but significant uptake of some other amino acids studied (Ala, Gly, Glu, and Asp); and with Ala, Glu, and Asp, uptake increased slightly in increasing times after injection. There was no significant uptake of Arg. The uptake of Leu could be decreased by the inclusion of nonradioactive Leu in the perfusate, and the Km and Vmax of the transport were 0.067 mM and 19.5 nmole/(g x min), respectively. The Km value is similar to that for transport into brain and much less than the values obtained in other tissues for the related amino acid Phe, which is transported by the same L system. The transport of L-Leu in the testis was also inhibited by L-Phe or D-Phe, D-Leu, and by the synthetic amino acid Bch, the characteristic marker for the L system, but was only slightly reduced if the perfusate was free of sodium, as is expected for the L system. By autoradiography after fixation by perfusing with glutaraldehyde, the transport of Leu could be localized to the endothelial cells of the larger vessels of the testicular microvasculature.

Arginine degradation by arginase in mitochondria of soybean seedling cotyledons

Arginase (EC 3.5.3.1) localization was studied in soybean (Glycine max L.) seedling cotyledons. Subcellular fractionation in a discontinuous Percoll gradient showed that arginase was localized in the mitochondrion. Arginine (Arg) uptake by mitochondria was demonstrated by co-sedimentation of [3H]Arg-derived label and the mitochondrial marker enzyme cytochrome c oxidase. Arginine uptake was complete in about 10 min. Since detergent but not NaCl released most label, we conclude that Arg was taken up and not bound to the organellar surface. Arginine transport was not saturable, at least up to 20 mM. Basic amino acids were the best inhibitors of Arg uptake. The uncoupler 2,4-dinitrophenol did not inhibit Arg uptake. At least 30% of L-[guanido-14C]Arg taken up by mitochondria was degraded by arginase in seedling cotyledons, while little or no degradation was detected in mitochondria from developing embryos, even though the Arg uptake level was similar in both mitochondrial preparations. These results are consistent with our previously reported pattern of arginase expression and urea accumulation during embryo development and seed germination (A. Goldraij and J.C. Polacco, 1999, Plant Physiol. 119: 297-303). The lack of Arg degradation allows developing embryos to conserve Arg, the main N-reserve amino acid utilized by germinating soybean.

L-Arginine uptake in rat cerebral mitochondria

The kinetics of L-[14C] arginine ( L-[14C] Arg) uptake and the effects of potential competitors on the uptake were analysed in nonsynaptic mitochondria isolated from rat cerebral hemispheres. Analysis of uptake kinetics revealed a high affinity component with a mean K m=0.08 mM, and V max=1.89 nmoles⧹min⧹mg, and a very low affinity component probably manifesting diffusion. The uptake of 25 mM L-Arg was strongly inhibited by a 20-fold excess of L-lysine ( L-Lys) and L-ornithine ( L-Orn), but not by D-Arg nor any neutral amino acid, which resembles the characteristics of the y+ transport system operating in the nerve- and glia cell-, and synaptic plasma membranes. Also in consistence with the other y + systems, L-Arg uptake to mitochondria was inhibited by a nitric oxide synthase (NOS) inhibitor L-N-monomethyl arginine ( L-NMMA), but not by another NOS inhibitor N G-nitro- L-arginine ( L-NNA). However, the uptake was very little affected by 20-fold excess of L-histidine ( L-His), L-glutamate ( L-Glu) or L-glutamine ( L-Gln), which is in contrast to the nonmitochondrial systems. The uptake was only marginally influenced by cytoplasmic L-Arg metabolites: ammonia, creatine, putrescine, or the mitochondrial L-Arg decarboxylation product, agmatine.

Metabolism of ornithine, α-ketoglutarate and arginine in isolated perfused rat liver

Ornithine (Orn; alpha-ketoglutarate (alpha KG) salt) and arginine (Arg) supplementation of enteral diets has been advocated in the treatment of hypercatabolism of trauma patients, but both compounds are subject to extensive hepatic metabolism. To compare the metabolism of these two compounds and to evaluate the possible influence of the alpha KG moiety, livers were perfused with alpha KG, Orn, ornithine alpha-ketoglutarate (OKG) or Arg (n 6 in each group) for 1 h. Arg uptake was nearly fourfold higher than Orn uptake (690 (SD 162) v. 178 (SD 30) nmol/min per g liver), and Orn uptake was not modified by alpha KG. Orn was totally metabolized by the liver, whereas Arg led to Orn release (408 (SD 159) nmol/min per g liver) and a threefold stimulation of urea production (Arg 1.44 (SD 0.22) v. Orn 0.45 (SD 0.09) mumol/min per g liver). alpha KG alone only increased hepatic aspartate uptake but, when associated with Orn as OKG, it led to an increase in glutamate release and in proline content in the liver and to a decrease in proline uptake. From these findings we conclude that (1) Arg load is extensively metabolized by the liver, inducing urea production, (2) in enteral use, Orn supplementation appears preferable to Arg as it is less ureogenic (as also recently demonstrated in vivo in stressed rats receiving isomolar amounts of Arg and Orn), (3) the liver participates in the Orn-alpha KG metabolic interaction, mostly in proline metabolism, which occurs in the splanchnic area.

Amino acids differentially inhibit the l-[ 3H]arginine transport and nitric oxide synthase in rat brain synaptosomes

The nitric oxide synthase (NOS) present in the cytosol obtained from rat cerebral cortex synaptosomes was inhibited by N G-nitro- l-arginine ( l-NOArg) with an IC 50 value of ∼0.06 μM. This compound did not affect the transport of l-arginine ( l-Arg) into synaptosomes at concentrations up to 100 μM but other potential inhibitors of NOS (N G-monomethyl- l-arginine, N G-amino- l-arginine and l-arginine methyl ester) inhibited l-Arg transport at a concentration <5 μM. We showed that concentrations of l-NOArg (0.001–3 μM) that did not block the uptake of tritiated arginine ( l-[ 3H]Arg) inhibited the catalytic activity of NOS in intact synaptosomes. l-NOArg at a concentration of 1 μM inhibited the cytosolic enzyme by 98.0 ± 2.0% of the total NOS activity whereas the enzyme studied in the intact synaptosomes was only inhibited by 75 ± 5% which suggested that the NOS in synaptosomes is not fully accessible to the external l-NOArg. On the other hand, l-Lysine did not inhibit the cytosolic NOS activity of ruptured synaptosomes but at a concentration that blocked 50.0 ± 4.5% of l-[ 3H]Arg uptake it inhibited the NOS activity in intact synaptosomes by 12.6 ± 3.6%, suggesting that the transport of l-Arg may be an important regulatory step in the pathway for nitric oxide generation.

Uptake of [3H]serotonin and [3H]glutamate by primary astrocyte cultures. II. Differences in cultures prepared from different brain regions.

Regional astrocyte cultures were derived by dissecting six regions; brain stem, cerebellum, mesencephalon, basal ganglia plus diencephalon, cerebral cortex, and hippocampus, from 3 to 4-day-old neonatal rat brains. Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to confirm the astrocyte composition of the cultures. The percentage of GFAP (+) cells between regions varied from 75% to 100%. Once confluent these cultures were incubated with radiolabeled serotonin or glutamate for uptake and autoradiographic studies. For the different brain regions Na(+)-dependent, [3H] L-glutamate, and fluoxetine-sensitive [3H] 5-HT uptake varied markedly. The relative order of uptake for [3H] 5-HT was MS (mesencephalon) greater than CC (cerebral cortex) greater than BG + DI (basal ganglia + diencephalon) greater than HP (hippocampus) greater than BS (brain stem) greater than CB (cerebellum). For [3H] L-glutamate the order was HP greater than CC greater than BG + DI greater than MS = BS greater than CB. For [3H] 5-HT this essentially corresponds to the reported order of binding in situ of the [3H] 5-HT-specific uptake ligand [3H] citalopram. For [3H] L-glutamate regional variation of the uptake for the different cultures corresponds to the regional uptake reported for different regions of rat brain. Double-label studies with GFAP and radiolabeled neurotransmitters were also used to study uptake into GFAP(+) astrocytes by autoradiography. Flat GFAP cells with or without processes comprised 65-98% of the cultures and represented most of the uptake. The percentage of all GFAP(+) cells that were positive for uptake of ARG varied from 50% to 90% and also showed differences in grain density both intra- and inter-regionally. These differences in transmitter uptake by GFAP(+) astrocytes in primary culture, which are dependent on the region of origin and correspond to regional differences in situ, suggest that such uptake in vitro may reflect uptake by astrocytes in vivo. Implied in this is that uptake by astrocytes represents a significant component of serotonin uptake in vivo.

Some aspects of arginine assimilation in a strain ofStreptococcus sanguis

Like many arginolytic streptococci,Streptococcus sanguis P4A7 is auxotrophic for arginine (Arg) and can also use this amino acid as an energy source; its dissimilation via the arginine deiminase (ADI) pathway is potentially important in dental plaque metabolism. Arg uptake was investigated in chemostat-grown cells; two systems were found: a low-affinity system (A) and a high-affinity system (B). Both systems (a) functioned as well as pH 5.5 and 8.0 as at 7.0; (b) were insensitive to proton-conducting uncouplers and metabolic inhibitors, and (c) were unaffected by prior starvation of cells or their pre-energization with glucose. Thus, Arg uptake appeared to be energy-independent. Inhibition studies with Arg structural analogues indicated that both the carboxyl and guanidino functional groups and their spatial relationship are important as recognition sites in system A, while all three functional groups appear important in system B. It is suggested that system A represents the ADI pathway, whereas system B is used to satisfy the organism's auxotrophic requirement.

FURTHER CHARACTERIZATION OF LYSOSOMAL SYSTEM y+

Using a trans-stimulation property associated with lysine exodus, we have demonstrated previously a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. By studying instead the uptake of arginine into highly purified flbroblast lysosomes, obtained by centrifuging through Percoll density gradients, we now examine additional characteristics of this system. For arg uptake it displays a broad pH optimum from pH 7.0-8.0. The rate of arg uptake is 10-fold greater at pH 7.0 than 5.0, thus favoring net entry of arg into lysosomes as a result of the low intralysosomal pH. In contrast, external MgATP accelerates lysosomal efflux of cationic amino acids while inhibiting their influx. Trans-stimulation of arg uptake is seen when lysosomes have been loaded with 2-aminoethyl-L-cysteine. Arg uptake (.03 mM) is strongly inhibited by the L-isomers of external 3.3 mM arg, lys, orn, 2, 4-diaminobutyrate, 2-aminoethylcysteine and his, whereas D-arg, neutral and anionic amino acids have little effect. In addition, lysosomal arg uptake is inhibited by α-N-methyl-L-arg (72%) and e-trimethyl-L-lys (49%), neither of which are recognized by the plasma membrane System y+. These observations indicate that lysosomal System y+ is structurally different from System y+ of the plasma membrane of the human fibroblast and various other cells. Thiocholine (TC) depleted cystinotic fibroblasts of their accumulated cystine to the same level and rate as produced by cysteamine supporting the view that TC may react with cystine to form a mixed disulfide recognized by lysosomal System y+ similar to the one formed by cysteamine. Support ackn. from Grant AM32281, NIH.