Biomedical subjects

L Hertz

Publications and source records attributed to L Hertz.

At least 55 records · Page 3Linked to original sources

PubMed1996

Pharmacological and developmental evidence that the potassium-induced stimulation of deoxyglucose uptake in astrocytes is a metabolic manifestation of increased Na(+)-K(+)-ATPase activity.

There is disagreement in the literature whether or not deoxyglucose accumulation, a measure of glycolytic activity, is increased in astrocytes during exposure to elevated concentrations of the potassium ion (K+). In the present work we have confirmed our previous finding that deoxyglucose accumulation in primary cultures of well-differentiated mouse astrocytes shows a robust increase when the K+ concentration in the incubation medium is raised to or above 12 mM. This response is inhibited by ouabain (with a half-maximum effect at approximately 0.1 mM), indicating that it is a metabolic manifestation of the activity of an Na(+)-K(+)-ATPase. The stimulation at this high level of K+ indicates a remarkably low K+ affinity of the Na(+)-K(+)-ATPase involved, enabling it to be activated by above-normal concentrations of K+. At a resting concentration of K+ (5.4 mM), at least one half of the deoxyglucose accumulation is also a reflection of Na(+)-K(+)-ATPase activity, as shown by its susceptibility to inhibition by ouabain. Ouabain has some effect even at a concentration of 0.1 microM, indicating participation of not only the alpha 1 isoform which has a low affinity to ouabain, but also of the alpha 2 isoform, which has a high affinity. The stimulatory effect of elevated K+ is absent in immature astrocytes and only develops after prolonged time in culture. It could not be evoked in a seemingly similar culture of rat astrocytes, which has previously been shown to lack Na(+)-K(+)-ATPase activity as well as the alpha 2 isoform of the ATPase. This isoform has unequivocally been found to be expressed by astrocytes in situ.

Animals↗

PubMed1996

Effects of benzodiazepines on potassium-induced increase in free cytosolic calcium concentration in astrocytes: interactions with nifedipine and the peripheral-type benzodiazepine antagonist PK 11195.

The benzodiazepines diazepam and midazolam at submicromolar concentrations potentiated the increase in free cytosolic calcium concentration in astrocytes in primary cultures evoked by an elevation of the extracellular potassium concentration ([K+]0), but they had little stimulatory effect at normal [K+]0 and none at maximally elevated [K+]0. Nifedipine, an inhibitor of the L-channel, counteracted both the effect of the elevated [K+]0 as such and the benzodiazepine modulation. PK 11195, an antagonist of the peripheral-type benzodiazepine receptor, counteracted the effect of the benzodiazepines, but had no effect on the increase in free cytosolic calcium evoked by the elevated [K+]0.

Animals↗

PubMed1995

Potentiation by K+ of anoxic release of newly synthesized neuronal glutamate.

Glutamate synthesis from [14C]glutamine, release of newly synthesized, labelled glutamate and cell death in primary cultures of the glutamatergic cerebellar granule cell neurones under anoxic conditions were increased by an elevation of the extracellular potassium concentration. Phenylsuccinate, an inhibitor of transmitochondrial transport and hence of glutamate synthesis from glutamine, decreased the potassium-enhanced glutamate synthesis and the release of newly synthesized glutamate and reduced cell death. Since the extracellular concentration of potassium is elevated during brain anoxia and glutamate neurotoxicity is thought to contribute to neuronal cell death under this condition, these observations may be of functional and potentially therapeutic relevance.

Animals↗

PubMed1995

Glycogenolytic response of primary chick and mouse cultures of astrocytes to noradrenaline across development.

Glycogen is the brain's largest energy store and it is mainly localised in astrocytes. Glycogen turnover is extremely rapid in the brain, especially during sudden increased demand when glucose supplies are insufficient. Previous culture studies have reported on the glycogenolytic effect of noradrenaline on 3--4 week-old primary mouse astrocyte cultures. This effect is believed to be mediated by the beta-adrenergic-cAMP signal transduction system. Recent evidence has shown a drop in forebrain glycogen levels at a specific time point during memory formation for a passive avoidance task in the day-old chick. This 'memory-related' glycogenolysis may be initiated by noradrenaline-induced rises in cAMP occurring around this point, but it is unknown whether astrocytic glycogenolysis is is stimulated by noradrenaline in day-old chicks. This question was approached in the present study and it was shown that noradrenaline is capable of stimulating both cAMP formation and glycogen breakdown in chick primary astrocyte cultures at developmental age (10-14 days in culture) comparable to the newborn chick. In contrast, noradrenaline did not have a corresponding glycogenolytic effect on 10-day-old mouse astrocyte cultures (equivalent to the 1-week mouse), although it induced a considerable amount of glycogen breakdown in older cultures (18 and 24-26 days).

Aging↗

PubMed1995

Pharmacological characteristics of potassium-induced, glycogenolysis in astrocytes.

Elevated extracellular concentrations of the potassium ion ([K+]o) stimulate glycogenolysis in primary cultures of mouse astrocytes that have been grown in the presence of dibutyryl cyclic AMP but not in corresponding cultures which have not been treated in this manner. The response is potently inhibited by nifedipine, suggesting that it is evoked by entry of calcium ions through voltage dependent L-channels. The benzodiazepine midazolam, which is known to enhance calcium entry at concentrations of [K+]o causing submaximum calcium entry, increases the glycogenolytic effect by such levels of [K+]o.

Animals↗

PubMed1995

Noradrenaline-induced stimulation of glutamine metabolism in primary cultures of astrocytes.

Effects of noradrenaline and of adrenergic subtype specific agonists on the uptake and metabolism of [14C]glutamine and [14C]glutamate in primary cultures of mouse astrocytes have been investigated. The total uptake of radioactivity from extracellular [14C]glutamine into the cells was enhanced during exposure to 100 microM noradrenaline, isoproterenol, or clonidine. This is partly due to an increased radioactivity in the glutamine pool and partly due to an increased formation of labeled glutamate from glutamine, which had become very marked (66%) after 240 min of incubation. The CO2 formation from labeled glutamine during 4 hr of incubation was enhanced about twofold in the presence of noradrenaline. Ten millimolar amino oxyacetic acid (AOAA), a transamination inhibitor, had no effect on CO2 formation from glutamine, indicating that the formation of alpha-ketoglutarate from glutamate occurs as an oxidative deamination. The stimulation of 14CO2 production from labeled glutamine was at least as large when glucose was deleted from medium, suggesting that the increased 14CO2 formation represents a stimulation of glutamine metabolism as such and is not only a reflection of an increase in oxidative metabolism of glucose and a bidirectional exchange between alpha-ketoglutarate and glutamate. The opposite process, incorporation of radioactivity from labeled glutamate into glutamine, was not enhanced in the presence of noradrenaline. The findings suggest that noradrenaline stimulates the rates of glutamine uptake, glutamate synthesis, and CO2 production from glutamine and thus increases energy supply to astrocytes but has no effect on the opposite reaction, i.e., glutamine formation from glutamate, a reaction of importance for neuronal-astrocyte interations.

Adrenergic alpha-Agonists↗

PubMed1995

Neuroprotective effect of phenylsuccinate, an inhibitor of cytosolic glutamate formation from glutamine, under anoxic conditions but not during exposure to exogenous glutamate.

Phenylsuccinate is an inhibitor of cytosolic glutamate formation from extracellular glutamine in cultured cerebellar granule cell neurons, a glutamatergic preparation. It prevents anoxic cell death in these cells as indicated by decreased lactate dehydrogenase (LDH) release and by the morphological appearance of the cells after the insult. In contrast, it does not prevent neurotoxicity by added glutamate because it is not an antagonist of the glutamate receptor.

Animals↗

PubMed1995

Further evidence that fluoxetine interacts with a 5-HT2C receptor in glial cells.

It is generally believed that the antidepressant drug fluoxetine (Prozac) exerts all its effects by inhibition of serotonin uptake into neurons and an ensuing increase in the extracellular concentration of serotonin. However, these studies have confirmed and expanded our previous observation that fluoxetine on its own exerts agonist effects on astrocytes (a glial cell type), which resemble those exerted by serotonin. Fluoxetine appears to act on a different subtype of receptor (the 5-HT2C receptor [in original terminology the 5-HT1C receptor]) than the one on which micromolar concentrations of serotonin are known to act in astrocytes (the 5-HT2A receptor [in original terminology the 5-HT2 receptor]). However, this study has shown that application of serotonin to these cells stimulates glycogenolysis and causes an increase in free cytosolic concentration of calcium that is not inhibited by the 5-HT2A selective antagonist, ketanserin. Moreover, both effects are pronounced at the low nanomolar level of serotonin and, therefore, by definition, act on the 5-HT2C receptor. The concentration/response correlation is identical for the serotonin effects on free cytosolic calcium concentration and on glycogenolysis. Fluoxetine exerts similar effects, but low nanomolar concentrations have no effect, and the concentration required to obtain half-maximum response is 1-3 microM, a concentration dependence that is consistent with the plasma levels of fluoxetine during treatment with this drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Astrocyte survival in the absence of exogenous substrate: comparison of immature and mature cells.

Astrocyte cultures prepared from newborn mouse neopallium were grown for either one or three weeks (representing, respectively, immature and mature astrocytes) and then exposed to deprivation of substrate (glucose and amino acids) for up to 48 hr. Cultures which had been deprived of metabolic substrates for either 24, 30, 36 or 48 hr were examined for lactate dehydrogenase efflux into the medium (an indicator of cell death) and ATP content. Significant cell death in mature astrocytes began after 30 hr of incubation in the substrate-deprived medium, a time when ATP had fallen to approximately 10% of its initial value. Immature astrocytes survived on a substrate-free medium for 48 hr before there was any indication at all of cell death, and this corresponded to a time when ATP values had fallen to 5% of the initial values. These findings are compared to previous observations during simulated ischemia (substrate deprivation plus anoxia) when (1) there was a faster cell death and (2) cell death occurred at higher ATP levels.

Adenosine Triphosphate↗

PubMed1994

High extracellular potassium concentrations stimulate oxidative metabolism in a glutamatergic neuronal culture and glycolysis in cultured astrocytes but have no stimulatory effect in a GABAergic neuronal culture.

Rates of deoxyglucose accumulation and of CO2 production from [U-14C]glucose, or from [U-14C]lactate or [2-14C]pyruvate (as a determination of tricarboxylic acid (TCA) cycle activity) were determined in primary cultures of either astrocytes, cerebellar granule cell neurons (utilizing glutamate as their transmitter) or cerebral cortical interneurons (utilizing GABA as their transmitter) during control ('resting') conditions and during exposure to an elevated extracellular potassium concentration, mimicking functional activity. The elevation of the extracellular potassium concentration increased the rate of deoxyglucose accumulation, but not of TCA cycle activity in astrocytes and both deoxyglucose accumulation and TCA cycle activity in cerebellar granule cells, but had no stimulatory effect in cerebral cortical neurons. Based on these observations it is suggested that the increase in energy metabolism in the CNS in vivo during functional activity mainly reflects increased active accumulation of potassium ions and extrusion of sodium ions in neurons receiving excitatory input and in adjacent astrocytes in order to re-establish pre-stimulus ion distribution across cell membranes.

Animals↗

PubMed1994

Correlation between content of high-energy phosphates and hypoxic-ischemic damage in immature and mature astrocytes.

The effect of 'simulated ischemia', i.e., combined anoxia and substrate deprivation, was studied in 1- and 3-week-old (i.e., immature and mature) primary cultures of mouse astrocytes. Cell survival, as indicated by retention of the high-molecular cytosolic protein lactate dehydrogenase was compared with retained high-energy phosphate compounds (ATP and phosphocreatine). A previously established longer survival of the immature cells during the metabolic insult was confirmed and found to correlate with a more complete maintenance of high-energy phosphates. However, in both the mature and immature cells, no death occurred as long as the ATP content remained at or above 25% of its control value. ATP concentrations below 10% of control were accompanied by almost complete cell death in both age groups. Thus, the better survival of immature astrocytes during simulated ischemia is correlated with better maintenance of the levels of high-energy phosphates and, regardless of age, cell death occurs only once a critically 'low' threshold of ATP has been reached.

Adenosine Triphosphate↗

PubMed1994

Effect of anoxia on glutamate formation from glutamine in cultured neurons: dependence on neuronal subtype.

Synthesis and release of glutamate formed from labeled glutamine were studied in primary cultures of the glutamatergic cerebellar granule cells and of the mainly GABAergic cerebral cortical neurons under anoxic conditions and under normoxic control conditions. Under both control and anoxic conditions cerebellar granule cells synthesized and released glutamate more intensely than cerebral cortical neurons, but this difference was enhanced under anoxic conditions. Thus, under normoxic conditions synthesis of intracellular labeled glutamate from glutamine was twice as high in cerebellar granule cell neurons as in cerebral cortical neurons during 30 min of incubation, but the release of newly synthesized labeled glutamate to the extracellular medium from cerebellar granule cell neurons was more than 4 times higher than the release from cerebral cortical neurons during 30 min of incubation. Based on these observations it is suggested that a major reason for the increase in extracellular glutamate concentration during brain ischemia may be enhanced production and release of glutamate, especially in glutamatergic neurons.

Animals↗

PubMed1994

Alteration in oxidative metabolism of alanine in cerebellar granule cell cultures as a consequence of the development of the ability to utilize alanine as an amino group donor for synthesis of transmitter glutamate.

Formation of 14CO2 from labeled alanine was measured in cultured cerebellar granule cells grown in the combined presence of alanine, alpha-ketoglutarate and glutamine or in the presence of glutamine alone. This was done in order to study whether the utilization of alpha-ketoglutarate plus alanine as precursors of transmitter glutamate, induced by culturing in the presence of these compounds, is reflected by an increase of CO2 production from alanine during stimulation with an elevated extracellular potassium concentration. Potassium stimulated CO2 production from alanine was present only in the cells grown in the combined presence of alanine, alpha-ketoglutarate and glutamine. This stimulation was abolished by glutamine, but not by ouabain, indicating that the depolarizing-induced stimulation of alanine metabolism is a consequence of increased release of transmitter glutamate formed from alanine, not a simple result of an increased metabolic rate.

Alanine↗

PubMed1994

Astrocytic glycogenolysis energizes memory processes in neonate chicks.

In previous pharmaco-behavioural experiments, we have shown that three sequential stages can be distinguished in discrimination memory for a single trial passive avoidance experience in neonate chicks: a short-term (STM) stage, available for 10 min following learning; an intermediate (ITM) stage, operating between 20 and 50 min (ITMB) post-learning; and a long-term (LTM) stage formed by 60 min after learning. The ITM stage can be divided into two parts: a first phase (ITMA) which is susceptible to inhibition by the uncoupler of oxidative phosphorylation (and thus of oxidative metabolism), 2,4-dinitrophenol (DNP), and a second DNP-insensitive phase (ITMB). ITMA occurs between 20 and 30 min post-training and ITMB between 30 and 50 min. In the present study we have shown: (1) that day-old chicks trained in the passive avoidance task and immediately thereafter injected with the glycolytic inhibitor iodoacetate show retention deficits that are first evident 30 min post-training, and (2) that glycogenolysis, i.e. breakdown of glycogen, a high-molecular carbohydrate energy store localized in astrocytes, occurs in the forebrains of trained, but otherwise untreated birds, between 35 and 55 min after learning. These findings strongly suggest that glycolysis, including astrocytically localized glycogenolysis, is essential to provide energy for active processes occurring during ITMB and that these processes are indispensable for subsequent development of long-term memory.

2,4-Dinitrophenol↗

PubMed1994

Cell death in primary cultures of mouse neurons and astrocytes during exposure to and 'recovery' from hypoxia, substrate deprivation and simulated ischemia.

Effects of hypoxia, substrate deprivation and simulated ischemia (combined hypoxia and substrate deprivation) on cell survival during the insult itself and during a 24 h 'recovery' period were studied in primary cultures of mouse astrocytes and in cerebral cortical neuronal-astrocytic co-cultures. Cell death was determined by release of the cytosolic high molecular enzyme lactate dehydrogenase (LDH) as well as morphologically (retention of staining with rhodamine 123 and lack of staining with propidium iodide as an indicator of live cells). Glutamate concentrations were measured in the incubation media at the end of the metabolic insults. Astrocytes were very resistant to hypoxia, but less so to simulated ischemia; under both conditions the glutamate concentrations in the media remained low. Cerebral cortical neurons were almost equally susceptible to damage by hypoxia and by simulated ischemia, although hypoxia had a faster deleterious effects on some of the neurons and simulated ischemia during a long-term insult (9 h) killed all neurons, whereas a non-negligible neuronal subpopulation survived 9 h of hypoxia. Neuronal cell death after long-term hypoxia (but not after simulated ischemia) was correlated with high concentrations of glutamate in the incubation media. After certain insults, most notably relatively short lasting simulated ischemia (3 h) in neurons (which caused no increased cell death during the insult), there was a large release of LDH during the 'recovery' period.

Analysis of Variance↗

PubMed1994

Clonidine enhances astrocytic glutamine uptake by authentic alpha 2-adrenoceptor stimulation.

The alpha 2-adrenergic agonist clonidine stimulates glutamine uptake and metabolism in primary cultures of mouse astrocytes. This is important because glutamine is a metabolic substrate for these cells. The alpha 2-antagonist yohimbine inhibits the clonidine stimulation, although not completely. The residual effect in the presence of yohimbine can be explained by the fact that this drug is also a serotonin agonist. Idazoxan, which specifically inhibits imidazoline preferring receptors, exerts no inhibition of glutamine uptake. These results suggest that the stimulatory effect of clonidine on glutamine accumulation in mouse astrocytes is due to its effect at an authentic alpha 2-adrenergic receptor site, not at the imidazoline preferring receptor site.

Adrenergic alpha-Agonists↗

PubMed1994

Effects of chronic exposure to ammonia on glutamate and glutamine interconversion and compartmentation in homogeneous primary cultures of mouse astrocytes.

Accumulation of radioactivity was studied in primary cultures of mouse astrocytes as a function of time of exposure (4-60 min) to 50 microM glutamate and 200 microM glutamine (initial concentrations), of which either glutamate or glutamine was 14C-labeled. Both the glutamate pool and the glutamine pool were compartmentalized. Initially, by far the major intracellular glutamate pool (> or = 90%) was derived from extracellular glutamate and could be converted to glutamine. This allowed a rather accurate determination of metabolic flux from glutamate to glutamine, which under control conditions amounted to 2.0-2.2 nmol/min per mg protein. After chronic exposure to 3 mM ammonia for 3 days this flux was significantly increased to 3.1-3.6 nmol/min per mg protein. Acute exposure to ammonia caused a smaller, apparent increase, which was not statistically significant. The glutamine content was compartmentalized at all stages of the incubation. It consisted of at least two different pools. One of these was accessible to extracellular glutamine and could be converted to intracellular glutamate (constituting a sizeable fraction of the total glutamate pool after longer incubation), whereas the other constituted endogenously derived glutamine, formed from accumulated glutamate. The specific activity of the precursor pool for glutamate synthesis could not be accurately determined and relatively exact fluxes therefore not be calculated. There was, however, no evidence that chronic exposure to ammonia decreases the rate of glutamine hydrolysis.

Ammonia↗

PubMed1994

Uptake, release, and metabolism of citrate in neurons and astrocytes in primary cultures.

Synthesis, uptake, release, and oxidative metabolism of citrate were investigated in neurons and astrocytes cultured from cerebral cortex or cerebellum. In addition, the possible role of citrate as a donor of the carbon skeleton for biosynthesis of neurotransmitter glutamate was studied. All cell types expressed the enzyme citrate synthase at a high activity, the cerebellar granule neurons containing the enzyme at a higher activity than that found in the astrocytes from the two brain regions or the cortical neurons. Saturable citrate uptake could not be detected in any of the cell types, but the astrocytes, and, in particular, those of cerebellar origin, had a very active de novo synthesis and release of citrate (approximately 70 nmol x h-1 x mg of protein-1). The rate of release of citrate from neurons was < 5% of this value. Using [14C]citrate it could be shown that citrate was oxidatively metabolized to 14CO2 at a modest rate (approximately 1 nmol x h-1 x mg-1 of protein) with slightly higher rates in astrocytes compared with neurons. Experiments designed to investigate the ability of exogenously supplied citrate to serve as a precursor for synthesis of transmitter glutamate in cerebellar granule neurons failed to demonstrate this. Rather than citrate serving this purpose it may be suggested that astrocytically released citrate may regulate the extracellular concentration of Ca2+ and Mg2+ by chelation, thereby modulating neuronal excitability.

Animals↗