ABSTRACT

The concentration of potassium in the extracellular fluid has been found to stimulate the rate of CO2 fixation by astroglial cells grown in primary culture. Raising the concentration of extracellular potassium increased both the initial rate of formation of the 14C-labeled products of 14CO2 fixation and the final steady-state level of these products within the cells. In contrast, neither veratridine nor L-glutamate affected the rate of CO2 fixation in astroglial cells. The very low rate of CO2 fixation found in primarily neuronal cultures was unaffected by increased extracellular potassium as was CO2 fixation in fibroblasts. When cultured alone, astroglial cells release a large fraction of the 14C-labeled products of CO2 fixation into the surrounding medium. Mixed cultures of astroglia and neurons also fix CO2 but, in contrast to astroglia cultured alone, release only a small fraction of the 14C-labeled products into the culture medium.

Studies of local glucose utilization in neural tissues in vivo with the autoradiographic [¹⁴C]deoxyglucose method have demonstrated that energy metabolism increases almost linearly with the degree of functional activation, i.e. spike frequency, in the terminal projection zones of activated pathways. The increased metabolism is found in neuropil and is minimal or undetectable in neuronal cell bodies. Electrical stimulation, increased extracellular [K⁺] ([K⁺]_o), or opening of Na⁺ channels with veratridine stimulates metabolism in neural tissues, and this increase is blocked by ouabain, a specific inhibitor of Na⁺,K⁺-ATPase. Activation of this enzyme to restore ionic gradients across cellular membranes appears to mediate the function-related increase in energy metabolism. The metabolic activation is, therefore, not directly related to the functional activity itself but to processes operating to recover from that activity. The limited spatial resolution of the [¹⁴C]DG method precludes identification of cellular elements in neuropil participating in the metabolic activation, e.g. axonal terminals, dendrites, or astrocytic processes enveloping the synapses. We have, therefore, attempted to simulate in vitro conditions to be expected from functional activation and increased spike activity in vivo, e.g. increased extracellular [K⁺], intracellular [Na⁺], or extracellular neurotransmitter levels, and examined their effects on glucose metabolism in neurons and astroglia in culture. Increased [K⁺]_o stimulated [¹⁴C]DG phosphorylation in neuronal and mixed neuronal-astroglial cultures, but not in astroglial cultures assayed in bicarbonate buffer; it did occasionally stimulate metabolism in astroglia when assayed in HEPES or phosphate buffers, but these effects were variable and inconsistent. Veratridine (75 μM) stimulated [¹⁴C]DG phosphorylation in neurons and astroglia; these stimulations were blocked by 1 mM ouabain or 10 μM tetrodotoxin (TTX), which blocks voltage-dependent Na⁺ channels. The Na⁺ ionophore monensin (10 μM) doubled the rate of metabolism, a stimulation that was only partially blocked by ouabain and unaffected by TTX. L-Glutamate (500 μM) stimulated [¹⁴C]DG phosphorylation in astroglia, but this stimulation was probably secondary to Na⁺ uptake into the cells via a sodium/glutamate co-transporter because it was not blocked by inhibitors of NMDA or non-NMDA receptors but was absent in Na⁺-free medium. These results indicate that astroglia contribute to the increased energy metabolism in neuropil during functional activation by mechanisms that promote Na⁺ entry into the cells.

Changing the medium of primary cell cultures of CNS origin causes severe damage that is mediated via the N-methyl-D-aspartate (NMDA)-type of glutamate receptors and dependent on the presence of glutamine in the medium. Data presented here show that glutamine has two roles in culture damage: glutamine is contaminated with a small amount of glutamate, which is responsible for initiating culture damage, and glutamine is the source of the glutamate that is produced extracellularly in damaged cultures. The NMDA receptor plays a critical role minutes after medium change when the glutamate contaminating the glutamine binds to NMDA receptors; during this time, addition of a low level (10-20 microM) of 2-amino-5-phosphonovaleric acid can block most culture damage and the appearance of extracellular glutamate. A higher level (300 microM) of 2-amino-5-phosphonovaleric acid can protect cultures when added at much later times (30-60 min). Between 3 and 6 h after medium change, the concentration of extracellular glutamate starts to rise and accumulates until the end of the culture period (20 h). Medium removed from cultures at 3 h or later after medium change and incubated alone (i.e., with no cells) also continues to generate glutamate; filtration (0.22 microns pore size) or centrifugation (18,000 g) stops the appearance of this glutamate. 6-Diazo-5-oxo-L-norleucine, an inhibitor of the mitochondrial enzyme glutaminase, blocks the generation of glutamate. Mitochondria or mitochondrial fragments are probably released from the damaged cells and then convert extracellular glutamine to glutamate, resulting in generation of a high extracellular glutamate concentration.

The metabolism of [1-¹³C]D-glucose by astrocytes, neurons and mixed astroglial/neuronal cultures derived from the striatum of fetal rats was studied using NMR. Metabolic activity was studied in resting and depolarized cells (55 mM K⁺), with dibutyryl cyclic-AMP added to the medium to promote cell differentiation, and with glutamate (0.1 mM) included in the medium. Due to sample limitations the accumulation of ¹³C label in-metabolites within the cells was not sufficient to quantitate. Of the metabolites released into the medium by the astrocyte cultures and the mixed astroglial/neuronal cultures, measurable amounts of label were present in lactate C-3 and C-2, glutamine C-2, C-3 and C-4, acetate C-2, citrate C-2 or C-4 and C-3, glycerol C-1 or C-3, succinate C-2 or C-3 and several unidentified metabolites. Of the labeled metabolites released into the medium, only succinate was markedly affected by K⁺-induced depolarization, dBcAMP, or glutamate. The label in succinate was increased, especially in the K⁺-depolarized astrocyte cultures (3- to 6-fold). The neuronal cultures consumed [1-¹³C]D-glucose much more slowly than the astrocyte cultures or the mixed cultures. Except for lactate C-3, there was no measurable ¹³C in metabolites in the medium of the neuronal cultures.