Absence Of Glycine Research Articles

The combinatorial effect of complexing agent and inhibitor on chemical–mechanical planarization of copper

Chemical–mechanical planarization (CMP) is a vital process for the fabrication of advanced copper multilevel interconnects schemes. The focus of this investigation was to understand the oxidation, dissolution and surface modification characteristics of Cu in slurries with varying pH. Hydrogen peroxide was used as the oxidizer, glycine as complexing agent and 3-amino-triazol (ATA) as inhibitor in the slurry. The electrochemical process involved in the oxidative dissolution of copper was investigated by potentiodynamic polarization studies. X-ray photoelectron spectroscopy was used to investigate the surface modification of copper and understand the interaction between Cu–H 2O 2–glycine–ATA during CMP. In the absence of glycine and ATA, copper removal rate was found to be high in the slurry with 5% H 2O 2 at pH 2. The removal rate then decreased and reached the minimum at pH 6 and started to increase in alkaline conditions. With the addition of 0.01 M glycine, the removal rates of copper were lowered in acidic slurries, but increased significantly in alkaline slurries. The addition of ATA lowered copper removal rates, however, better surface planarity was achieved. The present investigation provides an insight to the mechanism of Cu removal in the presence of oxidizer, complexing agent and inhibitor.

Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl-d-aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30min in the Mg2+-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24h later using propidium iodide staining. d,l-Homocysteine given alone appeared to be a weak neurotoxin, with EC50 of 17.4mM, whereas EC50 for l-glutamate was 0.17mM. Addition of 50μM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5μM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45Ca uptake by granule cells incubated in the presence of 25mM d,l-homocysteine with 50μM glycine was only 3% of the net uptake evoked by 1mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250μM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca2+ to neurones.

Chemical-Mechanical Planarization of Copper: The Effect of Inhibitor and Complexing Agent

AbstractThe present investigation was focused on understanding of the oxidation, dissolution and modification of Cu surface in slurries at various pH using hydrogen peroxide as oxidizer, glycine as complexing agent and 3-amino-triazol (ATA) as inhibitor during Cu-CMP. The electrochemical process involved in the oxidative dissolution of copper was investigated by potentiodynamic polarization studies. Surface modification of copper was investigated using Xray photoelectron spectroscopy to understand the interaction of Cu-H2O2-glycine-ATA during CMP. In the absence of glycine and ATA, the copper removal rate is found to be high in a slurry with 5% H2O2 at pH 2, then it decreases with increasing pH and reaches the minimum at pH 6, it continuously increases at alkaline condition. In the presence of 0.01M glycine, the removal rate of copper decreases in acidic slurries while increases significantly in alkaline slurries. With the further addition of ATA, the copper removal rate was reduced. However, better surface planarity was obtained. The present investigation enhanced understanding of the mechanism of Cu CMP in the presence of oxidizer, complexing agent and inhibitor for formulation of a highly effective CMP-slurry.

Effect of glycine and hydrogen peroxide on chemical–mechanical planarization of copper

Chemical–mechanical planarization (CMP) of copper is a vital process to produce sub-micron range and multilevel metallization to meet the demands of the current interconnect technology. The present investigation was focussed to understand the oxidation, dissolution and modification of Cu surface using hydrogen peroxide as oxidizer and glycine as inhibitor during Cu-CMP employing electrochemistry as well as dynamic and static removal rate measurements. Surface modification of copper was investigated in detail using X-ray photoelectron spectroscopy to understand the interaction of Cu–H 2O 2–glycine complex formation during CMP. Atomic force microscopy was employed to reveal any change in surface morphology during the CMP process. In the presence of 0.1 M glycine, copper removal rate was found to be high in the solution containing 2.5% H 2O 2 at pH 4 because of Cu 2+–glycine complexation reaction. In the absence of glycine, the removal rate of copper decreased with increasing H 2O 2 concentration due to the formation of a less soluble copper oxide film. The present investigation helped understanding the mechanism of Cu surface alteration in presence of oxidizers and glycine for formulation of highly effective CMP-slurry.

Anesthetic and ethanol effects on spontaneously opening glycine receptor channels.

Strychnine-sensitive glycine receptors mediate inhibitory neurotransmission occurring in the brain stem and spinal cord. Alcohols, volatile anesthetics and inhaled drugs of abuse are positive allosteric modulators of glycine receptor function, normally enhancing function only in the presence of glycine. A complication in studying allosteric actions on ligand-gated ion channels is in the dissection of their effects on neurotransmitter binding from their effects on channel opening. Mutation of an aspartate residue at position 97 to arginine in the glycine receptor alpha1 subunit simulated the effects of glycine binding, producing receptors that exhibited tonic channel opening in the absence of neurotransmitter; i.e. these receptors demonstrated a dissociation of channel opening from neurotransmitter binding. In these receptors, ethanol, enflurane, chloroform, halothane, 1,1,1-trichloroethane and toluene elicited inward currents in the absence of glycine. We previously identified mutations on ligand-gated ion channels that eliminate ethanol, anesthetic and inhalant actions (such as S267I on alpha1 glycine receptors). The double mutant (D97R and S267I) receptors were both constitutively active and resistant to the enhancing effects of ethanol and enflurane. These data demonstrate that ethanol and volatile anesthetics can affect glycine receptor channel opening independently of their effects on enhancing neurotransmitter binding.

Transition from GABAergic to glycinergic synaptic transmission in newly formed spinal networks.

The role of glycinergic and GABAergic systems in mediating spontaneous synaptic transmission in newly formed neural networks was examined in motoneurons in the developing rat spinal cord. Properties of action potential-independent miniature inhibitory postsynaptic currents (mIPSCs) mediated by glycine and GABA(A) receptors (GlyR and GABA(A)R) were studied in spinal cord slices of 17- to 18-day-old embryos (E17-18) and 1- to 3-day-old postnatal rats (P1-3). mIPSC frequency and amplitude significantly increased after birth, while their decay time decreased. To determine the contribution of glycinergic and GABAergic synapses to those changes, GlyR- and GABA(A)R-mediated mIPSCs were isolated based on their pharmacological properties. Two populations of pharmacologically distinct mIPSCs were recorded in the presence of glycine or GABA(A) receptors antagonists: bicuculline-resistant, fast-decaying GlyR-mediated mIPSCs, and strychnine-resistant, slow-decaying GABA(A)R-mediated mIPSCs. The frequency of GABA(A)R-mediated mIPSCs was fourfold higher than that of GlyR-mediated mIPSCs at E17-18, indicating that GABAergic synaptic sites were functionally dominant at early stages of neural network formation. Properties of GABA(A)R-mediated mIPSC amplitude fluctuations changed from primarily unimodal skewed distribution at E17-18 to Gaussian mixtures with two to three discrete components at P1-3. A developmental shift from primarily long-duration GABAergic mIPSCs to short-duration glycinergic mIPSCs was evident after birth, when the frequency of GlyR-mediated mIPSCs increased 10-fold. This finding suggested that either the number of glycinergic synapses or the probability of vesicular glycine release increased during the period studied. The increased frequency of GlyR-mediated mIPSCs was associated with more than a twofold increase in their mean amplitude, and in the number of motoneurons in which mIPSC amplitude fluctuations were best fitted by multi-component Gaussian curves. A third subpopulation of mIPSCs was apparent in the absence of glycine and GABA(A) receptor antagonists: mIPSCs with both fast and slow decaying components. Based on their dual-component decay time and their suppression by either strychnine or bicuculline, we assumed that these were generated by the activation of co-localized postsynaptic glycine and GABA(A) receptors. The contribution of mixed glycine-GABA synaptic sites to the generation of mIPSCs did not change after birth. The developmental switch from predominantly long-duration GABAergic inhibitory synaptic currents to short-duration glycinergic currents might serve as a mechanism regulating neuronal excitation in the developing spinal networks.

Human cytoplasmic serine hydroxymethyltransferase is an mRNA binding protein.

The 5' untranslated region (UTR) of the human cytoplasmic serine hydroxymethyltransferase (cSHMT) message is alternatively spliced, creating a full-length 5' UTR (LUTR) encoded within exons 1-3 and a shorter UTR (SUTR) that results from excision of exon 2. The role of the 5' UTRs in cSHMT expression was investigated by fusing the cSHMT 5' UTRs to the 5' end of the luciferase gene. Human cSHMT protein at 10 microM inhibits in vitro translation of cSHMT 5' UTR-luciferase fusion mRNA templates by more than 90%, but does not inhibit translation of the luciferase message lacking the UTR. Translation inhibition is independent of amino acid and folate substrate binding to the cSHMT enzyme. The cSHMT SUTR-luciferase mRNA binds to the cSHMT.glycine.5-formyltetrahydrofolate ternary complex with an apparent K(d) of 10 microM. Gel mobility shift assays demonstrate that the human cSHMT protein binds to the cSHMT LUTR-luciferase fusion mRNA in the presence and absence of glycine and 5-formyltetrahydrofolate pentaglutamate. The fusion cSHMT SUTR-luciferase message at 65 microM inhibits the cSHMT-catalyzed cleavage of allothreonine as a partial mixed type inhibitor, reducing both k(cat) and K(m) by 40 and 75%, respectively, while tRNA has no effect on cSHMT catalysis. These studies indicate that the cSHMT protein can bind mRNA, and displays increased affinity for the 5' untranslated region of its mRNA.

Net synthesis of acetate from CO 2 by Eubacterium acidaminophilum through the glycine reductase pathway

Eubacterium acidaminophilum combines the oxidation of amino acids such as alanine or valine with the reduction of glycine to acetate in a two-substrate fermentation (Stickland reaction). In the absence of glycine, dense cell suspensions oxidized alanine or valine only to a small extent, with limited production of hydrogen and acetate. Experiments with 14C-labeled carbonate revealed that acetate was formed under these conditions by net reduction of CO 2/HCO 3 −; 14C-labeled formate was formed as an intermediate. E. acidaminophilum did not grow with hydrogen plus CO 2; dense cell suspensions under H 2/CO 2 produced only very small amounts (<0.5 mM) of acetate. There was no activity of carbon monoxide dehydrogenase, indicating that the glycine pathway was used for acetate synthesis. The results are explained on the basis of biochemical and energetic considerations.

Net synthesis of acetate from CO2byEubacterium acidaminophilumthrough the glycine reductase pathway

Eubacterium acidaminophilum combines the oxidation of amino acids such as alanine or valine with the reduction of glycine to acetate in a two-substrate fermentation (Stickland reaction). In the absence of glycine, dense cell suspensions oxidized alanine or valine only to a small extent, with limited production of hydrogen and acetate. Experiments with 14C-labeled carbonate revealed that acetate was formed under these conditions by net reduction of CO2/HCO3−; 14C-labeled formate was formed as an intermediate. E. acidaminophilum did not grow with hydrogen plus CO2; dense cell suspensions under H2/CO2 produced only very small amounts (<0.5 mM) of acetate. There was no activity of carbon monoxide dehydrogenase, indicating that the glycine pathway was used for acetate synthesis. The results are explained on the basis of biochemical and energetic considerations.

Inducible expression of N-methyl-D-aspartate (NMDA) receptor channels from cloned cDNAs in CHO cells.

To develop a drug screening system, we introduced expression vectors carrying the mouse N-methyl-D-aspartate (NMDA) receptor channel epsilon1 and zeta1 subunit cDNAs under the promoter of the Drosophila heat shock protein hsp70 into Chinese hamster ovary (CHO) cells. We selected clonal cell lines by means of RNA blot hybridization and fura-2 fluorometry. One of these cell lines, ZE1-1, optimally expressed the epsilon1 and zeta1 subunit mRNAs when induced by an incubation at 43 degrees C for 2 h. Heated ZE1-1 cells exhibited the NMDA-induced intracellular Ca2+ elevation, whereas unheated they showed no such response. NMDA and L-glutamate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate, induced an increase in the intracellular Ca2+ concentration. The response to the agonists was marginal in the absence of glycine, and diminished by Mg2+ and NMDA receptor antagonists. Furthermore, exposure to agonists of ZE1-1 cells expressing the epsilon1/zeta1 NMDA receptor channel resulted in the release of lactate dehydrogenase (LDH) activity in the culture medium indicating agonist-induced cell death. NMDA receptor antagonists inhibited the LDH activity release. These results suggest that ZE1-1 cells will provide a useful screening system for novel drugs acting on the epsilon1/zeta1 NMDA receptor channel.

Molecular Cloning, Characterization, and Regulation of the Human Mitochondrial Serine Hydroxymethyltransferase Gene

The human mitochondrial serine hydroxymethyltransferase (mSHMT) gene was isolated, sequenced, and characterized. The 4.5-kilobase gene contains 10 introns and 11 exons, with all splice junctions conforming to the GT/AG rule. The 5' promoter region contains consensus motifs for several regulatory proteins including PEA-3, Sp-1, AP-2, and a CCCTCCC motif common to many genes expressed in liver. Consensus TATA or CAAT sequence motifs are not present, and primer extension and 5'-rapid amplification of cDNA ends studies suggest that transcription initiation occurs at multiple sites. The mitochondrial leader sequence region of the deduced mRNA contains two potential ATG start sites, which are encoded by separate exons. The intervening 891-base pair intron contains consensus promoter elements suggesting that mSHMT may be transcribed from alternate promoters. 5'-Rapid amplification of cDNA ends analysis demonstrated that the first ATG is transcribed in human MCF-7 cells. However, transfection of Chinese hamster ovary cells deficient in mSHMT activity with the human mSHMT gene lacking exon 1 overcame the cell's glycine auxotrophy and restored intracellular glycine concentrations to that observed in wild-type cells, showing that exon 1 is not essential for mSHMT localization or activity and that translation initiation from the second ATG is sufficient for mSHMT import into the mitochondria. Mitochondrial SHMT mRNA levels in MCF-7 cells did not vary during the cell cycle and were not affected by the absence of glycine, serine, folate, thymidylate, or purines from the media.

Activation of glycine decarboxylase in pea leaf mitochondria by ATP

Activity of glycine decarboxylase decreased by 60–70% after the isolated pea leaf mitochondria were aged for 5 h in the absence of glycine and was completely lost after 24 h. The reverse reaction, i.e., production of glycine from serine, ammonium, dihydrolipoate, and bicarbonate, was also inhibited in these aged mitochondria. Glycine decarboxylase could be reactivated by both exogenous and endogenous ATP. The latter was formed during the oxidation of succinate, malate, or oxoglutarate. Glycine decarboxylase consists of four subunits (P-, H-, L-, and T-proteins). The aged mitochondria were able to catalyze the exchange of [ 14C]-bicarbonate-glycine and the oxidation of dihydrolipoate, indicating the persistence of P-, H-, and L-protein activities. Serine hydroxymethyltransferase catalyzes the formation of serine from methylene tetrahydrofolate and another glycine molecule at the last reaction of glycine oxidation. The aged mitochondria were able to catalyze the formation of methylene tetrahydrofolate from [ 14C]serine and its reverse reaction. Therefore, it was concluded that the loss of glycine decarboxylase activity was due to an inhibition of the reaction catalyzed by T-protein, which required ATP for its activation.

Upstream Organization of and Multiple Transcripts from the Human Folylpoly-γ-glutamate Synthetase Gene

Folylpoly-gamma-glutamate synthetase (FPGS) is essential for the survival of proliferating mammalian cells and central to the action of all "classical" folate antimetabolites. We report the isolation of cDNAs corresponding to the 5' ends of FPGS mRNA from both human and hamster cells which include a start codon upstream of and in-frame with the AUG in the previously reported FPGS open reading frame. The predicted hamster and human amino-terminal extension peptides have features consistent with a mitochondrial targeting sequence. Ribonuclease protection and 5'-rapid amplification of cDNA ends assays indicated multiple transcriptional start sites consistent with the sequence of the promoter region of this gene, which was highly GC-rich and did not contain TATA or CCAAT elements. These start sites would generate two classes of transcripts, one including the upstream AUG and one in which only the downstream AUG would be available for translation initiation. Transfection of the full length human cDNA into cells lacking FPGS restored their ability to grow in the absence of glycine, a product of mitochondrial folate metabolism, as well as of thymidine and purines. Therefore, we propose that the mitochondrial and cytosolic forms of FPGS are derived from the same gene, arising from the use of the two different translation initiation codons, and that the translation products differ by the presence of a 42-residue amino-terminal mitochondrial leader peptide.

Modulation by Gly, Ca, and acidosis of injury-associated unesterified fatty acid accumulation in proximal tubule cells

We have examined the dependence of unesterified fatty acid accumulation by intact, freshly isolated proximal tubules on Ca2+, pH, and the cytoprotective amino acid, glycine, during injury induced by hypoxia, antimycin, or antimycin plus ionomycin. In the absence of glycine, similarly high levels of fatty acid accumulation were seen during all three injury conditions irrespective of whether tubules were incubated in normal 1.25 mM Ca2+ medium or in medium where Ca2+ was buffered to 0.1 microM, a maneuver which prevented injury-associated increase of cytosolic-free Ca2+ as measured with fura 2. In the presence of glycine, which strongly suppressed development of lethal membrane damage for at least 60 min and did not have any apparent direct effects on fatty acid accumulation, both Ca(2+)-independent and Ca(2+)-dependent components of fatty acid accumulation were discernible. The Ca(2+)-independent component accounted for approximately 2/3 of fatty acid accumulation and did not vary as Ca2+ ranged from 10 nM to 1 microM. Unequivocal Ca(2+)-dependent accumulation occurred when Ca2+ exceeded 10 microM. Lowering pH to 6.9 had a moderate, generalized suppressive effect on fatty acid accumulation, including the major Ca(2+)-independent component, irrespective of the presence of glycine. These data emphasize the role of Ca(2+)-independent fatty acid accumulation during proximal tubule cell injury, clarify the modulatory actions of the potent, intrinsic cytoprotective factors, glycine and reduced pH, and provide insight into the relationship between fatty acid accumulation and lethal membrane damage.

Efficient release of thermostable β-galactosidase by glycine addition and thermal treatment of Escherichia coli cells

Efficient release of thermostable β-galactosidase from a recombinant Escherichia coli by the addition of glycine to the culture broth and subsequent thermal treatment was investigated. The enzyme release rate was strongly dependent on glycine concentration. The enzyme release rate was almost proportional to glycine concentrations up to 2% in phosphate buffer; however, inactivation of the enzyme was not observed following incubation for up to 3 h at 70°C even in the presence of 10% glycine. In a preliminary experiment, severe thermal inactivation was observed in the presence of polyethylene glycol (PEG), but glycine was able to suppress the inactivation. Thermal treatment of the cell suspension was effective for the improvement of the enzyme release rate. In the absence of glycine, the enzyme release rate was low at 37 and 45°C, even though the initial release rate was high at 0.5 h and 60°C. The combination of thermal treatment and addition of glycine to the cell suspension significantly improved the initial enzyme release rate and the amount of enzyme released to the extracellular fraction at 37 and 45°C was as high as that at 60°C during a 2-h incubation.

Effect of morphine tolerance and abstinence on the binding of [ 3H]MK-801 to brain regions and spinal cord of the rat

The effect of chronic administration of morphine to rats on theN-methyl- d-aspartate (NMDA) receptors labeled with [ 3H]MK-801, a non-competitive antagonist, was determined in brain regions and spinal cord. Male Sprague-Dawley rats were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of 6 morphine pellets during a 7-day period. Each pellet contained 75 mg of morphine free base. Animals serving as controls were similarly implanted with placebo pellets. This procedure resulted in the development of a high degree of tolerance and physical dependence on morphine. Two sets of rats were used. In one, the pellets were left intact at the time of sacrifice (tolerant) and in the other the pellets were removed 16 h prior to sacrificing (abstinent). The binding constants, B maxandK d values of [ 3H]MK-801 were determined in cortex, hippocampus, hypothalamus, corpus striatum, midbrain and spinal cord. In the absence of glycine and glutamate, [ 3H]MK-801 bound to tissue membranes at a single high affinity site. TheB max andK d values of [ 3H]MK-801 were not altered in any of the tissues of the morphine abstinent rats. TheB max value of [ 3H]MK-801 was significantly decreased in cerebral cortex of morphine tolerant rats as compared to their placebo controls but theK d values did not change. In other brain regions and spinal cord of morphine tolerant rats and their placebo controls, theB max andK d values of [ 3H]MK-801 did not differ. Thus, for the first time these studies demonstrate that in the absence of glutamate and glycine, the [ 3H]MK-801 recognition sites are down-regulated selectively in the cerebral cortex of morphine tolerant rats.

Micellar behaviour of n-alkyl sulphates in a buffered medium at different temperatures

The CMC of sodium n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl sulphates in glycine buffer solutions at pH 3.2 were precisely determined at 15, 20, 25, 30 and 35°C by an electrical conductivity method. For each of the surfactant—solvent systems investigated the values of the CMC in each surfactant were lower than the equivalent values in the absence of glycine. CMC values decrease with the increase in the length of the alkyl chain, this variation being a linear function of the number of carbon atoms in the hydrocarbon chain. The CMC variation with temperature shows a minimum between 20 and 30°C. Thermodynamic data for micelle formation derived by application of the mass action model are presented.

Diazepam potentiation by glycine in pentylenetetrazol seizures is antagonized by 7-chlorokynurenic acid

This study evaluated a possible mechanism by which glycine potentiates the activity of diazepam (DZP) and valproic acid (VAL) against the clonic seizures induced by pentylenetetrazol (PTZ) in rats. Neither 7-chlorokyburenic acid (7-CLKYNA)_nor strychnine in doses of 10, 50, or 100 nmol ICV significantly altered the clonic seizure response to PTZ. However, 7-CLKYNA (100 nmol, ICV), but not strychnine (100 nmol, ICV), antagonized the anticonvulsant activity induced by coadministration of DZP (1.0 mg/kg, IP) and glycine (40 mmol/kg, PO). Neither 7-CLKYNA (100 nmol, ICV) nor strychnine (100 nmol, ICV) significantly altered the anticonvulsant activity induced by coadministration of VAL and glycine. 7-CLKYNA (100 nmol, ICV) had no effect on the anticonvulsant activity of DZP or VAL in the absence of glycine. These results provide evidence that the glycine potentiation of the anticonvulsant activity of DZP in clonic seizures induced by PTZ may be mediated by the strychnine-insensitive glycine receptor.

Equilibrium and kinetic study of glycine action on the N-methyl-D-aspartate receptor in cultured mouse brain neurons.

1. The characteristics of the activation of the N-methyl-D-aspartate (NMDA) response by glycine were studied using whole-cell and outside-out patch clamp recording techniques. 2. Glycine concentration-response (C-R) curves were measured in the presence of 10 microM-NMDA and fitted with the Hill equation modified to account for the response to NMDA observed in the absence of added glycine. The mean value of the apparent dissociation constant (KD) was 150 nM, and the mean value of the Hill coefficient (nH) was 1.1. When the KD was corrected for the concentration of contaminating glycine in nominally glycine-free solutions, estimated assuming that there is no response in the absence of glycine, the value was 130 nM. 3. The question of how many glycine binding sites there are on each NMDA receptor-channel complex was addressed by examining the curvature at the foot of the glycine C-R curve. An equation that allowed estimation of both the concentration of contaminating glycine and of the value of nH was fitted to glycine C-R data up to 50 nM. The mean value of nH was found to be 1.0, consistent with the idea that there is one glycine binding site. 4. The kinetics of the interaction of glycine with the NMDA receptor were measured by fitting single exponential curves to the current relaxation following a jump in glycine concentration in the presence of 10 microM-NMDA. The plot of the inverse of the relaxation time constant as a function of glycine concentration after the concentration jump was linear. The association rate constant was estimated from these data as 1.2 x 10(7) M-1 s-1 and the dissociation rate as 1.0 s-1. 5. Experiments were devised to allow the evaluation of the KD and dissociation rates of glycine in the absence of NMDA. They led to a value for KD of 80 nM, slightly but significantly lower than the value of 150 nM estimated in the presence of 10 microM-NMDA. The glycine dissociation rate in the absence of NMDA was found to be 0.7 s-1, not significantly different from that measured in the presence of 10 microM-NMDA. 6. The results are consistent with a model of the NMDA receptor with a single glycine binding site. The characteristics of glycine binding are similar in the absence and the presence of 10 microM-NMDA, although NMDA binding may cause a small increase in the glycine KD.(ABSTRACT TRUNCATED AT 250 WORDS)

N-methyl-D-aspartate increases cytosolic Ca2+ via G proteins in cultured hippocampal neurons.

The changes of the cytosolic Ca2+ concentrations ([Ca2+]i) induced by N-methyl-D-aspartate (NMDA) in fura-2-loaded cultured hippocampal neurons from rat embryos were investigated by the fast application method, using a fine pipe under extracellular Mg(2+)-free conditions. In the presence of Ca2+, NMDA, at concentrations in excess of 3 microM, induced a biphasic increase of [Ca2+]i, which consisted of an initial increase with a second rise that occurred after cessation of drug application. Under Ca(2+)-free conditions, NMDA (greater than 100 microM) in the absence of glycine or NMDA (greater than 50 microM) in the presence of glycine (greater than 10 microM) induced intracellular Ca2+ mobilization, which was blocked by 30 microM 2-amino-5-phosphonovaleric acid (APV) and reduced by islet-activating protein. When the neurons were superfused with Ca(2+)-free solution, the application of 3-10 microM NMDA, which had been dissolved in Ca(2+)-containing solution, induced the second phase [Ca2+]i increase, whereas application of kainate, quisqualate, or stimulation by 50 mM K+ did not. Islet-activating protein, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and D-sphingosine reduced the second phase [Ca2+]i increase. These results suggest that NMDA-induced intracellular Ca2+ mobilization is potentiated by the initial entry of Ca2+ into the cells and is regulated in an islet-activating protein-sensitive manner.