Angus Grieve

L-TRANs-Pyrrolidine-2,4-dicarboxylate and cis-1-aminocyclobutane-1, 3-dicarboxylate behave as transportable, competitive inhibitors of the high-affinity glutamate transporters

The ability of two conformationally restricted analogues of L-glutamate to function as non-transportable inhibitors of plasma membrane L-glutamate transport was investigated in primary cultures of cerebellar granule cells and cortical astrocytes. L-trans-Pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) and cis-1-aminocyclobutane-1,3-dicarboxylic acid (cis-ACBD) behaved as linear competitive inhibitors of the uptake of D-[3H]aspartate (used as a non-metabolizable analogue of L-glutamate) exhibiting Ki values between 40 and 145 microM; L-trans-PDC being the more potent inhibitor in each preparation. However, both L-trans-PDC and cis-ACBD, over a concentration range of 1 microM-5 mM, dose-dependently stimulated the release of exogenously supplied D-[3H]aspartate from granule cells maintained in a continuous superfusion system. The stimulated release was independent of extracellular calcium ions; essentially superimposable dose-response profiles being obtained in the absence and presence of 1.3 mM CaCl2 and yielding EC50 values of 16-25 microM and 180-220 microM for L-trans-PDC and cis-ACBD, respectively. Stimulated release of D-[3H]aspartate was unaffected by either 300 microM D-(-)-2-amino-5-phosphonopentanoic acid [D-APV; a selective antagonist of the N-methyl-D-aspartate (NMDA) receptor] or by 25 microM 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX; a selective antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor]. The release of D-[3H]-aspartate following stimulation by either L-trans-PDC or cis-ACBD was however markedly attenuated following substitution in the superfusion medium of sodium ions by choline ions. Taken together, these results support an action of L-trans-PDC and cis-ACBD consistent with that of being competitive substrates rather than non-transportable blockers of the plasma membrane L-glutamate uptake system.

Differential Changes in the Content of Amino Acid Neurotransmitters in Discrete Regions of the Rat Brain Prior to the Onset and During the Course of Homocysteine-Induced Seizures

Abstract: Changes in amino acid concentrations were investigated in selected regions of rat brain prior to the onset and during the course of epileptiform seizures induced by l‐homocysteine. The concentration of γ‐aminobutyric acid (GABA) decreased preictally in substantia nigra (‐ 18%), caudate putamen (‐ 26%), and inferior colliculus (‐ 46%). After seizure onset, the GABA content was further reduced in substantia nigra (‐ 31%) and additionally in hippocampus (‐ 18%). Preictal taurine levels were elevated in globus pallidus (+ 26%) and caudate putamen (+ 13%) but returned to normal after seizure onset. However, in hippocampus, taurine decreased both preictally (‐ 22%) and after seizure onset (‐ 56%). Glycine was reduced preictally only in globus pallidus (‐ 13%). After seizure onset the direction of its concentration change varied in the brain regions studied. Glutamate levels decreased preictally in hippocampus (‐ 10%) and hypothalamus (‐ 46%) but increased in globus pallidus (+ 14%). Normal levels were detectable after seizure onset in hypothalamus and globus pallidus but a further reduction in hippocampus (‐ 59%) and significant reductions in substantia nigra (‐ 15%) and caudate putamen (‐ 17%) were detected. Aspartate was elevated in hippocampus, both preictally (+ 49%) and after seizure onset (+ 21%) while at the same phases in globus pallidus a consistent reduction (‐ 30%) was observed. The glutamine content increased preictally in globus pallidus (+ 41%) and hypothalamus (+ 36%), and in all brain areas during the ictal phase of seizure, the hippocampus exhibiting a dramatic increase (∼300%). The contents of serine and alanine were altered in most regions studied only after seizure onset, with the exception of the hippocampus, where a decrease (‐ 41%) of serine was observed preictally.

Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[3H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (Km) ford-aspartate uptake being 6 μM, 21 μM and 84 μM, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (Ki∼Km) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (Ki>10–1000 times the appropriateKm value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.

Stimulation of γ-[3H]Aminobutyric Acid Release from Cultured Mouse Cerebral Cortex Neurons by Sulphur-Containing Excitatory Amino Acid Transmitter Candidates: Receptor Activation Mediates Two Distinct Mechanisms of Release

In primary cultures of mouse cerebral cortex neurons, sulphur‐containing excitatory amino acids (SAAs; namely, L‐cysteine sulphinate, L‐cysteate, L‐homocysteine sulphinate, L‐homocysteate, S‐sulphocysteine) at concentrations ranging from 0.1 μM to 1 mM evoked a saturable release of ‐γ‐[3H] aminobutyric acid ([3H] GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50), 100–150 nM) in releasing [3H] GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H] tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA‐stimulated release of [3H] GABA was shown to comprise two distinct components, calcium‐dependent and calcium‐independent, which occur after activation of N‐methyl‐B‐aspartate (NMDA) and non‐NMDA receptors. Thus, all SAA‐evoked responses were antagonized by the selective, competitive NMDA‐receptor antagonist, 3‐[(±)‐2‐carboxypiperazin‐4‐yl] propyl‐1‐phosphonic acid (IC50range, >50 μM) and the non‐NMDA‐receptor antagonist, 6, 7‐dinitroquinoxalinedione (IC50 range, 5–50 nM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA‐evoked responses without having any effect on basal levels of [3H] GABA efflux, a result consistent with an involvement of NMDA‐receptor activation. Calcium‐independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 pMexcitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 fiMSKF‐89976‐A, a non‐ transportable inhibitor of theGABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are dearly consistent with a receptor‐mediated, depolarization‐induced reversal of the GABA carrier.

Sulphur-containing excitatory amino acid-evoked Ca2+-independent release of d-[3H]aspartate from cultured cerebellar granule cells: The role of glutamate receptor activation coupled to reversal of the acidic amino acid plasma membrane carrier

Sulphur-containing excitatory amino acid transmitter candidates (500 microM) stimulated the Ca(2+)-independent efflux of exogenously-supplied D-[3H]aspartate from primary cultures of cerebellar granule cells superfused continuously with HEPES-buffered saline containing CoCl2 (1 mM) in place of CaCl2. The stimulated release of D-[3H]aspartate was markedly attenuated by 200 microM 6,7-dinitroquinoxalinedione, a concentration at which the antagonist inhibits both non-N-methyl-D-aspartate and N-methyl-D-aspartate ionotropic excitatory amino acid receptors. The Ca(2+)-independent component of evoked release was also markedly attenuated and, in some cases, abolished by removing NaCl from the superfusion medium. Furthermore, when 700 microM dihydrokainate (demonstrated herein as a mixed/non-competitive inhibitor of the high-affinity dicarboxylic amino acid transporter in cultured granule cells) was included in the superfusion medium, stimulated efflux of D-[3H]aspartate was reduced by between 15-78% of the control response; the extent of inhibition varying with the agonist employed. In constrast, agents which act as competitive inhibitors of the plasma membrane carrier in granule cells, e.g. beta-methylene-D,L-aspartate, potentiated the release of D-[3H]aspartate in a synergistic manner. Taken together, these findings are consistent with a mechanism for the Ca(2+)-independent release of D-[3H]aspartate that is mediated predominantly by activation of excitatory amino acid receptors resulting in a reversal of the high-affinity dicarboxylic amino acid transport system. Although the physiological relevance of such non-vesicular release from the cytosol remains obscure and is still a matter of some debate, this mode of release may be of pathological significance.

Neuroactive Sulphur Amino Acids Evoke a Calcium-Dependent Transmitter Release from Cultured Neurones That Is Sensitive to Excitatory Amino Acid Receptor Antagonists

Abstract: A dose‐dependent, saturable, and calcium‐dependent release of γ‐[3H]aminobutyrate ([3H]GABA) from cortical neurones and D‐[3H]aspartate from cerebellar granule cells following stimulation by a range of l‐enantiomers of neuroactive acidic sulphur amino acids has been demonstrated. Moreover, the sulphur amino acid‐evoked release of the transmitter amino acids was found to be sensitive to the presence of both selective N‐methyl‐D‐aspartate and quisqualate/kainate receptor antagonists. Following the recent demonstration of an endogenous location for several of the acidic sulphur amino acids and their excitotoxic involvement in several neuropathological states and coupled with the knowledge that many important CNS connections are still undefined as far as their excitatory transmitter or transmitters are concerned, the present findings are of immediate importance in the continued search for endogenous excitatory amino acid agonists in addition to glutamate and aspartate.

A Prototypic Intracellular Calcium Antagonist, TMB‐8, Protects Cultured Cerebellar Granule Cells Against the Delayed, Calcium‐Dependent Component of Glutamate Neurotoxicity

Abstract: The effect(s) of a prototypic intracellular Ca2+ antagonist, 8‐(N,N‐diethylamino)octyl‐3,4,5‐trimethoxybenzoate (TMB‐8), on glutamate‐induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free‐Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate‐induced toxicity, quantified by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an “early” Na+/Cl−‐dependent component observed within minutes of glutamate exposure, and a “delayed” Ca2+‐dependent component (ED50∼50 µM) that coincided with progressive degeneration of granule cells 4–24 h after a brief (5–15 min) exposure to 100 µM glutamate. Quantitative analysis of cell viability and morphological observations identify a “window” in which TMB‐8 (at >100 µM) protects granule cells from the Ca2+‐dependent, but not the Na+/Cl−‐dependent, component of glutamate‐induced neurotoxic damage, and furthermore, where TMB‐8 inhibits glutamate‐evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB‐8‐sensitive intracellular store may be a necessary step in the onset of glutamate‐induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB‐8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate‐evoked increases in [Ca2+]i and subsequently effect a pronounced time‐dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate‐evoked increases in [Ca2+]i and glutamate‐induced neurotoxicity.

Calcium influx via L‐type voltage‐gated channels mediates the delayed, elevated increases in steady‐state c‐fos mRNA levels in cerebellar granule cells exposed to excitotoxic levels of glutamate

The altered kinetics of steady‐state c‐fos mRNA production in cultured cerebellar granule cells under excitotoxic conditions was investigated in neurons subjected to depolarising stimuli, namely, high KCl and L‐glutamate (Glu), in which Ca2+ influx occurs by differing routes. Increases in intracellular‐free calcium levels ([Ca2+]i) stimulated by nontoxic or toxic levels of Glu were blocked by selective N‐methyl‐D‐aspartate (NMDA) receptor antagonism; were blocked only partially by the L‐type channel blocker, nifedipine; and were unaffected by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)/kainate receptor antagonists. Glu‐induced cell death was prevented only by NMDA receptor blockade. Exposure of cells to nontoxic levels of Glu resulted in a transient increase in c‐fos mRNA levels, whereas an excitotoxic dose produced a delay in the appearance of c‐fos mRNA but a subsequent, progressive, and sustained (>4 hr) increase. An excitotoxic dose of Glu in combination with either nifedipine or selective NMDA receptor antagonists resulted in the normal, transient increase of c‐fos mRNA levels. Chronic exposure to 55 mM KCl caused no cytotoxicity, although it resulted in a delayed, elevated increase in c‐fos mRNA levels that was unaffected by NMDA receptor blockade but reverted to the normal, transient profile of c‐fos mRNA formation when it was coadministered with nifedipine. The KCl‐induced increase in [Ca2+]i levels was inhibited dramatically by nifedipine but was unaffected by any of the ionotropic Glu receptor antagonists. The results support the notion that the appearance of a delayed but elevated increase in steady‐state c‐fos mRNA levels following exposure to excitotoxic doses of Glu is mediated specifically by calcium influx via L‐type voltage‐gated channels. J. Neurosci. Res. 52:641–652, 1998.

NMDA receptor‐mediated cGMP synthesis in primary cultures of mouse cerebellar granule cells appears to involve neuron‐astrocyte communication with NO operating as the intercellular messenger

The possibility that neuron‐astrocyte communication may be responsible for glutamate (Glu)‐stimulated cGMP formation even in relatively homogeneous primary cultures of mouse cerebellar granule cells (7 days in vitro) was investigated. Pharmacological analysis using selective excitatory amino acid (EAA) receptor antagonists showed that cGMP production, stimulated in these cultures by Glu and a variety of endogenous EAAs structurally‐related to Glu (namely, L‐aspartate, L‐cysteine sulphinate, L‐homocysteate, S‐sulpho‐L‐cysteine), was mediated wholly by N‐methyl‐D‐aspartate (NMDA) receptor activation. Moreover, EAA‐induced responses were dependent on the presence of extracellular calcium but unaffected by addition of the L‐type voltage‐sensitive calcium channel blockers nifedipine (10 μM) or verapamil (5 μM). The mode of calcium entry was also shown to be important since the calcium ionophore, Λ23187 (10 μM), was unable to stimulate cGMP levels above basal. cGMP formation was blocked by the competitive nitric oxide synthase inhibitor, L‐NG‐nitroarginine (100 μM), consistent with a role of nitric oxide (NO) in this signalling pathway. In the presence of added haemoglobin (1 μM), acting as a membrane‐impermeable NO scavenger, Glu‐stimulated cGMP formation was abolished implying that NO must act as an intercellular messenger. When the neuronal population was destroyed following a 24 hr exposure to the excitotoxin, S‐sulpho‐L‐cysteine (200 μM), Glu‐stimulated cGMP formation was abolished; whereas responses to the NO donor, sodium nitroprusside (SNP), although markedly reduced were still double that stimulated by Glu in the absence of the excitotoxin, suggesting the presence of non‐neuronal cells that can generate cGMP if supplied directly with NO. Consistent with this suggestion, low levels of the glial specific enzyme, glutamine synthetase, were detected in granule cell cultures. Furthermore, omission or delayed addition of the antimitotic agent, cytosine arabinoside (20 μM), to the growth medium caused a significant increase in the level of Glu‐stimulated cGMP formation.

Kinetic characterization of sulphur-containing excitatory amino acid uptake in primary cultures of neurons and astrocytes

Abstract The uptake of the neuroactive sulphur amino acids l -cysteine sulphinate, l -cysteate, l -homocysteine sulphinate and l -homocysteate was investigated in astrocytes cultured from the prefrontal cortex; in neurons, cultured from cerebral cortex; and, in granule cells, cultured from cerebellum. It was shown that each amino acid acted as a substrate for a plasma membrane transporter in both neurons and astrocytes. Astrocytes and neurons exhibited a high-affinity uptake for l -cysteine sulphinate and l -cysteate with K m values ranging from 14–100 μM, and a low-affinity uptake for l -homocysteine sulphinate and l -homocysteate, with K m values ranging from 225–1210 μM. The uptake of all transmitter candidates studied was partially sodium-dependent. This sodium-dependency was most evident at low ( μ M) concentrations of each substrate. The apparent uptake measured in the absence of sodium was included as a component in corrections made for non-saturable influx. With the exception of l -cysteine sulphinate, uptake of each sulphur amino acid was greatest in astrocytes, with V max values ranging between 15–32 nmol min −1 mg −1 cell protein. Moreover, the uptake of each sulphur amino acid in cerebellar granule cells ( V max values ranging between 10–25 nmol min −1 mg −1 cell protein) was consistently greater than that in cerebral cortex neurons ( V max values ranging between 1.5–6 nmol min −1 mg −1 cell protein).