Cerebral Cortical Astrocytes Research Articles

Methylmercury has a selective effect on mitochondria in cultured astrocytes in the presence of [U- 13C]glutamate

The effect of methylmercury on glutamate metabolism was studied by 13C magnetic resonance spectroscopy. Cerebral cortical astrocytes were pretreated with methylmercury, either 1 μM for 24 h, or 10 μM for 30 min, and subsequently with 0.5 mM [U- 13C]glutamate for 2 h. Labeled glutamate, glutamine, aspartate and glutathione were present in cell extracts, and glutamine, aspartate and lactate in the medium of all groups. HPLC analysis of these amino acids showed no changes in concentrations between groups. Surprisingly, the amounts of [U- 13C]glutamate and unlabeled glucose taken up by the astrocytes were unchanged. Furthermore, the amounts of most metabolites synthesized from [U- 13C]glutamate were also unchanged in all groups. However, formation of [U- 13C]lactate was decreased in the 10 μM methylmercury group. This was not observed for labeled aspartate. It is noteworthy that both [U- 13C]lactate and [U- 13C]aspartate can only be derived from [U- 13C]glutamate via mitochondrial metabolism. [U- 13C]glutamate enters the tricarboxylic acid cycle (located in mitochondria) after conversion to 2-[U- 13C]oxoglutarate and [U- 13C]aspartate is formed from [U- 13C]oxaloacetate, as is [U- 13C]lactate. [U- 13C]lactate can also be formed from [U- 13C]malate. This differential effect on labeled aspartate and lactate indicates cellular compartmentation and thus selective vulnerability of mitochondria within the astrocytes to the effects of methylmercury. The decreased lactate production from glutamate might be detrimental to surrounding cells since lactate has been shown to be an important substrate for neurons.

Oleate, linoleate and cholesterol differently modify aspartyl- and glutamyl-aminopeptidase activities in primary cultures of rat astrocytes

The intake of mono- and polyunsaturated fatty acids has been associated with a minor risk of cardiovascular diseases including hypertension. Changes in levels of fatty acids may also modify the cell activity and may be related with alterations in different regulatory processes. Aminopeptidases are zinc-metalloenzymes which metabolise circulating peptide hormones in several tissues. Glutamyl-aminopeptidase (GluAP) and to a lesser extent, aspartyl-aminopeptidase (AspAP), are related with angiotensin metabolism in the renin–angiotensin system. The present work was designed to study the effect of a range of concentrations (1–100 μM) of oleic and linoleic acids and cholesterol present in the culture medium on the activity of GluAP and AspAP in the culture of rat cerebral cortical astrocytes taken from 21-day-old fetuses. The results showed that oleic acid inhibits, while linoleic acid stimulates the activity of AspAP. Both fatty acids inhibit the activity of GluAP. Cholesterol stimulates the activity of both enzymes. On the basis of these results, a functional link may exit between the effects of fatty acids on hypertension and the modulation of aminopeptidase activity by these compounds in rat astrocytes, as an example of target cell type in the central nervous system.

Taurine and neural cell damage.

The inhibitory amino acid taurine is an osmoregulator and neuromodulator, also exerting neuroprotective actions in neural tissue. We review now the involvement of taurine in neuron-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress, and the presence of free radicals, metabolic poisons and an excess of ammonia. The brain concentration of taurine is increased in several models of ischemic injury in vivo. Cell-damaging conditions which perturb the oxidative metabolism needed for active transport across cell membranes generally reduce taurine uptake in vitro, immature brain tissue being more tolerant to the lack of oxygen. In ischemia nonsaturable diffusion increases considerably. Both basal and K+-stimulated release of taurine in the hippocampus in vitro is markedly enhanced under cell-damaging conditions, ischemia, free radicals and metabolic poisons being the most potent. Hypoxia, hypoglycemia, ischemia, free radicals and oxidative stress also increase the initial basal release of taurine in cerebellar granule neurons, while the release is only moderately enhanced in hypoxia and ischemia in cerebral cortical astrocytes. The taurine release induced by ischemia is for the most part Ca2+-independent, a Ca2+-dependent mechanism being discernible only in hippocampal slices from developing mice. Moreover, a considerable portion of hippocampal taurine release in ischemia is mediated by the reversal of Na+-dependent transporters. The enhanced release in adults may comprise a swelling-induced component through Cl- channels, which is not discernible in developing mice. Excitotoxic concentrations of glutamate also potentiate taurine release in mouse hippocampal slices. The ability of ionotropic glutamate receptor agonists to evoke taurine release varies under different cell-damaging conditions, the N-methyl-D-aspartate-evoked release being clearly receptor-mediated in ischemia. Neurotoxic ammonia has been shown to provoke taurine release from different brain preparations, indicating that the ammonia-induced release may modify neuronal excitability in hyperammonic conditions. Taurine released simultane ously with an excess of excitatory amino acids in the hippocampus under ischemic and other neuron-damaging conditions may constitute an important protective mechanism against excitotoxicity, counteracting the harmful effects which lead to neuronal death. The release of taurine may prevent excitation from reaching neurotoxic levels.

Multi-lipofection efficiently transfected genes into astrocytes in primary culture

This study demonstrated that liposome-mediated transfection — lipofection — is suitable for delivering genes into astrocytes. By repeatedly lipofecting the same astrocyte cultures, a process we call multi-lipofection, the transfection efficiency of the β-galactosidase (β-gal) gene was improved from 2.6±0.6 to 17.4±1.1%. This is the highest efficiency ever reported in gene-transfer with Lipofectin® in a primary culture of mouse cerebral cortical astrocytes. Furthermore, multi-lipofection did not cause observable disturbance to astrocytes as indicated by insignificant changes in the glial fibrillary acidic protein content in the cultures. In order to demonstrate that the transfected gene achieved a physiologically relevant expression level, a plasmid containing the pEF-hsp70 protein gene was lipofected into astrocytes. This produced colonies of astrocytes showing an increased resistance to heat-induced cell death. A similar experiment was performed with the glial-derived neurotrophic factor (GDNF) gene. Control astrocytes had no detectable GDNF. In the transfected astrocytes, the GDNF protein could be identified intracellularly by immunocytochemistry. Western blot analysis revealed, as compared to astrocytes with one lipofection, a 2.9-fold increase of GDNF with four lipofections. GDNF remained detectable in astrocytes 2 weeks after four lipofections. Thus, multi-lipofection provides a mild and efficient means of delivering foreign genes into astrocytes in a primary culture, making astrocytes good candidate vehicle cells for gene/cell therapy in the CNS.

Protection against ischemic injury in primary cultured astrocytes of mouse cerebral cortex by bis(7)-tacrine, a novel anti-Alzheimer's agent

The effects of bis(7)-tacrine, a novel acetylcholinesterase inhibitor, on ischemia-induced cell death and apoptosis were investigated in primary cerebral cortical astrocytes of mice. Following a 6 h in vitro ischemic incubation of the cultures, a marked decrease in the percentage of viable cells was observed by lactate dehydrogenase (LDH) release assay. Furthermore, using bisbenzimide staining, we determined that approximately 65% of the cells underwent apoptosis. Treatment with bis(7)-tacrine (1–10 nM) during ischemic incubation effectively inhibited the ischemia-induced apoptosis, as demonstrated by morphological and biochemical tests. Our results demonstrated that bis(7)-tacrine could protect astrocytes against ischemia-induced cell injury, indicating that the drug might be beneficial for the treatment of vascular dementia, in addition to Alzheimer's disease.

Expression of interleukin-1 alpha, tumor necrosis factor alpha and interleukin-6 genes in astrocytes under ischemic injury

Astrocytes form an integral part of the blood brain barrier and are the first cell type in the central nervous system to encounter insult if there is an ischemic attack. The immunologic reaction of astrocytes to an ischemic insult would be affective to the subsequent responses of other nerve cells. We previously showed that ischemia caused an increase in the levels of interleukin 1α (IL-1α), tumor necrosis factor α (TNFα), and interleukin 6 (IL-6) in the culture medium of mouse cerebral cortical astrocyte. We did not have evidence on the source of these cytokines. This study aimed to investigate the expressions of these cytokine mRNAs in the astrocytes under ischemia. Results demonstrated that ischemia could induce necrosis and apoptosis in astrocytes. By using the RT-PCR method, we demonstrated for the first time that the mRNA levels of IL-1α, TNFα and IL-6 in normal astrocyte was very low, but their expressions could be induced quickly under ischemia. These cytokines might be interactive as indicated by the difference in time course of their expressions, with IL-1α being the earliest and IL-6 being the latest. The result provided some understanding of the induction and progression of these immunologic responses in astrocytes under ischemia. It also supported our previous findings that astrocytes contributed to the cytokines released under ischemia.

A novel signaling molecule for neuropeptide action: activity-dependent neuroprotective protein.

The complete coding sequence of a novel protein (828 amino acids, pI 5.99), a potential new mediator of vasoactive intestinal peptide (VIP) activity was recently revealed. The expression of this molecule, activity-dependent neuroprotective protein (ADNP), was augmented in the presence of VIP, in cerebral cortical astrocytes. The mRNA transcripts encoding ADNP were enriched in the mouse hippocampus and cerebellum. The protein deduced sequence contained the following: (1) a unique peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor (ADNF) and exhibiting neuroprotection in vitro and in vivo; (2) a glutaredoxin active site; and (3) a classical zinc binding domain. Comparative studies suggested that the peptide, NAPVSIPQ (NAP), was more efficacious than peptides derived from ADNF. ADNP, a potential mediator of VIP-associated neuronal survival, and the new peptide, a potential lead compound for drug design, are discussed below.

Deep hypothermia and rewarming alters glutamate levels and glycogen content in cultured astrocytes.

Deep hypothermia has been associated with an increased incidence of postoperative neurologic dysfunction after cardiac surgery in children. Recent studies suggest an excitotoxic mechanism involving overstimulation of glutamate receptors. Extracellular glutamate uptake occurs primarily by astrocytes. Astrocytes also store glycogen, which may be used to sustain the energy-consuming glutamate uptake. Extracellular glutamate and glycogen content were studied during temperature changes mimicking cardiopulmonary bypass in vivo. Primary cultures of cerebral cortical astrocytes were used in a specially designed incubator allowing continuous changes of temperature and ambient gas concentrations. The sequence of events was as follows: normothermia, rapid cooling (2.8 degrees C/min) followed by 60 min of deep hypothermia (15 degrees C), followed by rewarming (3.0 degrees C/min) and subsequent 5 h of mild hyperthermia (38.5 degrees C). Two different conditions of oxygenation were studied: (1) normoxia (25% O2, 70% N2, 5% CO2); or (2) hyperoxia (95% O2, 5% CO2). The extracellular glutamate concentrations and intracellular glycogen levels were measured at nine time points. One hundred sixty-two cultures were studied in four independent experiments. The extracellular concentration of glutamate in the normoxic group increased significantly from 35+/-10 nM/mg protein at baseline up to 100+/-15 nM/mg protein at the end of 5 h of mild hyperthermia (P < 0.05). In contrast, extracellular glutamate levels did not vary from control in the hyperoxic group. Glycogen levels decreased significantly from 260+/-85 nM/mg protein at baseline to < 25+/-5 nM/mg protein at the end of 5 h in the normoxic group (P < 0.05) but returned to control levels after rewarming in the hyperoxic group. No morphologic changes were observed in either group. The extracellular concentration of glutamate increases, whereas the intracellular glycogen content decreases when astrocytes are exposed to a sequence of deep hypothermia and rewarming. This effect of hypothermia is prevented when astrocytes are exposed to hyperoxic conditions.

Decreased glutamate metabolism in cultured astrocytes in the presence of thiopental

The effect of thiopental on glutamate metabolism was studied by 13C magnetic resonance spectroscopy. Cerebral cortical astrocytes were incubated with 0.5 mM [U- 13C]glutamate for 2 hr in the presence of 0.5 or 1 mM thiopental. Labeled glutamate, glutamine, aspartate, and glutathione were observed in cell extracts, and glutamine, aspartate, and lactate in the medium. Not only present in the medium was uniformly labeled glutamate, but also glutamate derived from the tricarboxylic acid (TCA) cycle, and thus glutamate release could be detected. The amounts of [U- 13C]glutamate and unlabeled glucose taken up by astrocytes were unchanged in the presence of 0.5 mM thiopental and decreased to about 50% and 80%, respectively when the concentration was increased to 1 mM. The amounts of most metabolites synthesized from [U- 13C]glutamate were unchanged in the presence of 0.5 mM thiopental, but decreased [U- 13C]glutamine, [U- 13C]aspartate, and [U- 13C]lactate were observed in the 1 mM group. Surprisingly, the amounts of [1,2,3- 13C]glutamate, [2,3- 13C]aspartate, and [3,4- 13C]aspartate (2nd turn via the TCA cycle) were unchanged. However, this was not the case for [1,2- 13C]lactate and [2,3- 13C]lactate. Such variations indicate cellular compartmentation, possibly caused by a heterogeneous glutamate concentration within the cells affecting TCA cycle turnover rates differently.

Mitogenic Signaling by ATP/P2Y Purinergic Receptors in Astrocytes: Involvement of a Calcium-Independent Protein Kinase C, Extracellular Signal-Regulated Protein Kinase Pathway Distinct from the Phosphatidylinositol-Specific Phospholipase C/Calcium Pathway

Activation of ATP/P2Y purinergic receptors stimulates proliferation of astrocytes, but the mitogenic signaling pathway linked to these G-protein-coupled receptors is unknown. We have investigated the role of extracellular signal-regulated protein kinase (ERK) in P2Y receptor-stimulated mitogenic signaling as well as the pathway that couples P2Y receptors to ERK. Downregulation of protein kinase C (PKC) in primary cultures of rat cerebral cortical astrocytes greatly reduced the ability of extracellular ATP to stimulate ERK. Because occupancy of P2Y receptors also leads to inositol phosphate formation, calcium mobilization, and PKC activation, we explored the possibility that signaling from P2Y receptors to ERK is mediated by a phosphatidylinositol-specific phospholipase C (PI-PLC)/calcium pathway. However, neither inhibition of PI-PLC nor chelation of calcium significantly reduced ATP-stimulated ERK activity. Moreover, a preferential inhibitor of calcium-dependent PKC isoforms, Gö 6976, was significantly less effective in blocking ATP-stimulated ERK activity than GF102903X, an inhibitor of both calcium-dependent and -independent PKC isoforms. Furthermore, ATP stimulated a rapid translocation of PKCdelta, a calcium-independent PKC isoform, but not PKCgamma, a calcium-dependent PKC isoform. ATP also stimulated a rapid increase in choline, and inhibition of phosphatidylcholine hydrolysis blocked ATP-evoked ERK activation. These results indicate that P2Y receptors in astrocytes are coupled independently to PI-PLC/calcium and ERK pathways and suggest that signaling from P2Y receptors to ERK involves a calcium-independent PKC isoform and hydrolysis of phosphatidylcholine by phospholipase D. In addition, we found that inhibition of ERK activation blocked extracellular ATP-stimulated DNA synthesis, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y receptors.

Bilirubin toxicity and differentiation of cultured astrocytes.

To study the toxicity of bilirubin in primary cultures of newborn rat cerebral cortical astrocytes. Primary cultures of newborn rat astrocytes were incubated at bilirubin concentrations of 0, 1, 5, 10, 25, 50, 100, 200, and 2000 microM, at a bilirubin:albumin molar ratio of 1.7. Bilirubin toxicity was determined by changes in cellular morphology, trypan blue staining, and lactate dehydrogenase (LDH) release into the culture medium at various times of incubation. To determine if differentiation of astrocytes affects bilirubin toxicity, cultures were treated with dibutyryl cyclic adenosine monophosphate. All three indices of toxicity showed a bilirubin concentration dependence. LDH release in experimental cultures was significantly elevated (p < 0.05) above that of control cultures by 24 hours at bilirubin concentrations of > or = 100 microM. The absolute amount of LDH release differed significantly between the 200 and 2000 microM cultures from 1.5 to 24 hours, after which duration of exposure appeared to take over and all cultures approached maximum. LDH release for the lower concentrations all reached maximum by 120 hours, except for the 1 microM cultures, which showed no significant elevation above control throughout the study period. At 100 and 200 microM bilirubin, LDH release by untreated cells was significantly higher (p < 0.05) than release by treated cells by 36 hours. Undifferentiated astrocytes appeared to be more sensitive to bilirubin toxicity, which may correlate with the greater susceptibility of newborns to kernicteric injury. Studies with primary astrocyte culture may provide insight into how bilirubin sensitivity changes with brain development as well as the cellular and biochemical mechanisms of bilirubin encephalopathy.

Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide.

The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pI 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor; (2) a glutaredoxin active site; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuron-astrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the beta-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E-deficient mice accelerated the acquisition of developmental reflexes and prevented short-term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E-deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function.

Neurite outgrowth promotion by the alternatively spliced region of tenascin-C is influenced by cell-type specific binding.

We have investigated the impact of cellular environment on the neurite outgrowth promoting properties of the alternatively spliced fibronectin type-III region (fnA-D) of tenascin-C. FnA-D promoted neurite outgrowth in vitro when bound to the surface of BHK cells or cerebral cortical astrocytes, but the absolute increase was greater on astrocytes. In addition, different neurite outgrowth promoting sites were revealed within fnA-D bound to the two cellular substrates. FnA-D also promoted neurite outgrowth as a soluble ligand; however, the actions of soluble fnA-D were not affected by cell type. Therefore, we hypothesized that different mechanisms of cellular binding can alter the growth promoting actions of bound fnA-D. We found that fnA-D utilizes two distinct sequences to bind to the BHK cell surface as opposed to the BHK extracellular matrix. In contrast, only one of these sequences is utilized to bind to the astrocyte matrix as opposed to the astrocyte surface. Furthermore, Scatchard analysis indicated two types of receptors for fnA-D on BHK cells and only one type on astrocytes. These results suggest that active sites for neurite outgrowth within fnA-D are differentially revealed depending on cell-specific fnA-D binding sites. Therefore, the function of tenascin-C and its various domains must be considered in terms of cellular context.

Taurine release is enhanced in cell-damaging conditions in cultured cerebral cortical astrocytes.

The release of preloaded [3H]taurine from cultured cerebral cortical astrocytes was studied under various cell-damaging conditions, including hypoxia, ischemia, aglycemia and oxidative stress, and in the presence of free radicals. Astrocytic taurine release was enhanced by K+ (50 mM), veratridine (0.1 mM) and the ionotropic glutamate receptor agonist kainate (1.0 mM). Metabotropic glutamate receptor agonists had only weak effects on taurine release. Similarly to the swelling-induced taurine release the efflux in normoxia seems to be mediated mainly by DIDS-(diisothiocyanostilbene-2,2'-disulphonate) and SITS-(4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate) sensitive CI- channels, since these blockers were able to reduce both basal and K+ -stimulated release. The basal release of taurine was moderately enhanced in hypoxia and ischemia, whereas the potentiation in the presence of free radicals was marked. The small basal release from astrocytes signifies that taurine release from brain tissue in ischemia may originate from neurons rather than glial cells. On the other hand, the release evoked by K+ in hypoxia and ischemia was greater than in normoxia, with a very slow time-course. The enhanced release of the inhibitory amino acid taurine from astrocytes in ischemia may be beneficial to surrounding neurons, outlasting the initial stimulus and counteracting overexcitation.

Comparison of Lactate and Glucose Metabolism in Cultured Neocortical Neurons and Astrocytes Using 13C-NMR Spectroscopy

In cerebral cortical neurons, synthesis of the tricarboxylic acid (TCA) cycle-derived amino acids, glutamate and aspartate as well as the neurotransmitter of these neurons, γ-aminobutyrate (GABA), was studied incubating the cells in media containing 0.5 mM [U-¹³C]glucose in the absence or presence of glutamine (0.5 mM). Lyophilized cell extracts were analyzed by ¹³C nuclear magnetic resonance (NMR) spectroscopy and HPLC. The present findings were compared to results previously obtained using 1.0 mM [U-¹³C]lactate as the labeled substrate for the neurons. Regardless of the amino acids studied, incubation periods of 1 and 4 h resulted in identical amounts of ¹³C incorporated. Furthermore, the metabolism of lactate was studied under analogous conditions in cultured cerebral cortical astrocytes. The incorporation of ¹³C from lactate into glutamate was much lower in the astrocytes than in the neurons. In cerebral cortical neurons the total amount of ¹³C in GABA, glutamate and aspartate was independent of the labeled substrate. The enrichment in glutamate and aspartate was, however, higher in neurons incubated with lactate. Thus, lactate appears to be equivalent to glucose with regard to its access to the TCA cycle and subsequent labeling of glutamate, aspartate and GABA. It should be noted, however, that incubation with lactate in place of glucose led to lower cellular contents of glutamate and aspartate. The presence of glutamine affected the metabolism of glucose and lactate differently, suggesting that the metabolism of these substrates may be compartmentalized.

Metabotropic glutamate receptor agonists potentiate cyclic AMP formation induced by forskolin or beta-adrenergic receptor activation in cerebral cortical astrocytes in culture.

The metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD) potentiated the accumulation of cyclic AMP induced by either beta-adrenergic receptor stimulation (isoproterenol) or direct activation of adenylyl cyclase (AC) with forskolin in rat cerebral cortical astrocytes grown in a defined medium. In contrast, ACPD inhibits the cyclic AMP response in astrocytes cultured in a serum-containing medium. Pharmacological characterization indicated that a group I mGluR, of which only mGluR5 is detectable in these cells, is involved in the potentiation of cyclic AMP accumulation. Potentiation was elicited by mGluR I agonists [e.g., (R,S)-3,5-dihydroxyphenylglycine (DHPG)], but not by mGluR II or III agonists; it was pertussis toxin resistant and abolished by procedures suppressing mGluR5 function (phorbol ester pretreatment or DHPG-induced receptor down-regulation). Nevertheless, it appears that products generated through the mGluR5 transduction pathway, such as elevated [Ca2+]i or activated protein kinase C (PKC), are not involved in the potentiation as it was not influenced by either the intracellular calcium chelator BAPTA-AM or the PKC inhibitor Ro 31-8220. An inhibitor of phospholipase C, U-73122, markedly attenuated mGluR5-activated phosphoinositide hydrolysis but did not significantly affect the DHPG potentiation of the cyclic AMP response. A mechanism is proposed in which the potentiating effect on AC could be mediated by free betagamma complex that is liberated after the agonist-bound mGluR5 interacts with its coupled G protein.

Alpha-ketoisocaproate alters the production of both lactate and aspartate from [U-13C]glutamate in astrocytes: a 13C NMR study.

The present study determined the metabolic fate of [U-13C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mM alpha-ketoisocaproate (alpha-KIC). When astrocytes were incubated with 0.2 mM [U-13C]glutamate, 64.1% of the 13C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3-(13)C3]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [13C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [13C]glutamate into aspartate in astrocytes incubated in the presence of alpha-KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [13C]glutamate into the 1,2,3-(13)C3-isotopomer of lactate in cells incubated in the presence of alpha-KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of alpha-KIC. Altogether more of the [13C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of alpha-KIC than in control cells. Overall, the results demonstrate that the presence of alpha-KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate-aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.

Effect of orotic acid on the metabolism of cerebral cortical astrocytes during hypoxia and reoxygenation: an NMR spectroscopy study.

Astrocytes were incubated under normoxic or hypoxic conditions in Dulbecco's minimum essential medium containing [2-13C]acetate, unlabeled glucose and in some cases orotic acid, an intermediate in pyrimidine biosynthesis. After 12 hr the medium was replaced by fresh medium without drug and incubation was continued for 17 hr in a normal oxygen atmosphere (reoxygenation). Thereafter, medium was removed, cell extracts were prepared, and metabolism in the treatment group was compared to the untreated hypoxia group and to control. 13C and 1H NMR spectra revealed that 13C enrichment in citrate and glutamine C-4 in the initial medium were increased in the presence of orotic acid, compared to the untreated hypoxia group but lower than control. The drug increased acetate utilization during hypoxia to normoxic levels. Thus it appears that the treatment group had a more active mitochondrial metabolism, which was also reflected in higher intracellular uridine diphosphoryl sugars and ADP concentrations. Glutamine labeling was increased in the cell extracts in the presence of orotic acid. Thus it appears that, in the presence of the pyrimidine nucleotide precursor, astrocytes are capable of normal metabolism during hypoxia which might have implications for neuronal survival during low oxygen insults, since neurons are dependent on astrocyte produced precursors for their neurotransmitter synthesis.

Glutamate transport and metabolism in astrocytes.

Glutamate uptake and metabolism was studied in cerebral cortical astrocytes. The expression of the astrocytic glutamate transporter GLAST was found to be stimulated by extracellular glutamate through activation of kainate receptors on the astrocytes. Energy metabolism and ammonia homeostasis are two important aspects of glutamate handling in astrocytes. It is well known that glutamate transport into astrocytes and glutamine formation are energy consuming processes. Furthermore, ammonia is required for glutamine production. On the other hand, glutamate metabolism through the tricarboxylic acid cycle is an energy and ammonia producing pathway. In the present study it was shown that at an extracellular glutamate concentration of 0.5 mM, high energy phosphates were reduced, and more than 50% of the glutamate carbon skeleton entered the tricarboxylic acid cycle to yield products like lactate, aspartate, and additionally glutamate and glutamine derived from tricarboxylic acid cycle intermediates. Entry into the cycle was not affected by the transaminase inhibitor aminooxyacetic acid, indicating that deamination is the major route for 2-oxoglutarate formation from glutamate. Synthesis of glutamate from 2-oxoglutarate, however, proceeded via transamination. In an earlier study it was shown that at glutamate concentrations at and below 0.2 mM, glutamine appears to be the major product and entry of glutamate into the tricarboxylic acid cycle is decreased 70% by aminooxyacetic acid. In an attempt to unify the above mentioned results, it is suggested that availability of ammonia and energy demands are major factors determining the metabolic fate of glutamate in astrocytes.

Glutamine transport in cerebellar granule cells in culture

In the present study, uptake of glutamine by rat cerebellar granule cells, a predominantly glutamatergic nerve cell population, has been investigated. Glutamine is taken up by granule cells via at least three transport systems, A, ASC and L. The L-type low affinity system ( K m=2.6 mM) is the major transport system in the absence of Na +. The systems A and ASC represent the Na +-dependent transport routes, both with almost identical high affinity for glutamine ( K m=0.26 mM). Similar transport systems for glutamine are also found in cerebral cortical neurons, a predominantly GABAergic nerve cell population, and cerebral cortical astrocytes. The glutamine transport properties in granule cells, however, show a series of differences from that of cortical neurons and astrocytes: (1) uptake of glutamine by granule cells is primarily mediated by system A (54%), while contributions by system A in cortical neurons and astrocytes are less than 30%; (2) granule cells exhibit strikingly higher transport efficiency for glutamine ( V max/ K m=20 min −1 for system A as compared to the V max/ K m ratio of 5 min −1 in cortical neurons and astrocytes), and (3) the initial uptake rates and the steady-state accumulation levels of glutamine are two- to threefold higher in granule cells than that of cortical neurons and astrocytes. These results taken together suggest that in accordance with the important need to replenish the neurotransmitter pool of glutamate, glutamatergic neurons exhibit highly efficient transport systems to accumulate glutamine, one of the major precursors of glutamate.