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J Drejer

Publications and source records attributed to J Drejer.

At least 37 records · Page 2Linked to original sources
PubMed1989

Molecular target size analyses of the NMDA-receptor complex in rat cortex.

The molecular weights of different subunits of the NMDA-receptor complex were determined by high-energy radiation inactivation analyses of the binding of [3H]L-glutamate, [3H](3-(+/-)-2-(carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), [3H]N-(1-[2-thienyl]cyclohexyl)3,4-piperidine (TCP) and [3H]glycine to rat cortical membranes. The molecular target sizes of [3H]L-glutamate binding (the recognition site), [3H]TCP binding (the ionophore) and [3H]glycine (a modulatory unit) were similar: 121,000, 118,000 and 115,000 Da, respectively. These results suggest that the three subunits are on the same protein. The molecular weight of [3H]CPP binding was 209,000 Da. This suggests that in order to bind [3H]CPP (a competitive antagonist) with high affinity an additional macromolecule may be associated to the agonist site.

Animals↗
PubMed1989

Glycine reverses the effect of HA-966 on NMDA responses in cultured rat cortical neurons and in chick retina.

The effects of glycine on NMDA antagonism by a series of excitatory amino acid antagonists were tested in two functional in vitro models: NMDA induced [3H]GABA release from cultured mouse cortical neurons and NMDA evoked spreading depression in chick retina. In both models glycine reversed the NMDA antagonism by HA-966. Also NMDA block by kynurenic acid and by DNQX were partly reversed by glycine. However, CNQX, D-APV, ketamine and MK 801 showed the same NMDA antagonism in the absence and presence of glycine.

Animals↗
PubMed1989

Glutamate receptor activation in cultured cerebellar granule cells increases cytosolic free Ca2+ by mobilization of cellular Ca2+ and activation of Ca2+ influx.

The Ca2+ sensitive fluorescent probe, fura-2 has been used to monitor cytosolic free calcium levels in mature primary cultures of cerebellar granule cells during exposure to L-glutamate and other excitatory amino acids: quisqualate (QA) kainate (KA) and N-methyl-D-aspartate (NMDA). Glutamate at micromolar concentrations produced a prompt and dose-related increase in the intracellular concentration of free Ca2+, ([Ca2+]i), whereas QA, KA and NMDA had no effect. This increase was also seen in the absence of extracellular Ca2+, suggesting that L-glutamate promotes mobilization of Ca2+ from intracellular stores. In the presence of extracellular calcium, the elevation of [Ca2+]i was, in part, mediated by an increase in the plasma membrane permeability to Ca2+. This Ca2+ influx was not affected by the Ca2+-channel antagonist l-Verapamil. However, L-Verapamil did block the increase in [Ca2+]i seen after depolarization of the cells with potassium. The Ca2+ response elicited by glutamate was partially blocked by the excitatory amino acid antagonist glutamate diethyl ester (GDEE). Furthermore, glutamate stimulated the formation of inositol mono-, bis-, tris- and tetrakisphosphates (IP1, IP2, IP3, and IP4) suggesting a role for these compounds for the increase in [Ca2+]i.

Animals↗
PubMed1989

Evidence for evoked release of adenosine and glutamate from cultured cerebellar granule cells.

Evoked release of [3H]-D-aspartate which labels the neurotransmitter glutamate pool in cultured cerebellar granule cells was compared with evoked release of adenosine from similar cultures. It was found that both adenosine and [3H]-D-aspartate could be released from the neurons in a calcium dependent manner after depolarization of the cells with either 10-100 microM glutamate or 50 mM KCl. Cultures of cerebellar granule cells treated with 50 microM kainate to eliminate GABAergic neurons behaved in the same way. This together with the observation that cultured astrocytes did not exhibit a calcium dependent, potassium stimulated adenosine release strongly suggest that cerebellar granule cells release adenosine in a neurotransmitter-like fashion together with glutamate which is the classical neurotransmitter of these neurons. Studies of the metabolism of adenosine showed that in the granule cells adenosine is rapidly metabolized to ATP, ADP, and AMP, but in spite of this, adenosine was found to be released preferential to ATP.

Adenosine↗
PubMed1989

Selection of a pure cerebellar granule cell culture by kainate treatment.

Chronic exposure of dissociated cerebellar cultures to 50 microM kainate results in a complete loss of [3H]-GABA release which is a marker of GABAergic interneurons. No loss of granule cells was found and the glutamatergic nature of the granule cells appeared unaltered by the kainate treatment, since evoked release of [3H]-D-aspartate was maintained after kainate exposure. Glial cells in such cultures are virtually eliminated by treatment with an antimitotic such as cytarabine. In consequence a pure culture of cerebellar granule cells virtually free of stellate, basket and glial cells may be obtained by a combined chronic treatment of the cultures with kainate and cytarabine.

Animals↗
PubMed1989

Direct evidence that excitotoxicity in cultured neurons is mediated via N-methyl-D-aspartate (NMDA) as well as non-NMDA receptors.

Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L-glutamate, kainate (KA), N-methyl-D-aspartate (NMDA), or RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA). To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L-aspartic acid beta-hydroxamate was added at 500 microM to the cultures that were exposed to glutamate. Each of these EAAs was able to induce neurotoxicity. It was not possible to reduce or prevent glutamate-induced cytotoxicity by blocking only one of the glutamate receptor subtypes with either the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV) or with one of the specific non-NMDA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented. Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non-NMDA glutamate agonists with no effect on NMDA-induced cell death. Likewise, APV prevented NMDA-induced cell death without affecting the KA- or AMPA-induced neurotoxicity. It is concluded that EAA-dependent neurotoxicity is induced by NMDA as well as non-NMDA receptors.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1989

Glutamate-induced increase in intracellular Ca2+ in cerebral cortex neurons is transient in immature cells but permanent in mature cells.

The free cytosolic Ca2+ concentration ([Ca2+]i) of cultured cerebral cortex neurons was determined using a fluorescent Ca2+ chelator (Fluo-3) after exposure of the neurons to glutamate. Mature neurons (8 days in culture) responded within 45 s to 100 microM glutamate by an increase in [Ca2+]i from 75 to 340 nM, an increase that during the following 6 min of exposure reached 400 nM. This increase in [Ca2+]i could not be reversed by removal of glutamate. In the absence of extracellular CaCl2, only part of the initial, rapid, glutamate-induced increase in [Ca2+]i was observed in these neurons. In contrast to these findings, neurons cultured for only 2 days (immature neurons) exhibited only a small (from 75 to 173 nM) increase in [Ca2+]i after exposure to 100 microM glutamate, and this rapid increase in [Ca2+]i tended to decline on prolonged exposure to glutamate. Moreover, after removal of glutamate, the increase in [Ca2+]i was fully reversible. Pharmacological characterization of the response to glutamate in mature neurons showed that the N-methyl-D-aspartate (NMDA) receptor antagonists phencyclidine and D-2-amino-5-phosphonovalerate phosphonovalerate blocked 75 and 90%, respectively, of the response, whereas the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione had little effect.

Animals↗
PubMed1989

Glutamate-induced 45Ca2+ uptake into immature cerebral cortex neurons shows a distinct pharmacological profile.

Glutamate-induced 45Ca2+ uptake was studied in cerebral cortex neurons cultured for 4 days, i.e., at a developmental stage where the neurons are sensitive to the mixed agonist glutamate but not to the actions of N-methyl-D-aspartate or other excitatory amino acids. Using this experimental approach, allowing the investigation of effects elicited only by glutamate, it was demonstrated that the glutamate-stimulated Ca2+ influx could be completely antagonized by MK-801, phencyclidine, and cyclazocine in the nanomolar range, and by 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate and D-2-amino-5-phosphonopentanoate (APV) in the low micromolar range. However, the glutamate response was unaffected by variations in the Mg2+ concentration in the exposure media. In addition, the two quinoxalinediones 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione were equipotent with APV in blocking the glutamate-stimulated Ca2+ uptake. PK 26124 blocked the response in the high micromolar concentration range. Ketamine and gamma-glutamylaminomethylsulfonate were essentially without effect at concentrations up to 10 microM and 300 microM, respectively. These results may suggest the existence of a glutamate receptor with a pharmacological profile not compatible with the existent classification of glutamate receptor subtypes.

Animals↗
PubMed1988

Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists.

The N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors has been well described as a result of the early appearance of NMDA antagonists, but no potent antagonist for the "non-NMDA" glutamate receptors has been available. Quinoxalinediones have now been found to be potent and competitive antagonists at non-NMDA glutamate receptors. These compounds will be useful in the determination of the structure-activity relations of quisqualate and kainate receptors and the role of such receptors in synaptic transmission in the mammalian brain.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1988

New quinoxalinediones show potent antagonism of quisqualate responses in cultured mouse cortical neurons.

Two new quinoxalines, DNQX (6,7-dinitro-quinoxaline-2,3-dion = FG 9041) and CNQX (6-nitro-7-cyano-quinoxaline-2,3-dion = FG 9065), were tested as antagonists of excitatory amino acid responses in cultured neurons and brain slices. DNQX and CNQX showed potent competitive antagonism of quisqualate and kainate induced [3H]GABA release from cultured mouse cortical neurons with weaker effects on NMDA responses (Schild values for DNQX were 6.2, 5.9 and 5.4, respectively). Also in a model of excitatory amino acid induced 22Na efflux from striatal slices DNQX blocked responses to quisqualate and kainate more effectively than responses to NMDA.

Animals↗
PubMed1988

Energy metabolism in glutamatergic neurons, GABAergic neurons and astrocytes in primary cultures.

Several aspects of energy metabolism (glucose utilization, lactate production, 14CO2 production from labeled glucose, glutamate or pyruvate, oxygen consumption and contents of ATP and phosphocreatine) were measured in cerebellar granule cells (glutamatergic) in primary cultures and compared with corresponding data for cerebral cortical neurons (mainly GABA-ergic) and astrocytes. Cerebellar granule cells and astrocytes were metabolically more active than cerebral cortical neurons. Glutamate which is utilized as a major metabolic fuel as astrocytes and, to a lesser extent, in cerebral cortical neurons, was virtually not oxidized in cerebellar granule cells.

Animals↗
PubMed1988

Chaotropic ions affect the conformation of quisqualate receptors in rat cortical membranes.

Binding of [3H](R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) to quisqualate receptors in the presence of SCN- ions produced curvilinear Scatchard plots. Kinetic investigations of [3H]AMPA binding showed that the curvilinearity cannot be explained by assuming binding to two separate binding sites or by considering it due to cooperative interaction. A more likely explanation is that the quisqualate receptors exist in two states, one with high and one with low affinity for [3H]AMPA. Chaotropic ions change the relaxation constant between the two states.

Animals↗
PubMed1987

Phencyclidine analogues inhibit NMDA-stimulated [3H]GABA release from cultured cortex neurons.

The inhibition of [3H]TCP binding to rat cortical membranes by substances of the PCP/sigma-opiate type was compared with effects of the same compounds on N-methyl-D-aspartate (NMDA) responses in cultured mouse cortex neurons. A new technique of excitatory amino acid-induced [3H]GABA (gamma-amino-butyric acid) release in a continuous superfusion model was used for the purpose. A similar rank order of potencies was found for the substances whether as inhibitors of [3H]TCP binding (MK 801 greater than PCP greater than ketamine-cyclazocine) or as antagonists of NMDA (20 microM)-stimulated [3H]GABA release in the cultured neurons (phencyclidine = MK 801 greater than ketamine greater than cyclazocine).

Animals↗
PubMed1987

Different target sizes of the voltage-dependent Ca2+ channel and the [3H]nitrendipine binding site in rat brain.

High energy irradiation of frozen rat frontal cortex was used to determine the molecular target sizes of the [3H]nitrendipine binding site (93,500 +/- 9,700 daltons and of the voltage-dependent Ca2+ channel (340,500 +/- 34,000 daltons) determined as the K+-induced 45Ca2+ uptake into synaptosomes. The conclusion that could be drawn from the target size analysis was that 45Ca2+ flux occurs via a structure of molecular weight very different from that of the dihydropyridine binding site. Furthermore, it was found that [3H]nitrendipine binding to synaptosomes was not influenced by depolarization with K+.

Animals↗
PubMed1987

Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat.

Local cerebral glucose metabolism (LCMRglc) and local cerebral blood flow (LCBF) were studied following implantation of a microdialysis fiber in rat dorsal hippocampus. Recovery time after implantation varied from 0 to 24 h. All rats showed pronounced disturbances in LCMRglc and LCBF during the first 2 h of implantation. The changes consisted of (a) a general decrease in blood flow and glucose phosphorylation and (b) small areas (spots) around the fiber with increased glucose phosphorylation and decreased blood flow. Animals allowed to recover for 24 h demonstrated a near normalization of LCMRglu and LCBF, and the focal disturbances (spots) of glucose phosphorylation and blood flow disappeared. The slight reduction in blood flow and glucose metabolism at this time must be accepted, because extension of the recovery period beyond 24 h may give interpretation problems due to the developing gliosis.

Animals↗
PubMed1987

Excitatory amino acid-induced release of 3H-GABA from cultured mouse cerebral cortex interneurons.

A newly developed continuous superfusion model was used for studies of 3H-GABA release from cultured mouse cerebral cortex neurons. It was found that a series of excitatory amino acids (EAAs) representing all receptor subtypes evoked Ca2+- dependent release of 3H-GABA from the neurons. Quisqualate was the most potent agonist tested, with an EC50 value of 75 nM. L-Glutamate, N-methyl-D-aspartate (NMDA), and kainate showed EC50 values of 12, 16 and 29 microM, respectively. The EAA-evoked 3H-GABA release could be blocked by a series of EAA antagonists. The highly selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV) was found to block NMDA responses, whereas the nonselective antagonists cis-2,3-piperidine dicarboxylic acid (PDA) and gamma-D-glutamyl-aminomethyl sulphonic acid (GAMS) blocked responses to all agonists. NMDA responses were found to be sensitive to Mg+ blockade. EAA- as well as potassium-induced 3H-GABA release from the neurons could be detected as early as day 5 in culture. However, during the culture period up to 12 d, the responses to K+, quisqualate, and NMDA were increased. The ontogenetic development of binding sites for quisqualate, kainate, and NMDA in mouse cortex was studied using the radioligands 3H-alpha-amino-3-hydroxy-5-methyl-4-isoxasole propionate (3H-AMPA), 3H-kainate, and 3H-L-glutamate, respectively. The development of binding sites for the different EAA-receptor subtypes showed a good correlation with the development of neuronal 3H-GABA release evoked by the excitatory amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1986

Pharmacologically distinct glutamate receptors on cerebellar granule cells.

Cultured cerebellar granule cells were found to exhibit calcium-dependent release of 3H-D-aspartate when stimulated with excitatory amino acids. L-glutamate and L-aspartate were found to be potent stimulators of 3H-D-aspartate release, D-aspartate was weaker and only minor effects were seen with D-glutamate, quisqualate, kainate, N-methyl-D-aspartate (NMDA) and L-alpha-aminoadipate (L-alpha AA). It was also found that only L-glutamate and L-aspartate showed high affinity for the 3H-L-glutamate binding sites on granule cell membranes. Stimulation by L-glutamate of 3H-D-aspartate release could be blocked by various excitatory amino acid antagonists. From the relative potencies of agonists and antagonists on D-aspartate release it is suggested that cerebellar granule cells express functionally active glutamate receptors with pharmacological characteristics different from all known excitatory amino acid receptors.

2-Aminoadipic Acid↗