No abstract

The transport of lactate is an essential part of the concept of metabolic coupling between neurons and glia. Lactate transport in primary cultures of astroglial cells was shown to be mediated by a single saturable transport system with a K m value for lactate of 7.7 mM and a V max value of 250 nmol/(min ؋ mg of protein). Transport was inhibited by a variety of monocarboxylates and by compounds known to inhibit monocarboxylate transport in other cell types, such as ␣-cyano-4-hydroxycinnamate and p-chloromercurbenzenesulfonate. Using reverse transcriptase-polymerase chain reaction and Northern blotting, the presence of mRNA coding for the monocarboxylate transporter 1 (MCT1) was demonstrated in primary cultures of astroglial cells. In contrast, neuron-rich primary cultures were found to contain the mRNA coding for the monocarboxylate transporter 2 (MCT2). MCT1 was cloned and expressed in Xenopus laevis oocytes. Comparison of lactate transport in MCT1 expressing oocytes with lactate transport in glial cells revealed that MCT1 can account for all characteristics of lactate transport in glial cells. These data provide further molecular support for the existence of a lactate shuttle between astrocytes and neurons.The transport of lactate is an essential part of the concept of metabolic coupling between neurons and glia (1, 2). It has been demonstrated that glutamate at concentrations around 200 M strongly increases the rates of glycolysis and lactate release in cultured astroglial cells (3). It has further been shown that neurons are able to take up lactate and to use this compound as an energy substrate (1, 4, 5). In the mammalian retina, direct evidence has been provided for a transfer of lactate between Mü ller glial cells and photoreceptors (6). Besides its role as an exchangeable metabolic fuel, lactate also interferes with pH and volume regulation in neural cells (7).There is a considerable debate over the types of transporters involved in the uptake and release of lactate by astroglial cells.Nedergaard and Goldman (8) characterized lactate transport in cultured astrocytes and determined a low K m value of 0.4 mM. The carrier-mediated transport could not be inhibited by ␣-cyano-3-hydroxycinnamate or pCMBS, 1 both being typical inhibitors of monocarboxylate transport in other cell types. The transport process was reversible and accompanied by a cotransport of protons. Diffusion of protonated lactate could not be detected. In contrast to these results, Tildon et al. (9) identified two carrier-mediated processes for lactate uptake, characterized by K m values of 0.5 mM and 11 mM, respectively. The maximum velocity of the low-affinity transporter was 170 nmol/(min ϫ mg of protein), whereas only 10% of this value was found for the high affinity component. Transport was only partially inhibited by ␣-cyano-4-hydroxycinnamate and mersalyl. Acidic pH strongly increased transport activity, a finding consistent with a lactate/proton cotransport mechanism. Dringen et al. (10) detected solely non-saturable lactate transport ...

Safe and effective cell delivery remains one of the main challenges in cell-based therapy of neurodegenerative disorders. Graft survival, sufficient enrichment of therapeutic cells in the brain, and avoidance of their distribution throughout the peripheral organs are greatly influenced by the method of delivery. Here we demonstrate for the first time noninvasive intranasal (IN) delivery of mesenchymal stem cells (MSCs) to the brains of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. IN application (INA) of MSCs resulted in the appearance of cells in the olfactory bulb, cortex, hippocampus, striatum, cerebellum, brainstem, and spinal cord. Out of 1 × 10⁶ MSCs applied intranasally, 24% survived for at least 4.5 months in the brains of 6-OHDA rats as assessed by quantification of enhanced green fluorescent protein (EGFP) DNA. Quantification of proliferating cell nuclear antigen-positive EGFP-MSCs showed that 3% of applied MSCs were proliferative 4.5 months after application. INA of MSCs increased the tyrosine hydroxylase level in the lesioned ipsilateral striatum and substantia nigra, and completely eliminated the 6-OHDA-induced increase in terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine, 5'-triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining of these areas. INA of EGFP-labeled MSCs prevented any decrease in the dopamine level in the lesioned hemisphere, whereas the lesioned side of the control animals revealed significantly lower levels of dopamine 4.5 months after 6-OHDA treatment. Behavioral analyses revealed significant and substantial improvement of motor function of the Parkinsonian forepaw to up to 68% of the normal value 40-110 days after INA of 1 × 10⁶ cells. MSC-INA decreased the concentrations of inflammatory cytokines-interleukin-1β (IL-1β), IL-2, -6, -12, tumor necrosis factor (TNF), interferon-γ (IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF)-in the lesioned side to their levels in the intact hemisphere. IN administration provides a highly promising noninvasive alternative to the traumatic surgical procedure of transplantation and allows targeted delivery of cells to the brain with the option of chronic application.

The metabolism of [2-13C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using 13c NMR spectroscopy.After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-13C]glycine, cell extracts and incubation media were analyzed for 13C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-13C]glycine, the amount of de novo synthesized [2-13C]creatine was twofold increased, and in addition, 13C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-13C]glycine was utilized for the synthesis of glutathione in astroglial cells. 13Clabeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2~13C] serine, [3-13C] serine, and [2,3-13C] serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons. Key Words: Creatine-Glial metabolism-Glutathione-Glycine-NM R spectroscopy-Serine. Abbreviations used: DMEM, Dulbecco's modified Eagle's medium; GSx, amount of GSH plus twice the amount of GSSG; HSQC, heteronuclear single quantum coherence; MM, minimal medium; SHMT, serine hydroxymethyltransferase.