[1-(13)C]glucose metabolism in the tumoral and nontumoral cerebral tissue of a glioma-bearing rat.

C6 cells were used to establish a glioma-bearing rat model by stereotaxic injection in the left caudate nucleus. The tumor status was evaluated by magnetic resonance imaging and conventional histology. The glioma-bearing rats were infused for 1 h with a [1-(13)C]glucose solution. Perchloric acid extracts of the tumor and the ipsilateral and contralateral hemispheres were analyzed by 13C-NMR spectroscopy. The 13C-labeling patterns in compounds, mainly amino acids, indicated no drastic modification of carbon metabolism in both ipsilateral and contralateral hemispheres, as compared with control rats, whereas profound metabolic differences between brain tissue and tumor were observed. Glutamine C4 enrichment was lower in the glioma than in the brain [mean +/- SD values, 5.4 +/- 2.3 (n = 5) and 15.0 +/- 0.8% (n = 10), respectively] and also lower than the glutamate C4 enrichment in the glioma (mean +/- SD value, 22.6 +/- 4.2%; n = 5), indicating that tumor glutamine was neither synthesized inside the glioma nor taken up from the surrounding brain. The glutamine C4 enrichment in the serum (6.7 +/- 0.5%; n = 10) suggested that the glioma imported glutamine from the blood, a process probably connected with angiogenesis.

Compartmentation of lactate and glucose metabolism in C6 glioma cells. A 13c and 1H NMR study.

13C and 1H NMR spectroscopy was used to investigate the metabolism of L-lactate and D-glucose in C6 glioma cells. The changing of lactate and glucose concentration in the extracellular medium of C6 glioma cells incubated with 5.5 mM glucose and 11 mM lactate indicated a net production of lactate as the consequence of an active aerobic glycolysis. The 13C enrichments of various metabolites were determined after 4-h cell incubation in media containing both substrates, each of them being alternatively labeled in the form of either [3-13C]L-lactate or [1-13C]D-glucose. Using 11 mM [3-13C]L-lactate, the enrichment of glutamate C4, 69%, was found higher than that of alanine C3, 32%, when that of acetyl-CoA C2 was 78%. These results indicated that exogenous lactate was the major substrate for the oxidative metabolism of the cells. Nevertheless, an active glycolysis occurred, leading to a net lactate production. This lactate was, however, metabolically different from the exogenous lactate as both lactate species did not mix into a unique compartment. The results were actually consistent with the concept of the existence of two pools of both lactate and pyruvate, wherein one pool was closely connected with exogenous lactate and was the main fuel for the oxidative metabolism, and the other pool was closely related to aerobic glycolysis.

Use of spin-traps during warm ischemia-reperfusion in rat liver: comparative effect on energetic metabolism studied using 31P nuclear magnetic resonance.

Detection of free radicals by electron spin resonance (ESR) proves the involvement of reactive oxygen species (ROS) in reperfused organ injuries. Spin-traps are known to ameliorate hemodynamic parameters in an isolated postischemic heart. The effects of 5 mmol/L DMPO (5,5-dimethyl-1-pyrroline-N-oxide) or DEPMPO (5-(diethlphosphoryl)-5-methyl-1-pyrroline N-oxide) on intracellular pH (pHin) and ATP level were evaluated by 31P nuclear magnetic resonance on isolated rat liver submitted to 1 hour of warm ischemia and reperfusion. At the end of the reperfusion period, during which pHin recovered to its initial value (7.16 +/- 0.03) in all groups, the ATP recovery level (expressed in percentage of initial value) was similar in controls and DEPMPO (60% +/- 5%, n = 6 and 54% +/- 4%, n = 6, respectively), but only 37% +/- 1% in DMPO-treated livers (n = 6) (p < 0.05 versus controls and p < 0.05 versus DEPMPO). Oxidative phosphorylation was not affected by an addition of nitrones on isolated mitochondria extracted from livers not submitted to ischemia-reperfusion. In contrast, mitochondria extracted at the end of the ischemia-reperfusion showed an impairment in the phosphorylation parameters, particularly in the presence of DMPO. Mass spectrum of ischemic liver perchloric acid extracts evidenced probable catabolites in treated groups. The differences in the effect of the two nitrones on energetic metabolism may be explained by the production of deleterious catabolites by DMPO as compared to DEPMPO. Even though a specific radical scavenging effect could be operative in the liver, our results indicate that catabolic effects were predominant. The absence of deleterious effects of DEPMPO in contrast to DMPO on the liver energetic metabolism was evidenced, allowing the use of DEPMPO for ESR detection.

Myelin P0 glycoprotein: identification of the site phosphorylated in vitro and in vivo by endogenous protein kinases.

Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [32P]PO4(3-) from [gamma-32P]ATP into protein constituents. Among these, P0 glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P0 protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two 32P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P0 sequence revealed, besides a Lys172 to Arg substitution, that in the first peptide, two serine residues (Ser176 and Ser181) were phosphorylated, Ser176 appearing to be modified subsequently to Ser181. In the second peptide, Ser197, Ser199, and Ser204 were phosphorylated. All these serines are clustered in the C-terminal region of P0 protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P0. We found that, in vivo, Ser181 and Ser176 are not phosphorylated, whereas Ser197, Ser199, Ser204, Ser208, and Ser214 are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.

Pre-steady state study of beta-adrenergic and purinergic receptor interaction in C6 cell membranes: undelayed balance between positive and negative coupling to adenylyl cyclase.

Interactions between beta-adrenergic and ADP purinergic receptors in C6 glioma cell membrane preparations were investigated under steady state and then pre-steady state conditions of adenylyl cyclase (EC 4.6.1.1) activity, in order to determine how fast the second receptor antagonizes the transduction mechanism of the first. Cell membranes were washed to deplete them as thoroughly as possible of low molecular weight compounds, especially ATP and ADP, and to ensure better control of both substrate and agonist nucleotide concentrations. ATP concentrations were kept constant with the use of an ATP-regenerating system; the C6 cell line exhibited very active ectonucleotidases. The purinergic agonist ADP was replaced by its nonhydrolyzable congener adenosine 5'-O-(2-thio)diphosphate (ADP beta S), which was demonstrated, like ADP, to inhibit isoproterenol-stimulated adenylyl cyclase activity in intact cells (IC50 for ADP, 0.5 +/- 0.1 microM; IC50 for ADP beta S, 25 +/- 2 microM) and in membrane preparations (IC50 for ADP beta S, 79 +/- 20 microM). In the case of membrane preparations, ADP beta S did not compete with ATP, the substrate of the cyclase-catalyzed reaction, and behaved apparently as a non-competitive inhibitor of the enzyme. The pre-steady state kinetics of isoproterenol-stimulated adenylyl cyclase activity measured with a pulsed quenched-flow apparatus have previously been shown to include two steps, the first very rapid (taking place within 1-2 sec) and giving rise to a burst of cAMP synthesis and the second much slower and corresponding to the steady state reaction. ADP beta S inhibited the occurrence of both steps with comparable IC50 values (mean value, 55 +/- 20 microM). In the presence of increasing concentrations of the purinergic receptor agonist, the time constant of the exponential burst reaction was not affected, but its amplitude progressively decreased to zero. These results showed that the extinction of the beta receptor cAMP response by the purinergic ADP receptor occurred within the dead-time of the pulsed quenched-flow apparatus, which was 50 msec. Such a rapid inhibition of cAMP production excluded modulation of isoproterenol-stimulated adenylyl cyclase activity by the ADP receptor by a pathway other than its direct negative coupling to the cyclase via a Gi protein. In this respect, the P2 purinergic ADP receptor of the C6 glioma cell line appears comparable to the P2t receptor of platelets.

A quenched-flow study of a receptor-triggered second messenger response: cyclic AMP burst elicited by isoproterenol in C6 glioma cell membranes.

Fast kinetic studies of cAMP accumulation in C6 cell membranes show a burst of cAMP after beta-adrenergic receptor stimulation by isoproterenol. This burst is no longer observed when the ATP present in membrane preparations is hydrolyzed, but can be restored by their preincubation in the presence of ATP-Mg. The size of the burst is much larger than the number of beta-adrenergic receptors and is of the same order of magnitude as the value reported for G proteins. Further characterization of the burst will allow studies of the functional interaction of receptor-adenylate cyclase components in C6 membranes.