Monika Dolińska

Glutamine as a pathogenic factor in hepatic encephalopathy

Hepatic encephalopathy (HE) results from acute or chronic liver dysfunction and is associated with hyperammonemia. Ammonium ions penetrate from blood to brain, where they form glutamine (Gln) in the reaction with glutamate catalyzed by an astroglia‐specific enzyme, glutamine synthetase (GS). Experimental data suggest that many manifestations of HE can be ascribed to increased Gln synthesis and accumulation in the brain. In HE resulting from acute liver failure (“fulminant hepatic failure”), the osmotic action of Gln appears to be in a large degree responsible for cerebral edema and edema‐associated disturbances of cerebral blood flow and ionic homeostasis. In chronic HE not accompanied by cerebral edema, Gln contributes to impairment of cerebral energy metabolism, and its increased transport from brain to the periphery accelerates the blood‐to‐brain transport of aromatic amino acids, of which tryptophen (Trp) is converted to metabolites directly implicated in HE. Most of the evidence that Gln participates in pathological events has been derived from their disappearance or amelioration in HE rats in which the cerebral Gln content was reduced by treatment with a GS inhibitor, methionine sulfoximine. J. Neurosci. Res. 65:1–5, 2001.

Glutamine uptake and expression of mRNA’s of glutamine transporting proteins in mouse cerebellar and cerebral cortical astrocytes and neurons

The relative roles of the three sodium-dependent transport systems: A, ASC and N in the uptake of [3H]Gln, and the compatibility of the uptake characteristics with the expression of mRNAs coding for the Gln transporting molecules, were examined in primary cultures of astrocytes and neurons derived from mouse cerebellum, a glutaminergic system-enriched structure, and in cerebral cortex. Gln uptake activity (Vmax) was higher in cerebellar astrocytes or neurons than in their cerebral cortical counterparts. The N-methylamino-isobutyric acid (MeAiB)- and pH-sensitive, system A-mediated component of the uptake, and the uptake of [14C]MeAiB itself, was much more active in neurons than in astrocytes derived from either region. Also, the expression of mRNA for GlnT (SAT1), a system A isoform specific for Gln, was only expressed in neurons derived from both structures, while an alanine (Ala)-preferring system A transporter, SAT2, was expressed in neurons and astrocytes from either region. System ASC-mediated Gln uptake and expression of ASCT2 mRNA were in both structures more pronounced in astrocytes than in neurons, consistent with the postulated role of ASCT2 in the efflux of de novo synthesized Gln from astrocytes. System N-mediated (threonine+MeAiB-inhibitable) Gln uptake showed comparable activities in all four types of cells, which is compatible with the ubiquitous expression of NAT2 mRNA-a mouse brain-specific N-system isoform.

Glutamine transport in C6 glioma cells shows ASCT2 system characteristics.

Previous studies from this laboratory have shown that glutamine (Gln) uptake in a rat astrocytoma-derived C6 cell line shows characteristics similar with the uptake of a model ASC system substrate, threonine, whose pH-dependence and partial tolerance of Li(+) substitution for Na(+) resemble the ASCT2 variant of the system. In support of the previous findings, RT-PCR analysis revealed that C6 cells strongly express ASCT2 mRNA, but not at all GlnT mRNA or NAT2 mRNA, the A and N system variants specifically engaged in Gln transport in normal CNS. Other features of Gln transport in C6 cells indicating the involvement of ASCT2 system included its resistance to ouabain and stimulation of Gln efflux from the cells in the presence of excess Gln or cysteine (Cys), demonstrating that the system operates in the exchange mode. Replacement of NaCl in the incubation medium with isoosmotic sucrose did neither significantly affect the kinetics, nor any other major characteristics of Gln or Thr transport, including its pH-dependence, inhibition by ASCT system substrates or resistance to the model system A substrate-N-methylamino-isobutyric acid (MeAiB).

Ammonia stimulates glutamine uptake to the cerebral non-synaptic mitochondria of the rat.

The uptake of [3H]glutamine (GLN) to non-synaptic mitochondria isolated from rat cerebral hemispheres was measured in the absence or presence of 3 mM ammonium ion (ammonium chloride; ammonia). Ammonia increased Vmax of the saturable component of GLN uptake by > 20%, without affecting K(m), but did not change a non-saturable component of GLN transport representing diffusion or uptake mediated by a very low affinity carrier. Since GLN is an idiogenic osmole, its increased uptake may contribute to the swelling of astrocytic mitochondria and, subsequently, to a decrease in cerebral energy metabolism usually associated with acute hyperammonemic states. The result is consistent with the recent view that GLN accumulating in the brain in hyperammonemic conditions contributes to ammonia neurotoxicity.

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 Km = 0.08 mM, and Vmax = 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 gamma+ transport system operating in the nerve- and glia cell-, and synaptic plasma membranes. Also in consistance with the other gamma+ 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 NG-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.

Regulation of kynurenic acid synthesis in C6 glioma cells

Studies with brain slices have provided evidence that synthesis of kynurenic acid (KYNA) from kynurenine (KYN), which occurs in astrocytes, is modulated by changes in the ionic composition of the medium and the presence of depolarizing agents or the excitatory amino acid glutamate (Glu). The present study analyzed the effects of changes in incubation medium on KYNA synthesis in cultured C6 glioma cells. The synthesis was not affected by omission of Na+ and raising K+ concentration to 50 mM, conditions that in brain slices stimulate or inhibit KYNA formation, respectively. KYNA synthesis in C6 cells was inhibited by the absence of Ca2+, which contrasts with its Ca2+ independence in brain slices. Also, lack of Mg2+ and addition of a chloride channel blocker, 4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulfonate (SITS), did not affect the synthesis. KYNA synthesis in C6 cells was dose dependently inhibited by Glu. The inhibitory effect of Glu was not affected by GDPβS, an antagonist of metabotropic Glu receptors, the receptor class prevailing in C6 cells, suggesting that Glu acted intracellularly. NH4Cl and veratridine decreased KYNA production, mirroring the effects noted in brain slices. KYNA synthesis was strongly reduced in the presence of leucine (Leu), and the uptake of [14C]Leu was inhibited by the KYNA precursor KYN, which points to Leu as a potential endogenous modulator of KYNA formation in CNS cells.

Glutamine transport in C6 glioma cells: Substrate specificity and modulation in a glutamine deprived culture medium

A previous study has shown that glutamine (Gln) uptake in C6 cells grown in a standard medium containing 2 mM Gln, is predominantly mediated by a sodium‐dependent system that is inhibited by ASC system substrates alanine (Ala), serine (Ser), cysteine (Cys) and threonine (Thr), shows pH sensitivity and partial tolerance to substitution of Na+ by Li+, features compatible with system ASCT2 that is strongly expressed in cultured astrocytes. The uptake was not inhibited by the model system A substrate α‐(methylamino)isobutyric acid (MeAiB), and glycine (Gly) or proline (Pro), indicating that the substrate‐regulated system A as defined by routine criteria is relatively inactive in these cells (Dolińska et al., 2000 ). In this study we compared the uptake of radiolabeled Gln and a model ASC substrate ‐Thr in cells grown to the same density in Gln‐containing and Gln‐deprived media. Cells grown in the absence of Gln showed a reduced activity of system ASC‐mediated Gln uptake, and the system lost tolerance for Li+ and became somewhat more resistant to lowering pH of the medium. In contrast to cultured astrocytes deprived of Gln, the overall Gln uptake activity in C6 cells adapted to grow in a medium without Gln was lower than in cells grown in a Gln containing medium, and the uptake by system A remained inactive. C6 cells cultured both in the presence and absence of Gln expressed ASCT2 mRNA, indicating that system ASCT2‐mediated Gln uptake is modulated at a posttranscriptional level. In contrast to Gln uptake, Thr uptake was more active in cells cultured in the absence of Gln and showed neither pH dependence nor lithium tolerance in either medium, which is typical of an uptake mediated by the widespread ASCT1 isoform of system ASC. In C6 cells grown in the presence or absence of Gln alike, ≈20% of the sodium‐dependent Gln uptake was resistant to MeAiB+Thr, indicating contribution of system N. The N system‐mediated uptake in C6 cells grown in the absence, but not in the presence of Gln was not inhibited by glutamate (Glu) that conforms to the characteristics of the glial N system variant, SN1.

Glutamate uptake is inhibited by L-arginine in mitochondria isolated from rat cerebrum

Uptake of L-[14C]glutamate (L-[14C]GLU) into non-synaptic mitochondria isolated from rat cerebral hemispheres was measured in the presence of potential modulators of amino acid transport. The L-GLU carrier agonist 0.2 mM L-aspartate (L-ASP) virtually abolished L-GLU uptake (ASP/GLU concentration ratio, 1:1). L-Arginine (L-ARG) inhibited L-GLU uptake in a dose dependent manner over the concentration range 0.1–5 mM to maximum inhibition of 85%. Putrescine or ammonia had no effect, whereas 5 mM creatine and the NO generator, 5 mM sodium nitroprusside, increased the uptake by 73% and 57%, respectively. D-ARG was three times less effective in inhibiting L-GLU uptake than L-ARG at 5 mM concentration. The L-amino acids ornithine, lysine, histidine, tyrosine, phenylalanine, proline, leucine, isoleucine, tryptophan, glycine, methio-nine, valine, serine, taurine, alanine or cysteine did not affect the uptake when added in concentrations of 2–5 mM. A 14% inhibition of L-GLU uptake was noted in the presence of L-glutamine (L-GLN) (2 mM) or a dicarboxylate carrier ligand, α-ketoglutarate (α-KG) (5 mM), and a 30% inhibition with a dicarboxylate carrier inhibitor phenylsuccinate (PhSc) (5 mM). The results suggest that L-ARG functions as a specific endogenous modulator of cerebral mitochondrial L-GLU transport.

Glutamine transport in C6 glioma cells.

Glutamine transport across the cell membranes of a variety of mammalian tissues is mediated by at least four transport systems: a sodium-independent system L, and sodium-dependent systems A, ASC and N, the latter occurring in different tissue-specific variants. In this study we assessed the contribution of these systems to the uptake of [(3)H]glutamine in C6 rat glioma cells. The sodium-dependent uptake, which accounted for more than 80% of the total uptake, was not inhibited by 2-methylaminoisobutyric acid (MeAIB), indicating that system A was inactive, possibly being depressed by glutamine present in the culture medium. About 80% of the sodium-dependent uptake was mediated by system ASC, which differed from system ASC common to other CNS- and non-CNS tissues by its pH-dependence and partial lithium tolerance. The residual 20% of sodium-dependent uptake appeared to be mediated by system N, which was identified as a component resistant to inhibition by MeAIB+threonine. The system N in C6 cells appeared to be neither fully compatible with the neuronal system Nb, nor with the N system described in astrocytes: it differed from the former in being strongly inhibited by histidine and showing fair tolerance for lithium, and from the latter in its pH-insensitivity and strong inhibition by glutamate. The sodium-independent glutamine uptake differed from the astrocytic or neuronal uptake in its relatively weak inhibition by system L substrates and a strong inhibition by system ASC substrates, indicating a possible contribution of a variant of the ASC system.

Synaptosomal Uptake of Alpha-Ketoglutarate and Glutamine in Thioacetamide-Induced Hepatic Encephalopathy in Rats

The kinetics of uptake of two astroglia-derived glutamate (GLU) precursors, α-ketoglutarate (α-KG) and glutamine (GLN) were determined in synaptosomes derived from rats with acute hepatic encephalopathy (HE) induced with a hepatotoxin, thioacetamide (TAA). TAA treatment increased by 33% Vmax for high affinity, low capacity α-KG uptake, without influencing its Km. The increase of the uptake capacity for α-KG may represent a response of the GLUergic nerve terminals to the decreased cerebral α-KG content, which during HE is associated with depressed activity of pyruvate carboxylase, an enzyme that replenishes α-KG in astrocytes. The result is thus consistent with the notion that HE affects the astroglial control of GLUergic neurotransmission. The Km and Vmax for the low affinity, high capacity GLN uptake was not affected by TAA treatment.