Low Concentrations Of Glycine Research Articles

Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neurons.

Although glycine receptors (GlyRs) are responsible for the main spinal inhibitory responses in adult vertebrates, in the embryo they have been reported to mediate depolarizing responses, which can sometimes activate dihydropyridine-sensitive L-type calcium channels. However, these channels are not the only targets of dihydropyridines (DHPs), and we questioned whether GlyRs might be directly modulated by DHPs. By whole-cell recording of cultured spinal neurons, we investigated modulation of glycine responses by the calcium channel antagonists, nifedipine, nitrendipine, nicardipine and (R)-Bay K 8644, and by the calcium channel, agonist (S)-Bay K 8644. At concentrations between 1 and 10 microM, all these DHPs could block glycine responses, even in the absence of extracellular Ca2+. The block was stronger at higher glycine concentrations, and increased with time during each glycine application. Nicardipine blocked GABAA responses from the same neurons in a similar manner. In addition to their blocking effects, nitrendipine and nicardipine potentiated the peak responses to low glycine concentrations. Both effects of extracellular nitrendipine on glycine responses persisted when the drug was present in the intracellular solution. Thus, these modulations are related neither to calcium channel modulation nor to possible intracellular effects of DHPs. Another type of calcium antagonist, verapamil (10-50 microM), also blocked glycine responses. Our results suggest that some of the effects of calcium antagonists, including the neuroprotective and anticonvulsant effects of DHPs, might result partly from their interactions with ligand-gated chloride channels.

A Nuclear Magnetic Resonance (NMR) and Fourier-Transform Infrared (FTIR) Study of Glycine Speciation on a Cd-Rich Montmorillonite

AbstractAs a consequence of treatments with glycine solutions, glycine molecules enter the interlayer of both Ca- and Cd-rich montmorillonite. Measurements of d value suggest that at low glycine concentration (0.01 and 0.1 M glycine solutions) a “flat” arrangement of the glycine molecules occurs in the interlayer. In contrast, intercalation of more than one monolayer of glycine molecules occurs for the montmorillonite treated with a higher concentration of glycine (1 M glycine solution).Interlayer complexation of glycine occurs only for the Cd-rich form of montmorillonite, whereas no complexation is observed for Ca-rich montmorillonite. Both nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) results suggest that the adsorbed glycine, which fully protonates in the interlayer of montmorillonite to give the GlyH2− species, interacts with the interlayer Cd2+ to form the CdGlyx complex mainly through the carboxylate group. The interlayer cadmium, present as both Cd2+ and CdCl−, is complexed by the ligand glycine. In contrast, the cadmium adsorbed on the external surfaces of montmorillonite does not interact with the ligand. Complexation of CdCl+ only occurs for large amounts of adsorption of glycine (e.g., for samples treated with 1 M glycine solution).

Voltage dependence of the glycine receptor-channel kinetics in the zebrafish hindbrain.

Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were made on patches derived from the Mauthner cells of the 50-h-old zebrafish larva. As for glycinergic miniature inhibitory postsynaptic currents (mIPSCs), depolarizing the patch produced a broadening of the transient outside-out current evoked by short applications (1 ms) of a saturating concentration of glycine (3 mM). When the outside-out patch was depolarized from -50 to +20 mV, the peak current varied linearly with voltage. A 1-ms application of 3 mM glycine evoked currents that activated rapidly and deactivated biexponentially with time constants of approximately 5 and approximately 30 ms (holding potential of -50 mV). These two decay time constants were increased by depolarization. The fast deactivation time constant increased e-fold per 95 mV. The relative amplitude of the two decay components did not significantly vary with voltage. The fast component represented 64.2 +/- 2.8% of the total current at -50 mV and 54.1 +/- 10% at +20 mV. The 20-80% rise time of these responses did not show any voltage dependence, suggesting that the opening rate constant is insensitive to voltage. The 20-80% rise time was 0.2 ms at -70 mV and 0.22 ms at +20 mV. Responses evoked by 100-200 ms application of a low concentration of glycine (0.1 mM) had a biphasic rising phase reflecting the complex gating behavior of the glycine receptor. The time constant of these two components and their relative amplitude did not change with voltage, suggesting that modal shifts in the glycine-activated channel gating mode are not sensitive to the membrane potential. Using a Markov model to simulate glycine receptor gating behavior, we were able to mimic the voltage-dependent change in the deactivation time course of the responses evoked by 1-ms application of 3 mM glycine. This kinetics model incorporates voltage-dependent closing rate constants. It provides a good description of the time course of the onset of responses evoked by the application of a low concentration of glycine at all membrane potentials tested.

Molecular determinants of glycine receptor subunit assembly.

The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.

CAMP-dependent protein kinase modulation of glycine-activated chloride current in neurons freshly isolated from rat ventral tegmental area

Adenosine 3′,5′cyclic monophosphate-(cAMP)-dependent protein kinase (PKA) modulation of glycine-activated Cl − currents ( I Gly) in single neurons freshly isolated from the rat ventral tegmental area (VTA) was studied using whole-cell patch-clamp technique. In the majority of cells tested with Mg-ATP in the internal solution, I Gly induced by 3–10 μM glycine increased spontaneously (ran up). In the absence of internal ATP, I Gly remained stable in six of seven cells. External perfusion of 8-Br-cAMP, a PKA activator, potentiated I Gly only in cells showing run-up. 8-Br-cAMP potentiated I Gly induced by low concentrations of glycine, but had no effect on the maximal current. When added to the pipette solution, H-89, a PKA inhibitor, blocked ATP and 8-Br-cAMP induced run-up of I Gly. In contrast, dialysis with chelerythrine, a PKC inhibitor, did not alter the run-up of I Gly. These results suggest that the PKA pathway modulates the activity of the glycine receptor/channel complex via enhancing the affinity of the receptor for glycine.

Characterization of desensitization in recombinant N-methyl- d-aspartate receptors: comparison with native receptors in cultured hippocampal neurons

In the present study we have characterized the effect of Ca 2+, glycine, and agonist concentration on inactivation and desensitization in native and recombinant N-methyl- d-aspartate (NMDA) receptors. In agreement with earlier studies on neurons, we found that in the presence of saturating glycine concentrations, lowering [Ca 2+] o, will decrease inactivation of NMDA receptors in cultured hippocampal neurons. However, unlike native NMDA receptors under the same recording conditions, recombinant receptors did not exhibit Ca 2+-dependent inactivation. We also show that the glycine-insensitive desensitization observed in the recombinant receptors is subunit dependent, as NR1a2A and NR1a2B receptors significantly desensitized while the NR1a2C combination did not. Furthermore, we show this form of desensitization in NR1a2A receptors is due to classic agonist-induced desensitization. In addition, we demonstrate the presence of glycine-dependent desensitization in recombinant receptors. The ability of glycine to inhibit desensitization correlates to the rank order of glycine's affinity for potentiating the peak response for each subtype. Finally, using ifenprodil in the presence of high and low glycine concentrations, we present evidence that both 2A-like and 2B-like subtypes of receptors can independently coexist in single neurons.

Ternary complex formation kinetics involving (1,4,7,10-tetraazacyclododecane)nickel(II) and bidentate ligands

The kinetics of reactions of (1,4,7,10-tetraazacyclododecane)nickel(II), NiT 2+, with 1,10-phenanthroline, ethylenediamine, and glycine were studied at 25.0°C and μ = 0.10 mol dm −3 (NaCl), T is a tetradentate ligand. All reactions were first order in NiT 2+ and reversible. The reaction with 1,10-phenanthroline was first order in 1,10-phenanthroline with the formation rate constant k NiL phen = 1.31 × 10 4 dm 3 mol −1 s −1 and the dissociation rate constant k d = 19.6 s −1, followed the Eigen-Tamm mechanism. Ring closure was rate determining. The reaction involving ethylenediamine was resolved into the terms with formation rate constants k NiL en = 1.61 × 10 5 dm 3 mol −1, k NiL Hen = 4.64 × 10 3 dm 3 mol −1 s −1, and dissociation rate constants k NiT(en) = 4.55 s −1 and k H NiT(en) = 9.62 × 10 7 dm 3 mol −1 s −1. Ring closure was rate determining in the en pathway but not in the Hen + pathway. The reaction involving glycine varies in order as a function of the glycine concentration, being first order in glycine at low glycine concentration and shifting toward a zero-order dependence a high glycine concentration. The mechanism involves a singly O-bonded LNiON + intermediate in rapid equilibrium with reactants, K 1 = 1.10 × 10 3 dm 3 mol −1, followed by a slow ring closure, k 2 = 5.29 × 10 2 s −1. The dissociation rate constant is k d = 6.00 s −1. The important factors affecting the rates and the mechanisms of these reactions are the steric and inductive effects of the coordinated tetradentate ligand, the conformation angle distortion involving the “bite angle” of the two open coordination sites on NiL 2+, and the internal conjugate base effect of the attacking bidentate ligand.

Glycine receptor reduction within segmental gray matter in a rat model of neuropathic pain

Glycine is an amino acid neurotransmitter found in the spinal cord and is closely associated with interneurons that modulate afferent activity. We have previously shown that low segmental glycine concentrations or blockade of normal glycinergic activity lowers the threshold for pain thresholds. In addition, intrathecal glycine infusion increases the pain threshold in animal models of neuropathic pain. However, the role of the glycine receptor in neuropathic pain is not clear and is the basis for the current study. Using a unilateral sciatic nerve constriction injury model of neuropathic pain, the strychnine sensitive glycine receptor population was studied using immunohistochemical techniques. Glycine receptors are reduced in number in the dorsal horn bilaterally in injured animals. Glycine and related compounds are potentially valuable agents for treating chronic pain conditions in humans. A better understanding of glycine-receptor interactions should prove valuable as these compounds are studied in greater depth. [Neural Res 1998; 20: 161–168]

Neurotrophin Modulation of NMDA Receptors in Cultured Murine and Isolated Rat Neurons

Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons. J. Neurophysiol. 78: 2363-2371, 1997. Patch-clamp and calcium imaging techniques were used to assess the acute effects of the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF), on the responses of cultured and acutely isolated hippocampal and cultured striatal neurons to the glutamate receptor agonist N-methyl--aspartic acid (NMDA). The effects of BDNF on NMDA-activated currents were examined in greater detail. Currents evoked by NMDA, and the accompanying changes in intracellular calcium, were enhanced by low concentrations of the neurotrophins (1-20 ng/ml). The potentiation by the neurotrophins was rapid in onset and offset (<1 s). The neurotrophins also reduced desensitization of these currents in most cells. The enhancement of NMDA-activated currents by BDNF was observed using both perforated and whole cell patch recording techniques and could be demonstrated in outside-out patches. Furthermore, its effects were not attenuated by pretreatment with the protein kinase inhibitors genistein or 1-(5-isoquinolynesulfony)2-methylpiperazine (H7). Therefore, the actions of BDNF do not appear to be mediated by phosphorylation. Similar enhancements were observed with NT-3 and NT-4 and with NGF despite the fact that hippocampal neurons lack TrkA receptors. All together this evidence suggests that the enhancement of NMDA-evoked currents is unlikely to be mediated through the activation of growth factor receptors. Modulation of NMDA responses by BDNF was dependent on the concentration of extracellular glycine. The most pronounced potentiation by BDNF was observed at low concentrations, whereas no potentiation was observed in saturating concentrations of glycine, suggesting that BDNF may have increased the affinity of the NMDA receptor for glycine. However, the competitive glycine-site antagonist 7-chloro-kynurenic acid blocked the enhancement by BDNF without shifting the dose-inhibition relationship for this antagonist, and Mg2+ consistently depressed the potentiation of NMDA-evoked currents by BDNF, indicating that BDNF does not alter glycine affinity. BDNF also reversibly increased the probability of opening of NMDA channels recorded from outside-out patches taken from cultured hippocampal neurons. Other unrelated peptides including dynorphin and somatostatin also caused a glycine-dependent enhancement of NMDA currents and depressed the currents in saturating concentrations of glycine. In contrast, a shortened analogue dynorphin (6-17), which lacks N-terminus glycine residues, and another peptide met-enkephalin were without effects on NMDA currents recorded in low concentrations of glycine. Our results suggest that neurotrophins and other peptides can serve as glycine-like ligands for the NMDA receptor.

Potentiation of NMDA receptor-mediated responses by dynorphin at low extracellular glycine concentrations.

The effect of dynorphin A(1-13) on N-methyl-D-aspartate (NMDA)-activated currents was investigated in the presence of low extracellular glycine concentrations in Xenopus oocytes expressing recombinant heteromeric NMDA receptors and in cultured hippocampal neurons with the use of voltage-clamp techniques. At an extracellular added glycine concentration of 100 nM, dynorphin A(1-13) (10 microM) greatly increased the amplitude of NMDA-activated currents for all heteromeric subunit combinations tested; on average, the potentiation was: epsilon1/zeta1, 3,377 +/- 1,416% (mean +/- SE); epsilon2/zeta1, 1,897 +/- 893%; epsilon3/zeta1, 4,356 +/- 846%; and epsilon4/zeta1, 1,783 +/- 503%. Potentiation of NMDA-activated current by dynorphin A(1-13) was concentration dependent between 0.1 and 10 microM dynorphin A(1-13), with a half-maximal concentration value of 2.77 microM and an apparent Hill coefficient of 2.53, for epsilon2/zeta1 subunits at 100 nM added extracellular glycine. Percentage potentiation by dynorphin A(1-13) was maximal at the lowest glycine concentrations tested (0.01 and 0.1 microM), and decreased with increasing glycine concentration. No significant potentiation was observed at glycine concentrations > 0.1 microM for epsilon1/zeta1, epsilon2/zeta1, and epsilon4/zeta1 subunits, or at > 1 microM for epsilon3/zeta1 subunits. Potentiation of NMDA-activated currents by dynorphin A(1-13) was not inhibited by 1 microM of the kappa-opioid receptor antagonist nor-binaltorphimine, and potentiation was not observed with 10 microM of the kappa-opioid receptor agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzene-acetamide. Potentiation of NMDA-activated current by dynorphin A(1-13) was inhibited by the glycine antagonist kynurenic acid (50 microM). NMDA-activated current was also potentiated at low glycine concentrations by 10 microM dynorphin A(2-13) or (3-13), both of which have a glycine as the first amino acid, but not by 10 microM dynorphin A(4-13), which does not have glycine as an amino acid. In hippocampal neurons, 10 microM dynorphin A(1-13) or (2-13) potentiated steady-state NMDA-activated current in the absence of added extracellular glycine. The extracellular free glycine concentration, determined by high-performance liquid chromatography, was between 26 and 36 nM for the bathing solution in presence or absence of 10 microM dynorphin A(1-13), (2-13), (3-13), or (4-13), and did not differ significantly among these solutions. The observations are consistent with the potentiation of NMDA-activated current at low extracellular glycine concentrations resulting from an interaction of the glycine amino acids in dynorphin A(1-13) with the glycine coagonist site on the NMDA receptor. Because dynorphin A is an endogenous peptide that can be coreleased with glutamate at glutamatergic synapses, the potentiation of NMDA receptor-mediated responses could be an important physiological regulator of NMDA receptor function at these synapses.

Potentiation of chloride responses to glycine by three 5-HT3 antagonists in rat spinal neurones.

1. Modulations of Cl- responses to glycine by 5-hydroxytryptamine ligands were studied in cultured spinal neurones, by the whole-cell recording technique. 2. Three 5-HT3 antagonists were found to potentiate reversibly responses to low concentrations of glycine. Potentiations were induced by micromolar concentrations of LY-278,584 (1-10 microM) and by concentrations of MDL-72222 or ICS-205,930 between 10 nM and 1 microM. 3. Potentiations were observed over the whole voltage range without any change in the reversal potential of the glycine responses and without affecting the resting conductance. 4. The degree of potentiation was variable among cells. It increased with the concentration of the modulator, but only up to 100 nM for MDL-72222 and ICS-205,930. 5. The potentiation appeared to result from an increase in the affinity for glycine of glycine receptors. 6. Neither the blockade of glycine uptake by Na+ removal, nor the excision of membrane patches prevented the potentiation. 7. At high concentrations (10 microM), both MDL-72222 and ICS-205,930 had, in contrast, a blocking effect on glycine responses. 8. Potentiation by LY-278,584 and a dose-dependent modulation by MDL-72222 were also observed for taurine responses. 9. The effects on glycine responses of various ligands of 5-HT3 receptors (including agonists) are discussed. The ability of LY-278,584, MDL-72222 and ICS-205,930 to potentiate glycine responses appears to be independent of their known 5-HT3 receptor antagonist properties. It would be interesting to look for chemically related drugs that would be specific potentiators of glycine responses.

Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes.

1. Glycine responses were studied under voltage clamp in Xenopus oocytes injected with cDNA encoding mammalian glycine receptor subunits and in rat medullary neurones. Bath application of glycine gave strychnine-sensitive currents which reversed close to the expected equilibrium potentials for chloride ions. The peak currents for the receptors expressed in oocytes fitted a Hill equation with EC50 = 215 +/- 5 microM and Hill coefficient nH = 1.70 +/- 0.05 (means +/- s.e. means). The peak currents from the receptors in medullary neurones fitted a Hill equation with EC50 = 30 +/- 1 microM and Hill coefficient nH = 1.76 +/- 0.08. The current-voltage relationship for the receptors expressed in oocytes showed strong outward rectification (with Vrev = -21 +/- 2 mV), while that for the glycine responses from the medullary neurones in symmetrical Cl- was linear (with Vrev = 3.2 +/- 0.6 mV). 2. Inhalational general anaesthetics, at concentrations close to their human minimum alveolar concentrations (MACs), potentiated responses to low concentrations of glycine. The potentiation observed with the recombinant receptors (between 60-22%) was approximately twice that found with the medullary neurones (between 40-80%). For both the recombinant receptors and the receptors in medullary neurones, the degree of potentiation increased in the order of methoxyflurane approximately sevoflurane < halothane approximately isoflurane approximately enflurane. There was no significant difference between the potentiations observed for the two optical isomers of isoflurane. 3. For both the recombinant and native receptors, isoflurane potentiated the currents in a dose-dependent manner at low concentrations of glycine, although at high glycine concentrations the anaesthetic had no significant effect on the glycine-activated responses. The major effect of isoflurane was to cause a parallel leftward shift in the glycine concentration-response curves. The glycine EC50 concentration for the recombinant receptors decreased from a control value of 215 +/- 5 microM to 84 +/- 7 microM glycine at 610 microM isoflurane, while that for the medullary neurones decreased from a control value of 30 +/- 1 microM to 18 +/- 2 microM glycine at the same concentration of isoflurane. The potentiation was independent of membrane potential. 4. Isoflurane also potentiated responses to taurine, a partial agonist at the glycine receptor. This was observed for receptors expressed in oocytes at both low and saturating concentrations of taurine. The EC50 concentration decreased from a control value of 1.6 +/- 0.2 to 0.9 +/- 0.1 mM taurine in the presence of 305 microM isoflurane, while the maximum response to taurine increased from 47 +/- 2 to 59 +/- 2% of the maximum response to glycine. 5. Glycine receptors, like other members of the fast ligand-gated receptor superfamily, are sensitive to clinically relevant concentrations of inhalational general anaesthetics. Effects at these receptors may, therefore, play some role in the maintenance of the anaesthetic state.

Glycine enhances binding to the NMDA receptor complex in aged mice, but does not correct the aging change.

The purpose of this study was to determine whether glycine is effective in correcting the age-related change in the N-methyl-D-aspartate (NMDA) receptor complex. Autoradiographic analysis of [3H]MK801 and NMDA-displaceable [3H]glutamate binding was performed with 0, 10, 30 and 100 microM glycine on brain tissue from 3-, 10-, and 24-30-month-old C57B1 mice. Glycine, even at 10 microM, enhanced binding to both the NMDA and MK801 binding sites in all age groups. Glycine was not able to reduce the absolute difference in [3H]glutamate binding to NMDA sites between 3- and 24-month-olds. Low micromolar concentrations of glycine exacerbated the aging difference in [3H]MK801 binding between young and old mice, but 100 microM glycine produced smaller aging differences that were similar to those seen with no glycine. This study suggests that glycine may enhance NMDA receptor function in old animals, but it does not appear to correct the underlying problem with the NMDA receptor in aged animals.

Presynaptic NMDA Receptors Display Physiological Characteristics of Homomeric Complexes of NR1 Subunits that Contain the Exon 5 Insert in the N‐Terminal Domain

The subunit composition of the N-methyl-D-aspartate (NMDA) glutamate receptor affects both its channel activity and its sensitivity to modulation by a wide variety of substances. Expression studies in oocytes and physiological studies in neurons indicate that endogenous postsynaptic NMDA receptors are heterooligomeric complexes of NR1 and NR2 subunits. To deduce the subunit composition of the presynaptic NMDA receptor on noradrenergic nerve terminals, we examined the modulation of NMDA-evoked norepinephrine (NE) release from hippocampal synaptosomes. At high glycine concentrations, the NMDA-evoked release was not potentiated by reducing reagents, low micromolar Zn2+ or Ni2+, polyamines, or 100 microM histamine. It was also not inhibited by oxidizing agents or physiological concentrations of protons but was inhibited by high micromolar Co2+, Zn2+, and Ni2+, but not Fe3+, by high micromolar ifenprodil, and by 1 mM histamine. At low glycine concentrations, it was potentiated by spermine. These characteristics are similar to those displayed by homooligomeric complexes of NR1 subunits that contain in the N-terminal domain the 21-amino-acid insert encoded by exon 5. These data provide physiological evidence that some endogenous NMDA receptor complexes may contain only the NR1 (+ exon 5) subunits.

Functional N‐methyl‐D‐aspartate receptors in clonal rat phaeochromocytoma cells.

1. To characterize from a molecular and functional point of view the endogenous NMDA receptors expressed by phaeochromocytoma (PC12) cells, experiments involving polymerase chain reaction (PCR) amplification, Western blotting and patch-clamp analysis of undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells were performed. 2. Analysis of PC12 mRNA demonstrated the presence of NMDAR1 and NMDAR2C transcripts. The NMDAR1 subunits lack the amino terminal insert of twenty-one amino acid residues, where as transcripts with and without deletions I and II at the 3' end of the coding region were detected. Thus, NMDA receptors of the PC12 cells might include NMDAR1A, NMDAR1E, NMDAR1C and NMDAR1D subunits. 3. Differentiation by NGF treatment of PC12 cells did not alter mRNA expression for NMDA receptor subunits significantly but induced an increase in both the NMDAR1 protein and the total amount of functional receptors that correlated well with a parallel increase in membrane area. 4. NMDA receptors in differentiated PC12 cells had a high affinity for both glutamate and glycine. These were estimated kinetically as 0.59 microM and 74 nM, respectively. Responses to glutamate or NMDA were non-desensitizing in the presence of saturating glycine, but slowly desensitized with low concentrations of glycine. Currents were completely blocked by D-aminophosphonovalerate (APV), 7-Cl-kynurenate and phencyclidine, and showed a voltage-dependent magnesium blockade. Spermine did not potentiate but inhibited NMDA receptor-mediated responses in a voltage-independent manner. 5. With 0.5 mM Ca2+, single-channel analysis revealed very brief openings (mean open time (t(o)) = 0.42 ms), with at least two conductive states, 55 and 33 pS, both having markedly low open probability. At 2 mM Ca2+, conductances were reduced to 39 and 19 pS, without an effect in open probability or mean open time. 6. The functional properties of NMDA receptors in PC12 cells were very similar to those described for NMDAR1A-NMDAR2C heteromers recombinantly expressed. The PC12 cell line provides a simple and reproducible system to analyse some specific NMDA receptor properties.

Involvement of neuroexcitatory amino acids in the control of gonadotropin-releasing hormone release from the hypothalamus of the adult male guinea pig: predominantly inhibitory action of N-methyl-D-aspartate-mediated neurotransmission and its reversal after orchidectomy.

Excitatory amino acids (EAA) have been implicated in the control of LH secretion through facilitation of GnRH release, although disparate findings of an inhibitory effect of EAA on LH secretion in several species after gonadectomy have been reported. Using a static incubation system, we studied the effects of EAA receptor agonists and antagonists on in vitro GnRH release from the isolated medial basal hypothalamus (MBH) of the male guinea pig. In the presence of 0.4 mM glycine, the N-methyl-D-aspartate (NMDA)-specific receptor agonist N-methyl-D,L-aspartic acid (NMA) exerted a dose-dependent inhibition of GnRH output from the MBH of intact guinea pigs, which was significant (P < or = 0.01) at concentrations of 1 and 50 mM. The NMDA-specific receptor antagonist D,L-2-amino-5-phosphonovaleric acid (AP-5) significantly stimulated GnRH release at 10(-3) and 1 mM (P < or = 0.001) and was ineffective at 10(-6) mM. NMA (50 mM) and AP-5 (1 mM) produced similar effects when tested at a lower glycine concentration of 10 nM. The presence of the two compounds together resulted in overall unchanged GnRH output. The inhibitory effect of 50 mM NMA was also effectively blocked in the presence of 2 mM Mg2+ at both high and low glycine concentrations. When, for comparative purposes, isolated MBHs of 50-day-old intact male rats were exposed to 50 mM NMA, a response opposite to that seen in the intact guinea pig was observed, with a marked increase in GnRH output (P < or = 0.001). When tested at 10 nM glycine, 50 mM kainic acid, a non-NMDA-specific receptor agonist, on the other hand, had a marked stimulatory effect on GnRH output (P < or = 0.01) from intact guinea pig MBHs, an action that was prevented in the presence of the kainate/quisqualate receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (0.1 mM); 6,7-dinitro-quinoxaline-2,3-dione alone inhibited GnRH release (P < or = 0.01). In a separate series of experiments, the effects of EAA (in the presence of 10 nM glycine) on GnRH release from the MBHs of long term orchidectomized and sham-operated guinea pigs were compared. Orchidectomy led to a dramatic reversal of the NMDA-mediated inhibition of GnRH secretion observed in MBHs of sham-operated animals, with 50 mM NMA producing a marked increase (P < or = 0.0001) and 1 mM AP-5 resulting in a clear inhibition (P < or = 0.0001) of GnRH release. Kainic acid (50 mM), on the other hand, had a similar stimulatory action on GnRH release from the MBHs of both orchidectomized (P < or = 0.0001) and sham-castrated (P < or = 0.001) guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

分娩前後における乳牛蹄壁角質アミノ酸組成の変化

The changes in amino acid composition in the horny hoof wall of dairy cows were investigated during the periparturition period, in relation with actual milk yield, dry matter intake (DMI), total digestible nutrients (TDN) and crude protein (CP). The growth of the horny hoof wall was most rapid during the dry period as well as one month prior to and one monthfollowing calving. The horny hoof wall during the dry period was revealed to have lower concentrations of glycine, methionine and histidine and high levels of proline. The concentrations of aspartic acid, threonine, alanine, valline, isoleucine, tyrosine and arginine were closely correlated to milk yield and to DMI, TDN and CP, while those of serine, glycine, phenylalanine and lysine were not to DMI, TDN and CP. From the contents of hystidine, lysine, and arginine the horny hoof wall was considered to be most hardly keratinized during the dry period, while most softened following calving.

The hypoglycemic sulphonylurea tolbutamide increases [formula omitted]- but not kainate-activated currents in hippocampal neurons in culture

The effects of the hypoglycemic sulphonylurea tolbutamide, a marker of K +-ATP channels, on the N- methyl- D- aspartate -(NMDA) and kainate-activated currents were studied in rat hippocampal neurons in culture, using the patch-clamp technique in a whole-cell configuration. Tolbutamide (500 μM) reversibly increased the peak amplitude and the steady state level of NMDA- but not kainate-evoked currents. This effect was not glycine dependent as it was observed at low and saturated concentrations of glycine. The affinity of the NMDA receptor-channel complex for glycine did not change in the presence of tolbutamide. The action of tolbutamide on the NMDA-activated current was not mediated by K +-ATP channels since CsCl was added intracellularly at concentrations which completely blocked all K + channels. Possible mechanisms explaining the effect of tolbutamide via the modulation of intracellular messengers are discussed.

β‐Amyloid (25‐35) or Substance P Stimulates [3H]MK‐801. Binding to Rat Cortical Membranes in the Presence of Glutamate and Glycine

Micromolar concentrations of beta-amyloid (25-35) or substance P stimulated [3H]MK-801 binding in the presence of low concentrations of glutamate (1 microM) and glycine (0.02 microM). Unlike polyamines spermine and spermidine, neither beta-amyloid (25-35) nor substance P increased [3H]MK-801 binding in the presence of maximally stimulating concentrations of glutamate and glycine. 5,7-Dichloro-kynurenic acid, CGS-19755, and arcaine completely inhibited the stimulated [3H]MK-801 binding. There was an apparent decreased potency of the [3H]MK-801 binding inhibition curve for 5,7-dichlorokynurenic acid, but not CGS-19755 or arcaine, in the presence of either beta-amyloid (25-35) or substance P. The compounds do not appear to act through the strychnine-insensitive glycine binding site because neither beta-amyloid (25-35) nor substance P displaced [3H]glycine binding. Full-length beta-amyloid (1-40), up to 10 microM, did not stimulate [3H]MK-801 binding. Concentrations > 10 microM could not be tested because they formed large aggregate precipitates in the assay. The data indicate that beta-amyloid (25-35) or substance P does not stimulate [3H]MK-801 binding at either the N-methyl-D-aspartate, glycine, or polyamine binding sites. Furthermore, the nonpeptide substance P receptor (NK1) antagonist, CP-96,345, did not block beta-amyloid (25-35)- or substance P-stimulated [3H]MK-801 binding. Therefore, the effect is not due to an interaction between the substance P receptors and the N-methyl-D-aspartate receptor-operated ionophore. Finally, if these observations can be verified using single-channel recording techniques, they may have implications in the pattern of selective neuronal loss observed in patients with neurodegenerative processes such as Alzheimer's, Parkinson's, and Huntington's diseases.

Perturbations biologiques au cours des résections intra-utérines sous irrigation de glycocolle

This study was carried out to assess the relations between plasma glycine concentrations and the biochemical changes occurring during intra-uterine resections (IUR) under glycine irrigation. Sixty patients with benign uterine conditions were included. They were all ranked ASA 1 or 2. The biological parameters were assessed before surgery (T0), at the end of surgery (T1) and 60 min afterwards (T2). They included the blood count and blood concentrations of sodium, potassium, chloride, proteins, bicarbonates, glucose, urea nitrogen, creatinine and glycine. Plasma osmolarity was calculated. The irrigation of the uterine cavity resulted in an increase of glycine concentrations (28 % of cases), and a decrease of sodium (22 % of cases), proteins and haematocrit (32 % of cases). Mean osmolarity remained unchanged. Blood glycine concentrations were directly correlated with the volume of irrigating solution, as well as with the duration of surgery. Protidaemia was inversely related to the blood glycine concentration. Multiparous patients had lower glycine concentrations than nulliparous patients. This was probably due to the uterine cavity being less compliant in the latter. On the other hand, there was no correlation with the uterine pathological condition. In this series, five cases of uterine perforation occurred with very large biological variations, especially a decrease in haematocrit and osmolarity. In these cases a clinical and biological water intoxication syndrome may occur as a result of large volumes of irrigating fluid passing into the peritoneal cavity.