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Technology Update Reservoirs consisting of heterogeneous carbonates and shaly sands pose formation evaluation challenges for conventional logging-while-drilling (LWD) measurements. Widely used resistivity, density, neutron porosity, and sonic measurements are all sensitive to both the formation matrix and the fluids, which may create ambiguities in the evaluation of reservoir fluids. The Schlumberger proVision Plus magnetic resonance-while-drilling (MRWD) technology offers enhanced evaluation capabilities. By manipulating the hydrogen atoms in the fluids, nuclear magnetic resonance (NMR) methods provide a focused measurement of fluid properties and their interactions with the surrounding matrix at a fundamental level. The technology provides a more complete, real-time assessment of producibility in complex reservoirs. Evolution of the Technology The industry has used NMR technology for formation evaluation for more than 25 years, during which significant technical and operational developments have greatly enhanced its capabilities and applications. In porous media, NMR techniques reveal pore and fluid properties by exciting the nuclear states of hydrogen-containing molecules in the formation fluids and measuring the relaxation times for the induced excited energy states. The relaxation time for a molecule near a pore wall surface tends to be much faster than that for a molecule in the bulk fluid volume. A distribution of all the relaxation times, known as a T2 distribution, can be used to derive both irreducible and producible fluid volumes, and an improved estimation of permeability. These measurements are also important for identifying bypassed pay zones and estimating production rates. The first NMR tools were deployed on wireline after the hole had been drilled, which added rig time and cost to the operation and did not allow for real-time changes to the wellbore’s trajectory. Schlumberger shifted the service from wireline to a while-drilling application in 2002. This early work focused on redesigning the sensor array to make it more robust and capable of withstanding dynamic drilling environments. The design effort culminated with the introduction of the company’s latest MRWD system in 2012. Its new sensor makes reliable measurements under rapidly changing downhole temperatures and pressures and withstands the vibrations and drillstring shocks that are common during drilling. Further design enhancements allow the tool to be placed anywhere within the bottomhole assembly (BHA). A sleeve stabilizer minimizes tool motion without affecting the steerability of the BHA. Powered by a downhole electrical turbine, the tool does not need to be tripped to replace batteries.

The transport kinetics of γ-aminobutyric acid (GABA), taurine, and β-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (KmGABA= 24.9 ± 1.7 μM; KmTau= 20.0 ± 3.3 μM; Kmβ-Ala= 73.0 ± 3.6 μM) and astrocytes (KmGABA= 31.4 ± 2.9 μM, KmTau= 24.7 ± 1.3 μM; Kmβ-Ala= 70.8 ± 3.6 μM). The maximal uptake rates (Vmax) determined were such that, in neurons, VmaxGABA> Vmaxβ-Ala=VmaxTau, whereas in astrocytes, Vmaxβ-Ala > VmaxTau=VmaxGABA. Taurine was found to inhibit β-alanine uptake into neurons and astro cytes in a competitive manner, with Ki values of 217 μM in neurons and 24 μM in astrocytes. β-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 μM in neurons and 71 μM in astrocytes. However, β-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of β-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons. Taken together, the similarity between the results obtained for β-alanine and taurine uptake in addition to their competitive mode of mutual inhibition strongly suggest that these compounds share a common carrier in neurons and astrocytes and that neither amino acid in either cell type is transported appreciably, if at all, by the GABA carrier, and vice versa.

Young Technology Showcase Today’s drilling engineering teams are continually looking for better ways to mitigate risk and improve health, safety, and environmental conditions at the rig site. Many operators would prefer to acquire a comprehensive suite of petrophysical measurements while drilling—without incurring the risk and cost associated with deployment of chemical nuclear sources. Traditionally, both wireline and logging-while-drilling (LWD) tools have depended primarily on radioisotopes such as americium-beryllium to generate high-energy neutrons for porosity measurements, and cesium to generate gamma rays for bulk density measurements. However, transportation, storage, handling on the rig floor, and potential abandonment of radioactive sources at the wellsite should the tools become stuck require special safety precautions and costly, time-consuming procedures. In remote geographic regions, mobilizing chemical sources can be slow and logistically complex. If a bottomhole assembly (BHA) with a chemical source becomes stuck, complicated fishing procedures can waste precious rig time, and delay eventual production of oil and gas. To meet the growing industry need for an alternative to radioactive chemical sources, Schlumberger recently released NeoScope sourceless formation evaluation-while-drilling solution (Fig. 1). This technology is unique in three ways. It is the first LWD tool to introduce a completely new nuclear measurement to the industry—sourceless neutron gamma density (SNGD). It is the only tool in the industry that provides formation density and neutron measurements without the need for chemical nuclear sources. Finally, it is the first tool to provide collocated density and neutron measurements (Fig. 2). Development and Field Testing LWD tools have typically used two chemical nuclear sources to obtain neutron porosity and bulk density. In partnership with Japan Oil, Gas, and Metals National Corporation, Schlumberger first developed and deployed a pulsed neutron generator (PNG) for commercial standard neutron porosity, spectroscopy, and sigma measurements 7 years ago, eliminating the need for one of the chemical sources (241AmBe). However, development of SNGD was not yet mature enough for commercial use. The only way to acquire a reliable density measurement was still to use a cesium source. Hence, it remained impossible for operators to obtain a comprehensive sourceless suite of petrophysical data while drilling. Ongoing research and engineering led to modifications of existing technology, design of new detectors, and development of sophisticated characterization algorithms and calibrations to provide the first commercial SNGD measurement—a true breakthrough. Since 2005, extensive laboratory modeling and experimentation, as well as more than 200 field tests in more than 30 countries have been conducted to verify and refine the new SNGD measurement. Tests have been carried out in a wide range of formations under many different environmental conditions. Comparisons of the new PNG-based density measurement with conventional bulk densities derived from chemical sources showed good correlations both in carbonates and sandstone formations, onshore and offshore (Fig. 3). Field testing extended through early 2012, and the commercial solution was officially released in June.

Quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR) measurements were made to investigate the levels of c-fos mRNA as one measure of the expression of the c-fos gene. Exposure of mouse cerebellar granule cells to excitotoxic concentrations of glutamate (Glu) and aspartate (Asp) led to a changed time profile for mRNA expression, from a transient c-fos expression at 15-30 min to a delayed, elevated and sustained expression at later time points which was prevented by selective antagonism of the NMDA receptor but not of the AMPA/kainate receptor demonstrating that this c-fos induction was mediated through the specific activation of the NMDA Glu receptor subtype. The question as to whether c-fos expression changes could be used to predict excitotoxicity was addressed by testing the c-fos response of the cultures to several compounds, at low (and therefore non-toxic) and high (toxic) concentrations at two suitable time-points of exposure (30 and 240 min), in the presence and absence of Glu receptor antagonists. The compounds were divided into four groups, excitotoxins, neurotoxic but non-excitotoxic compounds, neuroactive but non-toxic compounds, and compounds that were toxic to other target organelles. The results of this study, using real-time RT-PCR, support the proposal that c-fos mRNA can be used as a specific biomarker of excitotoxicity and moreover encourage further studies to employ this highly sensitive, quantifiable and reproducible technique in a high throughput screen, with minimal use of animals for primary culture set-up. Furthermore, this test has the potential for application in screening newly-designed excitatory amino acid receptor antagonists in the search for clinically relevant drugs to treat a variety of neuropathologies.

The synthesis and pharmacological characterisation of (S)-CPW 399 as a novel, potent and subtype-selective agonist of the AMPA receptor was recently reported. Studies have been extended to investigate its excitotoxic action in primary cultures of mouse cerebellar granule cells. (S)-CPW 399 induced neuronal cell death in a time- and concentration-dependent manner (EC(50) approximately 70 microM) at 24-h exposure. (S)-CPW-induced neuronal death could be prevented by co-administration with either of the AMPA/kainate selective receptor antagonists 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) or by the 2,3-benzodiazepine, GYKI 53655 (a selective AMPA receptor antagonist); while no protection was afforded by either the NMDA receptor antagonist D,L(+/-)-2-amino-5-phosphonopentanoate (APV) or by nifedipine (an L-type calcium channel antagonist) when used alone or in combination. Cyclothiazide, which blocks AMPA receptor desensitisation, caused minimal potentiation of (S)-CPW 399-induced neuronal death, supporting accumulating evidence that (S)-CPW 399 is a full AMPA receptor agonist that markedly prevents a receptor desensitised conformation. (S)-AMPA, (S)-willardiine (a naturally-occurring heterocyclic excitatory amino acid) and its halogenated derivative, (S)-5-fluorowillardiine, had no deleterious effect on neuronal viability when used alone but each, in the presence of cyclothiazide, induced a concentration-dependent excitotoxic cell death with a rank order of potency (fluorowillardiineAMPA=willardiine). (S)-CPW 399 stimulated an increase in intracellular free-calcium levels ([Ca(2+)](i)) in a concentration-dependent fashion (EC(50) approximately 5 microM) attaining a value of six-fold that of 'resting' cells at maximum stimulation; achieved at approximately 100 microM (S)-CPW 399. The (S)-CPW 399-stimulated increase in [Ca(2+)](i) was virtually abolished by GYKI 53655, NBQX, CNQX and by cobalt ions; markedly inhibited by nifedipine and marginally affected by D-APV. These results suggest that (S)-CPW 399 may be used as a pharmacological tool to aid in the investigation of the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitisation.

Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca 2+ , and the demonstration of an increase in phospholipase A 2 (PLA 2 ) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA 2 in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCl inhibited both AA release and the increase in [Ca 2+ ] i triggered by Et-1, whereas valinomycin, which causes K + efflux, not only caused a rapid rise in [Ca 2+ ] i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA 2 in this cell type requires calcium influx dependent on K + efflux.

The effect of ionotropic excitatory amino acids and potassium on the formation of inositol phosphates elicited by the metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was studied in mouse cerebellar granule cells. In Mg(2+)-containing buffers, NMDA (50-100 microM), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 10-1,000 microM), and high potassium (10-30 mM) enhanced synergistically the response to a maximally effective concentration of 500 microM trans-ACPD. Potentiation of the trans-ACPD response was blocked by higher concentrations of NMDA (> 500 microM) and potassium (> 35 mM) but not by AMPA (up to 1 mM). The potentiation by NMDA of the trans-ACPD-stimulated phosphoinositide hydrolysis was blocked by D,L-2-amino-5-phosphonopentanoic acid (APV), a competitive NMDA-receptor antagonist. Under Mg(2+)-free conditions, the accumulation of inositol phosphates in the presence of trans-ACPD alone was equal to that attained by trans-ACPD in Mg(2+)-containing buffers when costimulated with maximally enhancing concentrations of NMDA (50 microM). trans-ACPD potentiated synergistically the NMDA-evoked increases in cytosolic free-Ca2+ levels in Mg(2+)-containing but not in Mg(2+)-free solutions, and moreover did not enhance the AMPA-evoked increases in cytosolic free-Ca2+ levels. The calcium ionophore A23187 caused a dose-dependent increase in inositol phosphate accumulation but did not enhance the response stimulated by trans-ACPD alone. These results demonstrate the existence of cross talk between metabotropic and ionotropic glutamate receptors in cerebellar granule cells. The exact mechanism remains unclear but appears to involve interplay of G protein-coupled phospholipase C activation and regulated elevation of cytosolic free-Ca2+ levels. This study may provide a framework for future investigations at the cellular and molecular level that clarify the functional relevance and molecular mechanisms that are described.

The L- and D-enantiomers of the sulphur-containing amino acids (SAAs)-homocysteate, homocysteine sulphinate, cysteate, cysteine sulphinate, and S-sulphocysteine-stimulated [3H] noradrenaline release from rat hippocampal slices in a concentration-dependent manner. The relative potencies of the L-isomers (EC50 values of 1.05-1.96 mM) were of similar order to that of glutamate (1.56 mM), which was 10-fold lower than that of NMDA (0.15 mM), whereas the D-isomers exhibited a wider range of potencies (0.75 to > 5 mM). All stimulatory effects of the SAAs were significantly inhibited by the voltage-sensitive Na+ channel blocker tetrodotoxin (55-71%) and completely blocked by addition of Mg2+ or Co2- to the incubation medium. All SAA-evoked responses were concentration-dependently antagonized by the selective NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (IC50 values of 3.2 - 49.5 microM). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, at 100 microM inhibited the [3H]noradrenaline release induced by glutamate and NMDA (65 and 76%, respectively) and by all SAAs studied (65-85%), whereas 10 microM CNQX only inhibited the effects of S-sulpho-L-cysteine and L- and D-homocysteate (33, 32, and 44%, respectively). However, the more selective AMPA/kainic acid receptor antagonist 6-nitro-7-sulphamoylbenzo (f) quinoxaline-2,3-dione (100 microM), which did not antagonize the [3H]noradrenaline release induced by glutamate and NMDA, reduced only the S-sulpho-L-cysteine-evoked response (25%). Thus, the stimulation of Ca2(+)-dependent [3H]noradrenaline release from hippocampal slices elicited by the majority of the SAAs appears to be mediated by the NMDA receptor.

The potent excitatory and neurotoxic actions of glutamate are known to influence the expression of a variety of genes, including those encoding the AP-1 transcription factor, which comprises proteins belonging to the Fos and Jun families. However, the precise role of Fos- and Jun-like transcription factors in these events remains elusive. Here we demonstrate, using primary cultures of mouse brain cerebellar granule cells as an in vitro model system, a possible involvement of the FosB/JunD heterodimer in excitotoxicity. Granule cells were grown for either 2 or 7 days in vitro (DIV) before exposure to varying concentrations (1-3000 microM) of the excitotoxin glutamate. In 7-DIV cells, glutamate induced a concentration-dependent neuronal death, whereas, in 2-DIV cells, no glutamate-induced neuronal damage was seen. We were particularly interested in comparing the protein composition of the AP-1 transcription factor complex in cells exposed to excitotoxic and to nontoxic conditions. AP-1 DNA binding activity was demonstrated by gel shift analysis in nuclear extracts derived from 7-DIV cells following exposure to either a nontoxic (10 microM) or an excitotoxic (250 microM) dose of glutamate and was similarly observed in extracts of 2-DIV cells exposed to the same levels of glutamate. Gel supershift analysis using antibodies against the different Fos and Jun family members allowed differentiation between AP-1 DNA binding in nuclear extracts as a function of both 1) viability status and 2) the stage of development. Of major significance was the finding that FosB could be detected as a component of AP-1 in 7-DIV cells only under excitotoxic conditions, whereas c-Fos, Fra-2, and JunD proteins were detectable under both excitotoxic and nontoxic conditions in cells of this age. In 2-DIV cells (in which glutamate is nontoxic), AP-1 comprised combinations of only Fra-1, Fra-2, c-Jun, and JunD. Because Fos family members are unable to form homodimers, this finding raises the possibility that the FosB/JunD heterodimer may have special significance in the mechanism of excitotoxic neuronal death.

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 alpha-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.

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 microM) or verapamil (5 microM). The mode of calcium entry was also shown to be important since the calcium ionophore, A23187 (10 microM), was unable to stimulate cGMP levels above basal. cGMP formation was blocked by the competitive nitric oxide synthase inhibitor, L-NG-nitroarginine (100 microM), consistent with a role of nitric oxide (NO) in this signalling pathway. In the presence of added haemoglobin (1 microM), 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 microM), 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 microM), to the growth medium caused a significant increase in the level of Glu-stimulated cGMP formation.

The stimulatory effect of excitatory sulphur-containing amino acids on inositol phosphate formation was investigated in primary cultures of cerebellar granule cells. L-Cysteine sulphinate (CSA), L-cysteate (CA), L-homocysteine sulphinate (HCSA), L-homocysteate (HCA) and S-sulpho-L-cysteine (SSC) dose-dependently stimulated the formation of [3H]inositol phosphates exhibiting EC50 values in the range 60-200 microM and maximal effects of six- to 17-fold that of basal [3H]inositol phosphate levels. Endogenous L-glutamate spontaneously released into the extracellular medium or following exposure of cells to HCSA, HCA or SSC did not contribute significantly to formation of [3H]inositol phosphates, whereas 10% of the total [3H]inositol phosphates accumulated following exposure to CSA and CA was due to released L-glutamate. The selective N-methyl-D-aspartate receptor antagonist, D,L-2-amino-5-phosphonopentanoic acid (APV, 500 microM) attenuated by 20% (HCSA) to between 80 and 100% (CSA, CA, SSC, HCA) the formation of [3H]inositol phosphates induced by 1 mM sulphur-containing amino acids. When, however, HCSA was used at 100 microM (a concentration near to its EC50 for phosphoinositide hydrolysis), APV inhibited induced responses by 70%. Sulphur-containing amino acid-stimulated [3H]inositol phosphate formation was unaffected by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM). Inhibition of sulphur-containing amino acid-stimulated [3H]inositol phosphate formation by co-administration of APV and CNQX was similar to that obtained in the presence of APV alone. CSA-, CA-, SSC- and HCA-stimulated [3H]inositol phosphate formation was markedly reduced by removal of Ca2+ from the extracellular medium whereas that stimulated by HCSA was less affected. A similar inhibitory profile was observed when the levels of sulphur-containing amino acid-induced increases in intracellular free calcium ([Ca2+]i) were measured in the presence of 500 microM APV; 1 mM HCSA-induced responses being inhibited by only 30% whereas responses to the remaining sulphur-containing amino acid (also at 1 mM) were inhibited by > 45%. When the sulphur-containing amino acids were used at concentrations approximating their EC50 values for phosphoinositide hydrolysis, APV inhibited the induced increases in [Ca2+]i by 70-100%. HCA and SSC co-administered with the less efficacious but selective metabotropic glutamate receptor agonist, (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) at maximally effective concentrations (1 mM) of each agonist stimulated [3H]inositol phosphate formation in an additive manner.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Exposure of mouse cerebellar granule cells to l-glutamate (Glu) at low non-excitotoxic concentrations results in a transient expression of the c-fos proto-oncogene. Higher excitotoxic concentrations of Glu result in a change in the temporal profile of c-fos expression to a delayed but sustained response. We have derived a ratio for the steady-state levels of c-fos mRNA after 30 min and 240 min of exposure to Glu, which suggests that a 240 min/30 min ratio of greater than 1 correlates with excitotoxicity (as measured by elevated LDH release), whereas a ratio of less than 1 correlates with a non-excitotoxic outcome.

The proposed action of β - p -chlorophenylglutamate (chlorpheg) as a selective blocker of l -homocysteate uptake was studied in primary cultures of mouse brain neurons and astrocytes and in rat brain synaptosomes. The preparations were incubated with 1 μM to 10 mM l -homocysteate and d -aspartate in the absence and presence of 7.5 mM chlorpheg. In each preparation uptake of the two amino acids comprised a saturable uptake and a non-saturable (passive diffusion) component. l -Homocysteate was of at least 100-fold lower affinity than d -aspartate as a substrate for the amino acid transporter. Chlorpheg was shown to be essentially equieffective as a weak competitive inhibitor of only the saturable uptake of l -homocysteate and d -aspartate. It is concluded that chlorpheg is not a selective blocker of l -homocysteate uptake.

The transport kinetics of the excitatory sulphur-containing amino acid (SAA) transmitter candidates, L-cysteine sulphinate (L-CSA), L-cysteate (L-CA), L-homocysteine sulphinate (L-HCSA), and L-homocysteate (L-HCA), together with their plasma membrane carrier specificity, was studied in cerebrocortical synaptosome fractions by a sensitive high performance liquid chromatographic assay. A high affinity uptake system could be demonstrated for L-CSA (Km = 57 +/- 6 microM; Vmax = 1.2 +/- 0.1 nmol/min/mg protein) and L-CA (Km = 23 +/- 3 microM; Vmax = 3.6 +/- 0.1 nmol/min/mg protein), whereas L-HCSA (Km = 502 +/- 152 microM; Vmax = 6.1 +/- 1.3 nmol/min/mg protein) and L-HCA (Km = 1550 +/- 169 microM; Vmax = 10.3 +/- 1.1 nmol/min/mg protein) exhibited much lower affinity as transport substrates. In all cases, only a single, saturable Na(+)-dependent component of uptake could be identified, co-existing with a non-saturable, Na(+)-independent influx component. Plasma membrane carrier specificity of the SAAs was established following comparison with other high-affinity neurotransmitter systems. High-affinity L-CSA and L-CA transport and low-affinity L-HCSA and L-HCA transport demonstrate strong positive correlations in inhibition profiles when compared against each other or individually against the high-affinity transport of L-[3H]glutamate, L-[3H]aspartate, or D-[3H]aspartate. Moreover, the transport systems for the excitatory SAAs exhibited a negative correlation when compared in inhibition profiles with the high affinity transport of both [3H] gamma-aminobutyric acid (GABA) and [3H]taurine.(ABSTRACT TRUNCATED AT 250 WORDS)

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 microM to 1 mM evoked a saturable release of gamma-[3H]aminobutyric acid ([3H]GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50, 100-150 microM) 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-D-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 (IC50 range, greater than 50 microM) and the non-NMDA-receptor antagonist, 6,7-dinitroquinoxalinedione (IC50 range, 5-50 microM). 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 microM excitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 microM SKF-89976-A, a non-transportable inhibitor of the GABA 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 clearly consistent with a receptor-mediated, depolarization-induced reversal of the GABA carrier.

There is sufficient evidence from both L1 and L2 research to in dicate that temporal variables (including speech rate and pause pheno mena) can be manipulated to facilitate listening comprehension. There is, however, no reliable evidence to indicate that rate modification is a widely occurring phenomena in NS-NNS interaction. This controlled study was designed to investigate whether 6 university professors would modify the temporal organization of their speech when deli vering identical lectures to NS, low-proficiency and high-proficiency NNS groups. Samples of the recorded lectures were spectrographi cally analysed and a large number of temporal variables were investi gated. Contrary to expectations derived from a reading of the L2/ESL literature no significant modifications to speech rate were found in deliveries to either the low- or high proficiency groups. Observed changes in articulation rate narrowly missed being significant, but differences were between the two NNS groups, not between the NS and NNS groups. Highly significant differences were, however, observed in the data on filled pauses. L1 pausological literature is drawn upon in suggesting explanations of these findings. Implica tions for L2 teaching are discussed.

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).

The study reported in this article describes an investigation into the effects of three speech rates: 200 words per minute [wpm] (3.8 syllables per second [sps]); 150 wpm (2.85 sps); and 100 wpm (1.93 sps) on the comprehension of three 350- to 400-word lexically and grammatically graded passages delivered to a group of lower-intermediate level adult NNSs(nonnative speakers). Results showed that moderately fast speech rates resulted in a significant reduction in comprehension, but that scores on passages delivered at slow rates did not significantly differ from those delivered at average rates. Subjective responses confirmed previous research findings showing that perception of speech rate variation is frequently inaccurate. Results of the investigation are discussed in relation to the practical recommendations that might be drawn from them, particularly in terms of time-benefit analysis, if future studies support the present tentative findings.

Rate-controlled recorded text can not only be used to facilitate listen ing comprehension through reducing speech rate but it can also help language learners become accustomed to increasingly rapid deliveries. Auditory material of this kind can be produced by speech compressors, temporal spacing or multi-recording of texts. This paper, therefore, briefly describes L1 and L2 research on temporal variables relevant to facilitating listening comprehension. This is followed by a consideration of practical applications of this research to L2 listening comprehension in preparing recorded materials, in teacher-talk and in testing.

A continuous superfusion system was used to study the release of [ 3 H] d -aspartate from primary cultures of mouse cerebellar granule cells. A number of neuroactive acidic sulphur amino acids (viz. cysteine sulphinate, cysteate, homocysteine sulphinate, homocysteate and S-sulpho-cysteine) of endogenous origin were shown to evoke a dose-dependent, saturable and stereospecific release of [ 3 H] d -aspartate. Substitution of Ca 2+ in the superfusion medium by Co 2+ (which blocks voltage-gated Ca 2+ -channels) resulted in a marked attenuation of evoked release following stimulation by l -enantiomers of all sulphur amino acids tested, thus demonstrating a “Ca 2+ -dependent” release of [ 3 H] d -aspartate. Cysteine sulphinate-evoked release of endogenous glutamate was shown to be Ca 2+ -dependent whereas negligible release of endogenous aspartate was observed. All sulphur amino acids inhibited to varying degrees, the intracellular accumulation of the [ 3 H]tetraphenylphosphonium cation, an observation consistent with either partial or complete depolarization of the plasma membrane. The sulphur amino acid-evoked release of [ 3 H] d -aspartate from the granule cells was inhibited by a variety of excitatory amino acid receptor antagonists. At a concentration range of antagonist between 0.1–500 μM, the sulphur amino acid-evoked release of [ 3 H] d -aspartate was inhibited by: (i) the selective, competitive N- methyl- d -aspartate receptor antagonists; 3-(±)-2-carboxypiperazin-4-yl) propyl-2-phosphonic acid and d -2-amino-5-phosphonopentanoic acid; (ii) the competitive quisqualate/kainate receptor antagonist, 6,7-dinitroquinoxalinedione; and (iii) the broad spectrum excitatory amino acid receptor antagonist, kynurenate. Such observations demonstrate that excitatory sulphur amino acid-evoked release of [ 3 H] d -aspartate from primary cultures of cerebellar granule cells occurs primarily by a receptor-mediated event in which both N- methyl- d -aspartate and quisqualate/kainate receptors are involved. The findings of this study provide additional evidence to support the transmitter candidacy of certain endogenous neuroactive sulphur amino acids and furthermore, suggest the mechanism by which their epileptogenic and neurotoxic actions may be explained.

In further exploring proposals for the measurement of early gene (c-fos mRNA) levels as a predictive index for in vitro excitotoxicity, this study, using immature (2 days in vitro) cultures of mouse cerebellar granule cells as an experimental model system, was undertaken to determine the effect of glutamate (Glu) i) in stimulating increases in intracellular free-calcium ([Ca2+]), ii) on cell viability and iii) on induction of steady-state c-fos mRNA levels. In parallel experiments the action of agents (viz. 55 mM KCl and the calcium ionophore, A23187) that mediate Ca2+ entry into cells via different routes was also evaluated. Glu was unable to induce excitotoxicity in granule cells at this stage of development in culture, but did stimulate a concentration-dependent and marked increase in [Ca2+], levels while also mediating a dramatic concentration-dependent perturbation in the kinetics of c-fos mRNA induction that appeared to arise solely from NMDA receptor-mediated Ca2+ influx. The results are presented in comparison to the actions of KCI and A23187 and considered in relation to earlier studies undertaken using mature (7 days in vitro) cultures of cerebellar granule cells.

(S)-CPW399 (2b) is a novel, potent, and subtype-selective AMPA receptor full agonist that, unlike (S)-willardiine and related compounds, in mouse cerebellar granule cells, stimulated an increase in [Ca(2+)](i), and induced neuronal cell death in a time- and concentration-dependent manner. Compound 2b appears to be a weakly desensitizing, full agonist at AMPA receptors and therefore represents a new pharmacological tool to investigate the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitization.

Two three-dimensional receptor interaction models for EAAT substrates and nontransportable inhibitors have been developed, and new glutamate (Glu) and aspartate (Asp) analogues have been synthesized. The analogues 1a and 3 represent novel lead compounds for the development of EAAT substrates and nontransportable inhibitors, selective for EAATs over iGluRs, as possible neuroprotective agents useful to minimize the progression of chronic or acute neurodegenerative diseases. The role played by the protonatable amine function in the interaction with EAATs has been discussed.

Using primary cultures of mouse cerebellar granule cells as an in vitro model system, it has been demonstrated that different profiles of temporal expression of the c-fos proto-oncogene are observed under non-excitotoxic and excitotoxic conditions. A ratio has been derived previously for the steady- state level of c-fos mRNA after 30 min and 240 min which suggests that a 240 min/30 min ratio of greater than 1 correlates with excitotoxicity, whereas a ratio of less than 1 correlates with a non-excitotoxic outcome. Moreover, a positive correlation is seen with abrogation of excitotoxicity in response to selective excitatory amino acid receptor antagonists. This test, proposed as a specific biomarker for excitotoxicity is undergoing prevalidation. Excitotoxicity is defined as neuronal cell death mediated by hyperactivation of glutamate receptor subtypes and therefore might be expected to be prevented by selective glutamate receptor antagonists. In preliminary evaluation studies, we have conducted work under the direction of the European Center for Validation of Alternate Methods (ECVAM) using compounds specified by ECVAM that have been subdivided into four groups based on known or presumed actions. These groups comprise: Group 1 - endogenous/synthetic excitotoxins; Group 2 - environmental, putative excitotoxins; Group 3 - neurotoxic but non-excitotoxic compounds, and Group 4 - non-toxic compounds. The results of this study support the proposal that the c-fos mRNA time-ratio test is a specific biomarker of excitotoxicity. Just as importantly, this test has the potential for application in screening newly-designed EAA receptor antagonists in the search for clinically relevant drugs to treat a variety of neuropathologies. (C) 2000 Elsevier Science Ltd. Chemicals/CAS: Excitatory Amino Acid Antagonists; Glutamic Acid, 56-86-0; Proto-Oncogene Proteins c-fos; RNA, Messenger

The effect of excitatory amino acids (EAAs) on c-fos mRNA expression was studied in primary cultures of mouse cerebellar granule cells and in neocortical neurons after 2 and 7 days in vitro (div). In cultured granule cells at 2 and 7 div, and in cortical neurons at 2 div, exposure to low levels (or = 10 microM) of a variety of EAAs (viz. glutamate [Glu], S-sulpho-L-cysteine [SC], N-methyl-D-aspartate [NMDA], alpha-amino-3-hydroxy-5-methyl-4-isoxazole [AMPA], and kainate [KA]) resulted in a transient increase in the level of c-fos mRNA which peaked at 30 min but returned to a basal level by 120 min. However, exposure of granule cells (7 div) to high levels (250 microM) of Glu, NMDA, KA, SC and of cortical neurons (7 div) to high levels (250 microM) of Glu, NMDA, KA, SC, or AMPA and to low levels (or = 10 microM) of Glu and AMPA resulted in a delay in c-fos mRNA induction but a subsequent, progressive increase that was sustained for at least 240 min. Furthermore, this effect was accompanied by a dose-related increase in the release of the cytosolic enzyme, lactate dehydrogenase, used as an indicator of excitotoxicity. A ratio (Q240/30) for the steady-state levels of c-fos mRNA after 30 min and 240 min of exposure to EAAs was determined which showed that Q240/302 correlated reproducibly with excitotoxic cell death, whereas a ratio ofor = 1 correlated with a nonexcitotoxic event. In both cell types at 7 div, coadministration of the selective NMDA receptor antagonist, DL(+/-)-2-amino-5-phosphonopentanoic acid (APV) with cytotoxic levels of Glu 1) protected against EAA-induced neurotoxicity and 2) exhibited a transient c-fos mRNA expression (Q240/30 values approximately 1). In contrast, the AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), provided no protection against excitotoxicity and had no significant effect on the Glu-induced delay in c-fos mRNA expression. These results suggest that the Q240/30 c-fos mRNA ratio may 1) be used as a predictive index for excitotoxic neuronal death, 2) provide information on the identity of the receptor subtype mediating excitotoxicity in different brain cell types, and 3) aid in establishing the role of excitotoxicity during the development of neurons in vitro.

Summary o 1. Glutamate is the predominant endogenous EAA at fast-acting synapses in the CNS but also serves an equally important function as an intermediate in carbohydrate and nitrogen metabolism. 2. Other endogenous EAAs such as aspartate, cysteine sulphinate, and homocysteate may also function as excitatory transmitters. 3. Information transfer between neurons using the EAAs is mediated by at least four subtypes of receptor, namely, the NMDA, AMPA, KA, and metabotropic receptors. 4. The NMDA, AMPA, and KA receptor subtypes are directly linked to cation channels, whereas the mGluR is coupled via a G protein to various intracellular second messengers. 5. The NMDA receptor exhibits special pharmacological properties consistent with an involvement in higher neural functions. 6. Paradoxically, the same properties of the NMDA receptor may be responsible for initiating neuronal cell damage evident in a number of acute and chronic neuropathologies. 7. AMPA and KA receptors contribute to the faster excitatory neurotransmission; AMPA receptor activation appears to provide the initial depolarizing stimulus necessary for removing the voltage-block of the NMDA receptor-coupled ion channel. 8. The slower-acting mGluRs may serve a modulatory role in neurotransmission. At present, their role in normal and abnormal brain function is uncertain. 9. A number of highly potent and selective EAA receptor antagonists have been synthesized; unfortunately, their inability to permeate the blood-brain barrier following systemic administration means that none are currently in clinical use. It is expected, however, that suitable drugs will soon be developed.

During evaluation of potential end-points for in vitro neurotoxicity screening we investigated what influence changes in cyclic GMP (cGMP) levels might exert on the degree of glutamate (Glu)-induced neurotoxicity in primary cultures of mouse cerebellar granule cells. Depletion of Glu-stimulated cGMP levels by N-methyl-D-aspartate receptor antagonists fully protected against Glu-induced toxicity. However, when Glu-stimulated cGMP levels were either depleted or elevated by the use of a variety of pharmacological agents acting intracellularly at various points of the NO/cGMP signalling pathway the degree of cytotoxicity exerted by Glu was unaltered. These results imply that cGMP and NO do not modulate the toxic effects of Glu and are therefore unsuitable as biomarkers of excitotoxicity in these cells.

The effect(s) of a prototypic intracellular Ca 2+ 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-Ca 2+ levels ([Ca 2+ ] i ) that was dependent on the extracellular concentration of Ca 2+ ([Ca 2+ ] o ). In addition, this increase in [Ca 2+ ] i correlated with a decrease in cell viability that was also dependent on [Ca 2+ ] 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 Ca 2+ -dependent component (ED 50 ∼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 Ca 2+ -dependent, but not the Na + /Cl - -dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca 2+ ] i . These findings suggest that Ca 2+ 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 [Ca 2+ ] i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca 2+ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca 2+ ] i and glutamate-induced neurotoxicity.

An elevated, sustained expression of c-fos mRNA was found in primary cultures of mouse cerebellar granule cells following exposure to toxic concentrations of the excitatory amino acids, L-glutamate, L-homocysteate, S-sulpho-L-cysteine and N-methyl-D-aspartate (NMDA), using leakage of lactate dehydrogenase (LDH) as an indicator of cytotoxicity. In contrast, when used at non-toxic concentrations these compounds induced a rapid and transient increase in c-fos mRNA levels. Both LDH release and elevated, sustained c-fos mRNA induction were blocked (in the case of L-homocysteate) or reduced (in the case of L-glutamate and S-sulpho-L-cysteine) by the selective NMDA receptor antagonist (DL(+/-)-2-amino- 5-phosphonopentanoic acid) whereas 6-cyano-7-nitroquinoxaline-2,3-dione (a selective antagonist at non-NMDA ionotropic receptors) had no effect. These data suggest a role for altered c-fos mRNA expression in excitotoxic mechanisms.

The action of TMB-8 [8-N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate] on glutamate (Glu)- and 55 mM KCl (K+)-induced increases in intracellular free calcium levels ([Ca2+]i) was studied in cultured mouse cerebral cortical neurones. Glu-induced responses were mediated by two mechanisms, one independent of and the other dependent on extracellular Ca2+, both being inhibited by TMB-8 in a dose-dependent manner. TMB-8 had no effect on [3H]dizocilpine binding kinetics in cortical membrane preparations. The transient and sustained phases of K(+)-induced increases in [Ca2+]i were also inhibited by TMB-8 in a dose-dependent manner. The Ca2+ channel antagonist, verapamil, blocked K(+)-induced responses but had no effect on Glu-induced responses. Although TMB-8 displays multiple effects, its action at voltage-gated Ca2+ channels does not appear to contribute significantly to its inhibition of the Glu-induced increase in [Ca2+]i.

Effects of the sulphur-containing acidic amino acids (SAAs) cysteic acid (CA), homocysteic acid (HCA), cysteine sulphinic acid (CSA), homocysteine sulphinic acid (HCSA), and S-sulphocysteine (SC) on intracellular concentrations of Ca2+ ([Ca2+]i) and cGMP ([cGMP]i) as well as their cytotoxic actions were investigated in cultured cerebral cortical neurons. The glutamate receptor subtype selective antagonists APV (D-(-)-2-amino-5-phosphonopentanoate) acting on N-methyl-D-aspartate (NMDA) receptors and DNQX (6,7-dinitroquinoxaline-2,3-dione) acting on non-NMDA receptors were employed to obtain information about the involvement of glutamate receptor subtypes in these actions of the SAAs. It was found that all SAAs exerted a cytotoxic action on the neurons. The ED50 values for CSA, CA, HCSA, and HCA were around 30 to 50 microM and that for SC was about 150 microM. The glutamate transport blocker L-aspartate-beta-hydroxamate increased the efficacy of CSA and CA but had no effect on the cytotoxic actions of the remaining SAAs. In case of CA, HCA, and SC the cytotoxicity could be prevented by APV alone and for HCSA, DNQX could block the toxic action. DNQX reduced the toxicity of HCA somewhat but the presence of APV was required for complete protection. CSA toxicity could only be blocked by the combination of APV and DNQX. All SAAs induced an increase in [cGMP]i and [Ca2+]i and with regard to [Ca2+]i SC was the most potent and CA the least potent SAA. The effect of all SAAs on [cGMP]i could be blocked by APV alone whereas DNQX had no effect except in the case of HCSA where the response was blocked completely and HCA where the response was inhibited by 75%.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary cultures of mouse cerebral cortex neurons, cerebellar granule cells and cortical astrocytes were maintained in vitro for respectively 8-10, 7-10 and 21-24 days. Following these times, amino acids were extracted from the cells by use of ice-cold 70% (v/v) ethanol and the extracts lyophilised. The lyophilised extracts when resuspended were subjected to reverse-phase high performance liquid chromatographic (HPLC) analysis for detection of free amino acids. Samples of cell culture growth medium and water blanks were treated in a similar manner. Identification of L-homocysteate (HCA) and L-homocysteine sulphinate (HCSA) was undertaken by matching retention times with regard to external standards and by 'spiking' cell extracts with authentic compounds. On this basis, HCA and HCSA were consistently detectable in astrocytes at levels of, respectively, 72.3 +/- 33.7 pmol/mg protein (n = 24) and 49.4 +/- 28.7 pmol/mg protein (n = 24). However, in neurons, a peak corresponding to HCSA could not be detected above the background noise, while the area of the peak corresponding to HCA was always greater than, but not significantly different from, that of the background noise present in water blanks. HCA and HCSA were not detectable in the serum used for preparation of the cell culture growth medium. Taken together, these findings indicate a predominant localisation of HCA and HCSA in astrocytes which, at least in culture, appear to possess the metabolic machinery necessary for synthesising and storing these amino acids without any neuronal influence.

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.

The effects of dl -homocysteine, and dl -homocysteate, on extracellular levels of amino acids in the rat hippocampus have been studied using brain microdialysis. Hippocampal electroencephalogram activity was monitored simultaneously using an electrode attached to the dialysis probe. dl -Homocysteine (1200 mg/kg; i.p. injection) produced epileptic activity in hippocampus in an inconsistent manner. Alterations in electroencephalogram activity were not observed in urethane anaesthetized animals, whereas 50% of Hypnorm anaesthetized animals exhibited epileptic activity, dl -Homocysteate (2 μmol; i.e.v.) induced epileptic activity in a majority of animals anaesthetized using urethane. Dialysate levels of aspartate were significantly elevated by homocysteine in both groups of animals. Conversely, dialysis levels of GABA were reduced. Dialysate levels of other amino acids measured (glutamate, glutamine, taurine, alanine and valine) were not affected significantly. Dialysate levels of taurine were increased significantly in animals injected with homocysteate. These data suggest that the imbalance in excitatory: inhibitory neurotransmission in the hippocampus caused by these alterations in extracellular levels of neuroexcitatory (i.e. aspartate) and neuroinhibitory (i.e. GABA) transmitters could underly the epileptic effect of homocysteine.

In an attempt to investigate the apparent absolute selectivity of the synaptic vesicle L-glutamate carrier, L- and D-enantiomers of excitatory sulphur-containing amino acid (SAA) transmitter candidates (which are close structural analogues of L-glutamate) were tested for their capacity to compete for vesicular L-[3H]-glutamate uptake. All SAAs inhibited, to varying degrees (52-86%), the vesicular uptake of L-[3H]-glutamate. A similar level of inhibition was exerted by SAAs with either a shorter or equal carbon chain length to L-glutamate. Moreover, inhibition was stereospecific in favour of the D-enantiomers. These studies indicate an appreciable interaction of the SAAs with the recognition site of the vesicular L-glutamate carrier. Further investigations are required to establish the substrate potential of the SAAs.