Katarzyna A. Nałęcz

The monocarboxylate carrier from bovine heart mitochondria: partial purification and its substrate-transporting properties in a reconstituted system.

The monocarboxylate (pyruvate) carrier from bovine heart mitochondria was extracted from submitochondrial particles with Triton X-114 in the presence of cardiolipin. By a single hydroxylapatite chromatography step a 125-fold purification of the carrier protein could be achieved. High pyruvate/pyruvate-exchange activity was recovered, when the protein was reconstituted into phospholipid vesicles. No transport activity was observed, when the isolation occurred in the absence of phospholipids. The 2-cyano-4-hydroxycinnamate sensitive pyruvate exchange reaction was strongly temperature sensitive and dependent on the amount of protein reconstituted. Other 2-ketoacids caused competitive inhibition of the pyruvate uptake. Inhibitors of other mitochondrial carries, however, had very low or no effect on the monocarboxylate exchange. The influence of different -SH group reagents on the measured pyruvate/pyruvate-exchange in the reconstituted system was similar to the one observed with intact mitochondria. It is concluded that the described procedures for extraction, purification and reconstitution of the mitochondrial monocarboxylate carrier conserved the functional properties of the protein.

Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood–brain barrier

Carnitine β‐hydroxy‐γ‐(trimethylammonio)butyrate – a compound necessary in the peripheral tissues for a transfer of fatty acids for their oxidation within the cell, accumulates in the brain despite low β‐oxidation in this organ. In order to enter the brain, carnitine has to cross the blood–brain barrier formed by capillary endothelial cells which are in close interaction with astrocytes. Previous studies, demonstrating expression of mRNA coding two carnitine transporters – organic cation/carnitine transporter 2 (OCTN2) and B0,+ in endothelial cells, did not give any information on carnitine transporters polarity in endothelium. Therefore more detailed experiments were performed on expression and localization of a high affinity carnitine transporter OCTN2 in an in vitro model of the blood–brain barrier by real‐time PCR, western blot analysis, and immunocytochemistry. The amount of mRNA was comparable in endothelial cells and kidney, when referred to house‐keeping genes, it was, however, significantly lower in astrocytes. Polarity of OCTN2 localization was further studied in an in vitro model of the blood–brain barrier with use of anti‐OCTN2 antibodies. Z‐axis analysis of the confocal microscope pictures of endothelial cells, with anti‐P‐glycoprotein antibodies as the marker of apical membrane, showed OCTN2 localization at the basolateral membrane and in the cytoplasmic region in the vicinity of nuclei. Localization of OCTN2 suggest that carnitine can be also transported from the brain, playing an important role in removal of certain acyl esters.

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.

Organic cation/carnitine transporter OCTN3 is present in astrocytes and is up-regulated by peroxisome proliferators-activator receptor agonist.

In the brain beta-oxidation, which takes place in astrocytes, is not a major process of energy supply. Astrocytes synthesize important lipid metabolites, mainly due to the processes taking place in peroxisomes. One of the compounds necessary in the process of mitochondrial beta-oxidation and export of acyl moieties from peroxisomes is l-carnitine. Two Na-dependent plasma membrane carnitine transporters were shown previously to be present in astrocytes: a low affinity amino acid transporter B(0,+) and a high affinity cation/carnitine transporter OCTN2. The expression of OCTN2 is known to increase in peripheral tissues upon the stimulation of peroxisome proliferators-activator receptor alpha (PPARalpha), a nuclear receptor known to up-regulate several enzymes involved in fatty acid metabolism. The present study was focused on another high affinity carnitine transporter-OCTN3, its presence, regulation and activity in astrocytes. Experiments using the techniques of real-time PCR, Western blot and immunocytochemistry analysis demonstrated the expression of octn3 in rat astrocytes and, out of two rat sequences ascribed as similar to mouse OCTN3, XM_001073573 was found in these cells. PPARalpha activator-2-[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]acetic acid (WY-14,643) stimulated by 50% expression of octn3, while, on the contrary to peripheral tissues, it did not change the expression of octn2. This observation was correlated with an increased Na-independent activity of carnitine transport. Analysis by transmission electron microscopy showed an augmented intracellular localization of OCTN3 upon PPARalpha stimulation, mainly in peroxisomes, indicating a physiological role of OCTN3 as peroxisomal membrane transporter. These observations point to an important role of OCTN3 in peroxisomal fatty acid metabolism in astrocytes.

Involvement of OCTN2 and B 0,+ in the transport of carnitine through an in vitro model of the blood-brain barrier

Carnitine is known to accumulate in brain, therefore transport of carnitine through the blood–brain barrier was studied in an in vitro system using bovine brain capillary endothelial cells (BBCEC) grown on filter inserts in a co‐culture system with glial cells. Long‐term exposure of BBCEC to carnitine resulted in a high accumulation of long‐chain acyl carnitines, which decreased dramatically upon removal of carnitine. Kinetic analysis of carnitine accumulation indicated a possibility of functioning of more than one transporter. BBCEC were incubated in the presence of substrates and inhibitors of known carnitine transporters added from either apical or basolateral side. Inhibition by replacement of sodium and expression of OCTN2 (RT‐PCR) were in agreement with earlier reports on the functioning of OCTN2 in apical membrane. For the first time, functioning of OCTN2 was demonstrated in the basolateral membrane, as well as functioning in both membranes of a low affinity carnitine transporter B0,+. Expression of B0,+ in BBCEC was confirmed by RT‐PCR. These results suggest that OCTN2 and B0,+ could be involved in carnitine transport in both the apical and basolateral membrane.

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 γ‐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 α‐ketoisocaproate (α‐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 α‐KIC‐induced change in neuronal excitability. Leucine and α‐KIC decreased the concentration of glutamate in thalamus and cortex without affecting GABA concentrations. Thus, BCAA and α‐KIC, by decreasing glutamatergic neurotransmission, could favor GABAergic neurotransmission, which is known to increase the occurrence of seizures in GAERS. Finally, the transport of [1‐14C]α‐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 [1‐14C]α‐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.

The activity of pyruvate carrier in a reconstituted system: substrate specificity and inhibitor sensitivity.

The pyruvate carrier, of molecular mass 34 kDa, was purified from mitochondria isolated from rat liver, rat brain, and bovine heart, by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate. Its activity after reconstitution in phosphatidylcholine vesicles was measured either as uptake of [1-14C]pyruvate or as exchange with different 2-oxoacids. All preparations exhibited similar apparent Km values for pyruvate, but somewhat different V(max) values. The ability to exchange different anions of physiological significance, including branched-chain 2-oxoacids, confirmed the known substrate specificity described for the pyruvate carrier in mitochondria. The sensitivity of pyruvate transport toward phenylglyoxal suggested an important role of arginyl residues in the transport activity, while a role of lysyl and histidyl residues was not confirmed.

High affinity carnitine transporters from OCTN family in neural cells.

Neurons are known to accumulate l-carnitine—a compound necessary for transfer of acyl moieties through biological membranes, apart from very low β-oxidation of fatty acids in adult brain. Present study demonstrates expression of octn2 and octn3 genes coding high affinity carnitine transporters, as well as presence of both proteins in neurons obtained from suckling and adult rats, and also in mouse transformed neural cells. Measurements of carnitine transport show activity of both transporters in neural cells, pointing to their importance in physiological processes other than β-oxidation.

A polarized localization of amino acid/carnitine transporter B0,+ (ATB0,+) in the blood–brain barrier

Brain capillary endothelial cells control the uptake and efflux from the brain of many hydrophilic compounds due to highly specialized transporters often localized in a polarized way. Localization of Na(+)- and Cl(-)-dependent amino acid and carnitine transporter B(0,+) (ATB(0,+)) was studied in a co-culture of bovine brain capillary endothelial cells (BBCEC) grown on filters above astrocytes (an in vitro blood-brain barrier model). Immunoblotting and three-dimensional immunocytochemistry analysis with anti-B(0,+)antibodies demonstrated the presence of this transporter and its prevalent co-localization with P-glycoprotein i.e. at the apical side. The sensitivity of leucine uptake through the apical membrane to 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH), D-serine as well as sodium and chloride replacement confirm the functioning of ATB(0,+) and suggests an important physiological role of ATB(0,+) in controlling the delivery of amino acids and carnitine to the brain.

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.