Metabolic approach of absence seizures in a genetic model of absence epilepsy, the GAERS: study of the leucine-glutamate cycle.

We suggest that a dysregulation of energy metabolism in the brain of genetic absence epilepsy rats from Strasbourg (GAERS) could create a specific cerebral environment that would favor the expression of spike-and-wave discharges (SWD) in the thalamocortical loop, largely dependent on glutamatergic and gamma-aminobutyric acid (GABA)-ergic neurotransmissions. We tested several aspects of metabolic activity in the brain of GAERS compared to a genetic strain of nonepileptic (NE) rats. Glucose metabolism was higher in all brain regions of GAERS compared to those of NE rats along the whole glycolytic and aerobic pathways, as assessed by regional histochemical measurement of lactate dehydrogenase and cytochrome oxidase activities. Branched-chain amino acids (BCAA) and alpha-ketoisocaproate (alpha-KIC), the ketoacid of leucine, when injected intraperitoneally, increased the number of SWD in GAERS but had only a slight effect on their duration. These data speak in favor of a BCAA- or alpha-KIC-induced change in neuronal excitability. Leucine and alpha-KIC decreased the concentration of glutamate in thalamus and cortex without affecting GABA concentrations. Thus, BCAA and alpha-KIC, by decreasing glutamatergic neurotransmission, could favor GABAergic neurotransmission, which is known to increase the occurrence of seizures in GAERS. Finally, the transport of [1-(14)C]alpha-KIC in freshly isolated cortical neurons was lower in GAERS than in NE rats, and this difference was shown to be of metabolic origin. The addition of gabapentin, a specific inhibitor of BCAA transaminase (BCAT), reduced the transport of [1-(14)C]alpha-KIC in GAERS and NE rats to a level that became identical in both strains. This strain-dependent change was not related to a difference in the activity of BCAT, which was identical in GAERS and NE rats. The exact origin of this apparent metabolic dysregulation of energy metabolism in GAERS that could underlie the origin of seizures in that strain remains to be explored further.

Modulation of absence seizures by branched-chain amino acids: correlation with brain amino acid concentrations.

The occurrence of absence seizures might be due to a disturbance of the balance between excitatory and inhibitory neurotransmissions in the thalamo-cortical loop. In this study, we explored the consequences of buffering the glutamate content of brain cells on the occurrence and duration of seizures in Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a genetic model of generalized non-convulsive epilepsy. Branched-chain amino acids (BCAAs) and alpha-ketoisocaproate (alpha-KIC), the ketoacid of leucine were repeatedly shown to have a critical role in brain glutamate metabolism. Thus, GAERS were injected by intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) route with these compounds, then the effects on seizures were evaluated on the electroencephalographic recording. We also measured the concentration of amino acids in thalamus and cortex after an i.p. injection of leucine or alpha-KIC. Intracerebroventricular injections of leucine or alpha-KIC did not influence the occurrence of seizures, possibly because the substances reached only the cortex. BCAAs and alpha-KIC, injected intraperitoneally, increased the number of seizures whereas they had only a slight effect on their duration. Leucine and alpha-KIC decreased the concentration of glutamate in thalamus and cortex without affecting GABA concentrations. Thus, BCAAs and alpha-KIC, by decreasing the effects of glutamatergic neurotransmission could facilitate those of GABAergic neurotransmission, which is known to increase the occurrence of seizures in GAERS.

Transport of L-carnitine in isolated cerebral cortex neurons.

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH-group reagents (N-ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4-aminobutyrate (GABA) in a competitive way (Ki = 20.9 +/- 2.4 mM). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1-[2-([(diphenylmethylene)amino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711) acid decreased carnitine accumulation while betaine, taurine and beta-alanine had no effect. The GABA transporters expressed in Xenopus laevis oocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nalecz, D. Dymna, J. E. Mroczkowska, A. Broër, S. Broër, M. J. Nalecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.

[The 1999 Nobel Prize for physiology or medicine]. / Nagroda Nobla 1999 w dziedzinie fizjologii lub medycyny.

It has been awarded to Günter Blobel, for a so-called signal hypothesis. In this research Günter Blobel has proven that information about the final localization of a protein in the cell is given in its sequence. Such a targeting signal can be either cut off (presequence) or can be localized within the final sequence of a protein. The mechanisms responsible for secretion, the movement of proteins to or from nucleus, and guiding of proteins to mitochondria, chloroplasts and peroxisomes have been described. In particular, proteins involved in interactions with the signal sequences were reviewed. The channels responsible for transfer of polipeptides through the membranes were also presented. A contribution of Günter Blobel and his coworkers in the foundation and development of a new field of research on protein guidance has been emphasized.

Transport of alpha-ketoisocaproate in rat cerebral cortical neurons.

Transport of alpha-ketoisocaproic acid (KIC), the product of leucine transamination, was studied in the cerebral cortex cells isolated from the adult rat brain. The process of [(14)C]KIC accumulation was followed in the presence of aminooxyacetate, an inhibitor of transaminases. Accumulation of KIC was not affected by Na(+) replacement, its initial velocity was observed to be higher upon lowering of external pH. Addition of KIC promoted acidification of cytoplasmic pH, monitored with 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The detected inhibition of KIC accumulation by alpha-cyano-4(OH)cinnamate pointed to an involvement of one of monocarboxylate transporters (MCT), although 4,4'-diisothiocyano-2,2'-stilbenedisulphonate was without effect. Accumulation of KIC was inhibited by lactate; the effect of pyruvate was detected to be much weaker. Other branched-chain alpha-ketoacids (ketoisovalerate, keto-methylvalerate), as well as beta-hydroxybutyrate and valproate decreased the transport of KIC by 30, 60, and 80%, respectively. The observed characteristics of KIC accumulation in the cortical neurons indicate an involvement of one of the MCT transporters. A crucial role of SH group(s) in the process of KIC accumulation, excluding MCT2, indicates the MCT1, although an involvement of another isoform of MCT in the process of KIC transport in neurons from cerebral cortex of adult brain has not been definitely excluded.

Blood-brain barrier controls carnitine level in the brain: a study on a model system with RBE4 cells.

Transport of carnitine was studied with immortalized rat brain endothelial cells (RBE4), an in vitro model of the blood-brain barrier. The experiments on uptake and efflux through the luminal membrane excluded any involvement of choline and amino acids transporters, as well as that of glycoprotein P. Acetyl-, octanoylcarnitine, and betaine were without any effect; the only compound decreasing both processes was butyrobetaine. An exposure of the abluminal membrane resulted in a 40% inhibition of carnitine uptake by the substrates of neutral amino acid transporter L, while its efflux through the basolateral membrane, occurring in a form of free carnitine, was sensitive to SH group reagent, mersalyl, and was diminished by butyrobetaine. These features of carnitine transport did not fully correspond to the known characteristics of the proteins transporting carnitine in other tissues (OCTN2 and CT1); however, they did not exclude an involvement of a transporter belonging to the same superfamily. Moreover, such a protein in brain endothelium would fulfill a regulatory role in the transport of carnitine through the blood-brain barrier.

Modulation of pentylenetetrazol-induced seizure activity by branched-chain amino acids and alpha-ketoisocaproate.

Branched-chain amino acids, and mainly leucine act as nitrogen donors in the cerebral glutamate-glutamine cycle, thereby reducing brain excitability. Rats equipped with cortical electrodes received 300 mg/kg of leucine, isoleucine, valine or the ketoacid of leucine, alpha-ketoisocaproate at 2 h before the induction of seizures by 40 mg/kg pentylenetetrazol. Control groups received saline or a commercial mixture of amino acids, Vamine(R). Leucine and isoleucine increased the latency to absence-like and tonic-clonic seizures but did not influence the duration of the tonic-clonic seizure. Vamine(R), valine and alpha-ketoisocaproate had no effect. These data are consistent with the role of leucine in buffering brain glutamate concentration.

Transport of alpha-ketoisocaproate in neuroblastoma NB-2a cells.

Transport of alpha-ketoisocaproate (KIC), a ketoacid originating from leucine and proposed to be involved in the buffering of glutamate in neurones, was studied in neuroblastoma NB-2a cells. The accumulated KIC was mostly transaminated to leucine, while free keto-acid was detectable either only after prolonged times or after inhibiting transaminase with aminooxyacetate. Accumulation of KIC was found to be inhibited by other branched-chain ketoacids, while lactate and beta-hydroxybutyrate were ineffective. The transport of KIC, resembling a facilitated diffusion, was decreased by phloretin, alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyano-2,2'-stilbenedisulphonate, and p-chlorimercuribenzoate. The process of accumulation did not resemble a symport with protons; therefore an involvement of the known proton-coupled monocarboxylate transporters (MCT) was excluded. Distribution of KIC suggests a mechanism involving a cotransport with 2 [Na+].

Effect of palmitoylcarnitine on the cellular differentiation, proliferation and protein kinase C activity in neuroblastoma nb-2a cells.

Palmitoylcarnitine is synthesized through the action of palmitoylcarnitine transferase I--an enzyme specifically inhibited by etomoxir. An increase of the intracellular content of palmitoylcarnitine in neuroblastoma NB-2a cells after administration of carnitine was correlated with an inhibition of cell proliferation and a concomitant promotion of differentiation processes. The activity of protein kinase C was measured in vivo, with cells permeabilized through the use of streptolysin O and a peptide substrate. Palmitoylcarnitine inhibited the phorbol ester stimulated reaction of the peptide phosphorylation in a concentration dependent way. The degree of protein kinase C inhibition was correlated with intracellular increase of the palmitoylcarnitine content, pointing to this compound as a natural modulator of protein kinase C activity.

Evidence for an asymmetrical uptake of L-carnitine in the blood-brain barrier in vitro.

The transport of L-carnitine (4-N-trimethylammonium-3-hydroxybutyric acid) was studied with a primary culture of porcine brain capillary endothelial cells (BCEC) as an in vitro model of the blood-brain barrier. The measurements with suspended cells and cell monolayers allowed to distinguish a polarized transport phenomena. The part of the BCEC cells exposed to the medium (apical membrane) accumulated carnitine by a sodium-independent, saturable (Km=28 microM) system, with k=0.018 min-1. Exposure of the basolateral part revealed a presence of a facilitated diffusion process. Carnitine uptake through the saturable system was inhibited by butyrobetaine. Acylcarnitines and choline have no effect on the carnitine accumulation in suspended cells, a process diminished by phenylalanine, leucine, and L system inhibitor. This points to the possibility that carnitine enters through the basolateral membrane using amino acid transporting systems. A different, novel system is postulated to operate in the apical part of the plasma membrane of BCEC.

Carnitine--a known compound, a novel function in neural cells.

Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid) seems to fulfill in the brain a different role than in peripheral tissues. Carnitine is accumulated by neural cells in a sodium-dependent way. The existence of a novel transporter in plasma membrane, specific to compounds with a polar group in the beta-position with respect to carboxyl group, has been postulated. The presence of a carnitine carrier in the inner mitochondrial membrane has been proven and the protein has been purified. It is postulated that its major role in adult brain would be translocation of acetyl moieties from mitochondria into the cytoplasm for acetylcholine synthesis. The latter process is stimulated by carnitine and choline in a synergistic way in cells utilizing glucose as the main energetic substrate. Carnitine metabolism in neural cells leads to accumulation of different acyl derivatives of carnitine. Palmitoylcarnitine can influence directly the activity of protein kinase C. An involvement of carnitine in a decrease of palmitate pool used for palmitoylation of regulatory proteins has been postulated.

Transport of carnitine in neuroblastoma NB-2a cells.

Carnitine accumulation was measured in cultured neuroblastoma NB-2a cells. This process was found partially sodium dependent and its kinetics to be a sum of a saturable transport (Km = 123 +/- 13 microM) and diffusion (D = 63 +/- 7 pmol/mg protein/min/mM). On the contrary to previous reports on neural cells, the accumulation of carnitine was found insensitive to gamma-aminobutyric acid (GABA). Measurements of carnitine accumulation in the presence of different compounds resulted in the conclusion that carnitine transport does not occur through the known systems specific toward choline and/or amino acids. For instance, an observed inhibition of carnitine transport by serine and cysteine, without any effect of alanine, excluded a possible role of ASC amino acid transport system. An involvement of a new transporter is thus postulated, specific toward compounds with a polar group in the beta position with respect to the carboxylic group.

Effect of gamma-aminobutyric acid on the carnitine metabolism in neural cells.

Isolated rat cerebral cortex cells were able to accumulate L-carnitine and this process was competitively inhibited by 1 mM gamma-aminobutyric acid (GABA) with a shift of Km from 7.8 +/- 1.9 mM to 14.6 +/- 4.0 mM. Addition of GABA also affected distribution of carnitine derivatives. The decrease of acetylcarnitine level by 1.6 fold was correlated with the inhibition of carnitine acetyltransferase (1.77 times). A postulated involvement of this enzyme in delivering acetyl moieties for acetylcholine synthesis would suggest a negative feedback between GABA and the level of acetylcholine.

Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells.

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 microM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of beta-oxidation might be correlated with the lower level of acetylcholine synthesis.

Mechanism of carnitine transport catalyzed by carnitine carrier from rat brain mitochondria.

The transport mechanism of reconstituted carnitine carrier purified from rat brain mitochondria was studied kinetically. Short and medium chain acyl carnitine derivatives had much higher affinity to the carnitine carrier in comparison with long chain acyl carnitine derivatives, therefore both homologous (carnitine/carnitine) and heterologous (carnitine/acetylcarnitine) antiports were analysed. A complete set of half-saturation constants was established for various substrate concentrations on both the external and the internal side of the membrane. Bisubstrate initial velocity analyses of the exchange reaction resulted in a kinetic pattern which is consistent with a sequential antiport mechanism. This type of mechanism implies formation of a ternary complex of the carrier with one internal and one external substrate molecule before the transport reaction occurs.

Synergistic effect of choline and carnitine on acetylcholine synthesis in neuroblastoma NB-2a cells.

An influence of carnitine on acetylcholine synthesis from radiolabeled glucose was monitored in neuroblastoma NB-2a cells. Upon addition of carnitine the distribution of its derivatives was found significantly different than the values published for brain, the level of long-chain acyl derivatives being much higher and reaching 60%. Carnitine itself did not change acetylcholine level. Together with choline (20 microM), carnitine was observed to stimulate (by 36%) acetylcholine synthesis in a synergistic way, which indicated that both substrates could be limiting factors of this process in NB-2a cell line of neuroblastoma.

Purification of carnitine carrier from rat brain mitochondria.

Brain mitochondria isolated from rats of different age were solubilized with Triton X-100 and the detergent extract was subjected to chromatography on dry hydroxyapatite and celite. The highest specific activity (110 mumol/10 min per g protein) measured after reconstitution of isolated proteins into phosphatidylcholine vesicles correlated with the appearance of a polypeptide with a molecular mass of 33,000. Activity of the carnitine carrier, both in isolated mitochondria and in the reconstituted system, varied with animal age, being twice higher in suckling rats than in adults. After reconstitution, the carnitine exchange showed sensitivity to SH groups modifying reagents, N-ethylmaleimide and mersalyl. Acetyl, propionyl and palmitoyl esters of carnitine decreased carnitine/carnitine exchange. Short and medium chain acyl derivatives were more potent inhibitors, pointing to a different substrate specificity of carnitine carrier in brain, in comparison with other tissues.

Isolation of tocopherol-binding proteins from the cytosol of smooth muscle A7r5 cells.

Cultured smooth muscle A7r5 cells were able to take up alpha-tocopherol (32 +/- 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol-based affinity chromatography and alpha-, beta-, gamma-, and delta-tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had alpha-[3H]tocopherol binding capability. The 58-kDa polypeptide could also be eluted by chromanol and the 81-kDa polypeptide could be eluted also by phytol. The 81-kDa polypeptide had the unique P-E-E-D-Q-X-Q-Y N-terminal sequence.