The plasma membranes of oat normally resemble those of other eukaryotes in containing mainly phospholipids and sterols. We here report the novel ¢nding that the galactolipid digalactosyldiacylglycerol (DGDG) can constitute a substantial proportion of oat plasma membrane lipids, in both shoots and roots. When oat was cultivated under severe phosphate limitation, up to 70% of the plasma membrane phosphoglycerolipids were replaced by DGDG. Our ¢nding not only re£ects a far more developed potential for plasticity in plasma membrane lipid composition than often assumed, but also merits interest in the context of the limited phosphate availability in many soils. ß

Key points• Rapid exchange of metabolites like glucose and lactate between different cell types is crucial for energy supply to the brain.• Carbonic anhydrase 2 (CAII) enhances lactate transport in mouse cerebellar and cerebral astrocytes.• Enhancement of transport activity is independent of the enzyme's catalytic function, but requires binding of CAII to the C-terminal tail of the monocarboxylate transporter MCT1.• CAII could enhance lactate flux by acting as a 'proton collecting antenna' for MCT1.• By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide neurons with an increased supply of energy substrate.Abstract Rapid exchange of metabolites between different cell types is crucial for energy homeostasis of the brain. Besides glucose, lactate is a major metabolite in the brain and is primarily produced in astrocytes. In the present study, we report that carbonic anhydrase 2 (CAII) enhances both influx and efflux of lactate in mouse cerebellar astrocytes. The augmentation of lactate transport is independent of the enzyme's catalytic activity, but requires direct binding of CAII to the C-terminal of the monocarboxylate transporter MCT1, one of the major lactate/proton cotransporters in astrocytes and most tissues. By employing its intramolecular proton shuttle, CAII, bound to MCT1, can act as a 'proton collecting antenna' for the transporter, suppressing the formation of proton microdomains at the transporter-pore and thereby enhancing lactate flux. By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide the neurons with an increased supply of energy substrate. Abbreviations 4-MI, 4-methylimidazole; aCSF, artificial cerebrospinal fluid; AE1, anion exchanger isoform 1; BCA, bichionic acid; CA, carbonic anhydrase; Car2, carbonic anhydrase II coding gene; CD147, cluster of differentiation 147; Co-IP, co-immunoprecipitation; EAAT, excitatory amino acid transporter; GFAP, glial fibrillary acidic protein; GST, glutathione S-transferase; gp70, glycoprotein 70; MCT, monocarboxylate transporter; m/z, mass-to-charge ratio; NBCe1, electrogenic sodium bicarbonate cotransporter isoform 1; NHE1, sodium/hydrogen exchanger isoform 1; PDB, protein data bank; PLA, proximity ligation assay.

The cellular distribution of transporters and enzymes related to glutamate metabolism led to the concept of the glutamate-glutamine cycle. Glutamate is released as a neurotransmitter and taken up primarily by astrocytes ensheathing the synapses. The glutamate carbon skeleton is transferred back to the presynaptic neurons as the nonexcitatory amino acid glutamine. The cycle was initially thought to function with a 1:1 ratio between glutamate released and glutamine taken up by neurons. However, studies of glutamate metabolism in astrocytes have shown that a considerable proportion of glutamate undergoes oxidative degradation; thus, quantitative formation of glutamine from the glutamate taken up is not possible. Oxidation of glutamate is initiated by transamination catalyzed by an aminotransferase, or oxidative deamination catalyzed by glutamate dehydrogenase (GDH). We discuss methods available to elucidate the enzymes that mediate this conversion. Methods include pharmacological tools such as the transaminase inhibitor aminooxyacetic acid, studies using GDH knockout mice, and siRNA-mediated knockdown of GDH in astrocytes. Studies in brain slices incubated with [ N]glutamate demonstrated activity of GDH in astrocytes in situ. These results, in conjunction with reports in the literature, support the conclusion that GDH is active in astrocytes both in culture and in vivo and that this enzyme plays a significant role in glutamate oxidation. Oxidative metabolism of glutamate, primarily mediated by GDH, but also by transamination by aspartate aminotransferase, provides considerably more energy than is required to maintain the activity of the high-affinity glutamate transporters needed for efficient removal of glutamate from the synaptic cleft. © 2016 Wiley Periodicals, Inc.

Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids.

Omission of extracellular Ca2؉ for 15 min from the incubation medium of cultured hippocampal slices stimulated the efflux of glutathione, phosphoethanolamine, hypotaurine, and taurine. The efflux was reduced by several blockers of gap junctions, i.e. carbenoxolone, flufenamic acid, and endothelin-1, and by the connexin43 hemichannel blocking peptide Gap26 but was unchanged by the P2X 7 receptor inhibitor oxidized ATP, a pannexin1 hemichannel blocking peptide and an inactive analogue of carbenoxolone. Pretreatment of the slices with the neurotoxin N-methyl-D-aspartate left the efflux by Ca 2؉ omission unchanged, indicating that the stimulated efflux primarily originated from glia. Elevated glutamate efflux was detected when Ca 2؉ omission was combined with the glutamate uptake blocker L-trans-pyrrolidine-2,4-dicarboxylate and when both Ca 2؉ and Mg 2؉ were omitted from the medium. Omission of Ca 2؉ for 15 min alone did not induce delayed toxicity, but in combination with blocked glutamate uptake, significant cell death was observed 24 h later. Our results indicate that omission of extracellular Ca 2؉ stimulates efflux of glutathione and specific amino acids including glutamate via opening of glial hemichannels.