Purification and characterization of the glycine receptor of pig spinal cord

A large-scale purification procedure was developed to isolate the glycine receptor of pig spinal cord by affinity chromatography on aminostrychnine agarose. After an overall purification of about 10 000-fold, the glycine receptor preparations contained three major polypeptides of Mr 48 000, 58 000, and 93 000. Photoaffinity labeling with [3H]strychnine showed that the [3H]strychnine binding site is associated with the Mr 48 000 and, to a much lesser extent, the Mr 58 000 polypeptides. [3H]Strychnine binding to the purified receptor exhibited a dissociation constant KD of 13.8 nM and was inhibited by the agonists glycine, taurine, and beta-alanine. Gel filtration and sucrose gradient centrifugation gave a Stokes radius of 7.1 nm and an apparent sedimentation coefficient of 9.6 S. Peptide mapping of the [3H]strychnine-labeled Mr 48 000 polypeptides of purified pig and rat glycine receptor preparations showed that the strychnine binding region of this receptor subunit is highly conserved between these species. Also, three out of six monoclonal antibodies against the glycine receptor of rat spinal cord significantly cross-reacted with their corresponding polypeptides of the pig glycine receptor. These results show that the glycine receptor of pig spinal cord is very similar to the well-characterized rat receptor protein and can be purified in quantities sufficient for protein chemical analysis.     

Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels.

Functional maturation of the nicotinic acetylcholine receptor is executed by its gamma-to-epsilon subunit switching. The glycine receptor also has fetal (alpha 2) and adult (alpha 1) isoforms. However, whether subunit switching is responsible for developmental changes in glycine receptor function is not known. We recorded single-channel currents from homomeric glycine receptors expressed in Xenopus oocytes with cRNAs encoding the alpha 2 or alpha 1 subunits and compared them with those recorded from native glycine receptors in rat spinal neurons at various ontogenic periods. The mean channel life times of the alpha 1 and mature glycine receptors were equally short, whereas both the alpha 2 and fetal receptors showed a significantly longer open time. Consistent with these results, the decay time of the glycinergic inhibitory postsynaptic currents (IPSCs) in spinal neurons became shorter during postnatal development. We conclude that developmental switching of alpha subunits may accelerate the kinetics of IPSCs.     

Molecular evidence for a glycine-gated chloride channel in macrophages and leukocytes

Recent studies have demonstrated that glycine blunts the response of Kupffer cells to endotoxin. Based on pharmacological evidence, it was hypothesized that Kupffer cells and other macrophages contain a glycine-gated chloride channel similar to the glycine receptor expressed in neuronal tissues. Moreover, glycine stimulates influx of radiolabeled chloride in Kupffer cells in a dose-dependent manner. RT-PCR was used to identify mRNA of both alpha- and beta-subunits of the glycine receptor in rat Kupffer cells, peritoneal neutrophils, and splenic and alveolar macrophages, similar to the sequence generated from rat spinal cord. Importantly, the sequence of the cloned Kupffer cell glycine receptor fragment for the beta-subunit was >95% homologous with the receptor from the spinal cord. Membranes of these cells also contain a protein that is immunoreactive with antibodies against the glycine-gated chloride channel. These data demonstrate that Kupffer cells, as well as other macrophages and leukocytes, express mRNA and protein for a glycine-gated chloride channel with both molecular and pharmacological properties similar to the channel expressed in the central nervous system.     

Functional chloride channels by mammalian cell expression of rat glycine receptor subunit

Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.     

Direct Effect of Remifentanil and Glycine Contained in Ultiva? on Nociceptive Transmission in the Spinal Cord: In Vivo and Slice Patch Clamp Analyses

Background

Ultiva® is commonly administered intravenously for analgesia during general anaesthesia and its main constituent remifentanil is an ultra-short-acting μ-opioid receptor agonist. Ultiva® is not approved for epidural or intrathecal use in clinical practice. Previous studies have reported that Ultiva® provokes opioid-induced hyperalgesia by interacting with spinal dorsal horn neurons. Ultiva® contains glycine, an inhibitory neurotransmitter but also an N-methyl-D-aspartate receptor co-activator. The presence of glycine in the formulation of Ultiva® potentially complicates its effects. We examined how Ultiva® directly affects nociceptive transmission in the spinal cord.

Methods

We made patch-clamp recordings from substantia gelatinosa (SG) neurons in the adult rat spinal dorsal horn in vivo and in spinal cord slices. We perfused Ultiva® onto the SG neurons and analysed its effects on the membrane potentials and synaptic responses activated by noxious mechanical stimuli.

Results

Bath application of Ultiva® hyperpolarized membrane potentials under current-clamp conditions and produced an outward current under voltage-clamp conditions. A barrage of excitatory postsynaptic currents (EPSCs) evoked by the stimuli was suppressed by Ultiva®. Miniature EPSCs (mEPSCs) were depressed in frequency but not amplitude. Ultiva®-induced outward currents and suppression of mEPSCs were not inhibited by the μ-opioid receptor antagonist naloxone, but were inhibited by the glycine receptor antagonist strychnine. The Ultiva®-induced currents demonstrated a specific equilibrium potential similar to glycine.

Conclusions

We found that intrathecal administration of Ultiva® to SG neurons hyperpolarized membrane potentials and depressed presynaptic glutamate release predominantly through the activation of glycine receptors. No Ultiva®-induced excitatory effects were observed in SG neurons. Our results suggest different analgesic mechanisms of Ultiva® between intrathecal and intravenous administrations.     

Glycine receptor heterogeneity in rat spinal cord during postnatal development.

Two different isoforms of the inhibitory glycine receptor were identified during postnatal development of rat spinal cord. A neonatal form characterized by low strychnine binding affinity, altered antigenicity, and a ligand binding subunit differing in mol. wt (49 kd) from that of the adult receptor (48 kd) predominates at birth (70% of the total receptor protein). Separation from the adult form could be achieved by either use of a selective antibody or glycine gradient elution of 2-aminostrychnine affinity columns. Both isoforms co-purify with the mol. wt 93 kd peripheral membrane protein of the postsynaptic glycine receptor complex.     

Asymmetric cross-inhibition between GABAA and glycine receptors in rat spinal dorsal horn neurons

Presynaptic nerve terminals of inhibitory synapses in the dorsal horn of the spinal cord and brain stem can release both GABA and glycine, leading to coactivation of postsynaptic GABAA and glycine receptors. In the present study we have analyzed functional interactions between GABAA and glycine receptors in acutely dissociated neurons from rat sacral dorsal commissural nucleus. Although the application of GABA and glycine activates pharmacologically distinct receptors, the current induced by a simultaneous application of these two transmitters was less than the sum of currents induced by applying two transmitters separately. Sequential application of glycine and GABA revealed that the GABA-evoked current is more affected by glycine than glycine-evoked responses by GABA. Activation of glycine receptors decreased the amplitude and accelerated the rate of desensitization of GABA-induced currents. This asymmetric cross-inhibition is reversible, dependent on the agonist concentration applied, but independent of both membrane potential and intracellular calcium concentration or changes in the chloride equilibrium potential. During sequential applications, the asymmetric cross-inhibition was prevented by selective GABAA or glycine receptor antagonists, suggesting that occupation of binding sites did not suffice to induce glycine and GABAA receptors functional interaction, and receptor channel activation is required. Furthermore, inhibition of phosphatase 2B, but not phosphatase 1 or 2A, prevented GABAA receptor inhibition by glycine receptor activation, whereas inhibition of phosphorylation pathways rendered cross-talk irreversible. Taken together, our results demonstrated that there is an asymmetric cross-inhibition between glycine and GABAA receptors and that a selective modulation of the state of phosphorylation of GABAA receptor and/or mediator proteins underlies the asymmetry in the cross-inhibition.     

Sensitive Immunoassay Shows Selective Association of Peripheral and Integral Membrane Proteins of the Inhibitory Glycine Receptor Complex

The inhibitory glycine receptor of mammalian spinal cord is a ligand-gated chloride channel that, on affinity purification, contains two subunits of 48-kilodalton (kD) and 58-kD molecular mass in addition to an associated 93-kD protein. Ligand-binding 48-kD subunit and 93-kD protein were quantified in the CNS of the adult rat using a newly developed dot receptor assay (detection limit less than or equal to 1 fmol/assay) which employs monoclonal antibodies specific for glycine receptor polypeptides. The 93-kD protein was found to codistribute at a fixed stoichiometry with the 48-kD subunit throughout the CNS of the rat. Moreover, the 93-kD protein cofractionated with the ligand-binding subunit on solubilization and affinity chromatography or immunoprecipitation. However, both proteins were separated on sucrose gradient centrifugation of detergent extracts of spinal cord membranes in accord with earlier observations on purified receptor. These data prove that the 93-kD polypeptide is selectively associated with the membrane core of the strychnine-sensitive glycine receptor. The regional distribution of glycine receptor polypeptides was also determined in the CNS of the spastic rat mutant. In contrast to hereditary spasticity in mouse and cattle, no reduction of glycine receptors was found in the spastic rat.     

The general anesthetic pentobarbital slows desensitization and deactivation of the glycine receptor in the rat spinal dorsal horn neurons

Although many general anesthetics have been found to produce anesthetic and analgesic effects by augmenting GABA(A) receptor (GABA(A)R) function, the role of the glycine receptor (GlyR) in this process is not fully understood at the neuronal level in the spinal cord. We investigated the effects of a barbiturate general anesthetic, pentobarbital (PB), on the glycinergic miniature inhibitory postsynaptic currents (mIPSCs) and the responses to exogenously applied glycine, or taurine, a low affinity GlyR agonist, by using the whole-cell patch-clamp technique in the rat spinal dorsal horn neurons isolated using a novel mechanical method. Bath application of 30 microm PB significantly prolonged the decay time constant of the spontaneous glycinergic mIPSC without changing its amplitude and frequency. Co-application of 0.3 mm PB reduced the peak amplitude, affected the macroscopic desensitization and deactivation of the response to externally applied Gly in a concentration-dependent manner. In addition, the recovery of Gly response from desensitization was also prolonged by PB. However, PB did not change the desensitization and deactivation kinetics of the taurine-induced response. The GABA(A)R antagonist bicuculline (10 microm) did not affect the effect of PB on the Gly response. Thus, PB prolonged the spinal glycinergic mIPSCs by slowing desensitization and deactivation of GlyR. Two other structurally different intravenous anesthetics, i.e. propofol (10 microm) and etomidate (3 microm), prolonged the duration of the glycinergic mIPSC in the rat spinal dorsal horn neurons. In conclusion, on GlyR-Cl(-) channel complexes there may exist action site(s) of intravenous general anesthetics. GlyR and glycinergic neurotransmission may play an important role in the modulation of general anesthesia in the mammalian spinal cord.     

Functional expression in Xenopus oocytes of the strychnine binding 48 kd subunit of the glycine receptor.

The inhibitory postsynaptic glycine receptor (GlyR) of rat spinal cord is an oligomeric transmembrane protein which forms an agonist-gated anion channel. Expression in Xenopus oocytes of its mol. wt 48,000 subunit generated glycine-gated chloride channels which were analysed by voltage clamp. The agonist and antagonist response properties as well as the desensitization characteristics of these 48 kd subunit receptors resembled GlyRs expressed from spinal cord poly(A)+ RNA. These data indicate that the 48 kd subunit is capable of assembling into a functional receptor homo-oligomer which displays the pharmacology characteristic of the spinal cord GlyR.     

Strychnine-Sensitive, Glycine-Induced Release of [3H]Norepinephrine from Rat Hippocampal Slices

The amino acid glycine is the primary inhibitory neurotransmitter of the mammalian spinal cord. Glycine has also been shown to facilitate the excitatory actions of glutamate at the N-methyl-D-aspartic acid receptor subtype. In this article, glycine is shown to increase the Ca2(+)-dependent release of [3H]norepinephrine from preloaded slices of the rat hippocampus. This effect was inhibited noncompetitively by nanomolar concentrations of strychnine, which differentiates it from the glycine site associated with the N-methyl-D-aspartate receptor. Glycine also released [3H]acetylcholine, but was without effect on the efflux of [3H]serotonin or gamma-[3H]aminobutyric acid from the same tissue preparation. The release of [3H]norepinephrine was reversibly blocked by tetrodotoxin, indicating the effect is not initiated at the noradrenergic terminals, but requires propagation of an action potential. The results suggest that a glycine site that is pharmacologically similar to that found in the spinal cord exists in the rat hippocampus. We suggest that this site may participate in modulating the release of specific neurotransmitters in the brain.     

Glycinergic ligands modulate the rate of phosphorylation of the glycine receptor by protein kinase C.

The alpha subunit of the glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C (Ruiz-Gómez et al., (1991) J. Biol. Chem. 266, 559-566). We report here that the rate of phosphorylation of the glycine receptor by this kinase is higher in the presence of agonists (glycine, beta-alanine) than in the presence of antagonists (strychnine, RU-5135). These results suggest that activated glycine receptors would be a preferential target for functional regulation through phosphorylation mechanisms.     

Single-channel currents underlying glycinergic inhibitory postsynaptic responses in spinal neurons.

Single-channel properties of glycine receptors have been characterized so far only in cultured neurons. To characterize the glycine receptor channels in situ, we applied the patch-clamp technique to spinal neurons in slice preparations. Glycine-gated, single-channel currents were recorded in outside-out patches excised from spinal neurons. In the falling phase of glycinergic inhibitory synaptic currents, single-channel currents were resolved as discrete steps. In both cases, the glycine-gated channels showed similar multiple conductance levels. These results suggest that the receptor channel properties are indistinguishable in the synaptic and extrasynaptic sites. We conclude that multiple conductance states of a receptor channel are the native feature of the glycine receptor in situ.     

Book Review

1-Aminocyclopropane carboxylic acid (ACPC) competitively inhibited (IC50, 38 +/- 7 nM) [3H]glycine binding to rat forebrain membranes but did not affect [3H]strychnine binding to rat brainstem/spinal cord membranes. Like glycine, ACPC enhanced 3H-labelled (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate ([3H]MK-801) binding to N-methyl-D-aspartate receptor-coupled cation channels (EC50, 135 +/- 76 nM and 206 +/- 78 nM for ACPC and glycine, respectively) but was approximately 40% less efficacious in this regard. The maximum increase in [3H]MK-801 binding produced by a combination of ACPC and glycine was not different from that elicited by glycine, but both compounds potentiated glutamate-stimulated [3H]MK-801 binding. These findings indicate that ACPC is a potent and selective ligand at the glycine modulatory site associated with the N-methyl-D-aspartate receptor complex.     

Modulation of Glycinergic Transmission in the Rat Spinal Dorsal Commissural Nucleus by Ginkgolide B

The action of ginkgolide B (GB), a powerful compound of Ginkgo biloba extract, on glycine-mediated spontaneous currents in rat spinal sacral dorsal commissural nucleus (SDCN) neurons was examined. IPSCs evoked in spinal cord slices were inhibited in a dose-dependent manner by the addition of GB to the superfusion solution. The amplitude of eIPSCs was reduced to 61 ± 6.4% by 10 μM GB with acceleration of the kinetics of the currents, indicating the effect of GB on channel pores. Both the amplitude and success ratio (R_suc) of eIPSC induced by electrical focal stimulation of single glycinergic nerve endings (boutons) also changed in the presence of 1 μM GB. These data suggest that GB modulates not only post-synaptic glycine receptors but also the pre-synaptic glycine release machinery.     

Glycine Binding to Rat Cortex and Spinal Cord: Binding Characteristics and Pharmacology Reveal Distinct Populations of Sites

Glycine is the principal inhibitory neurotransmitter in posterior regions of the brain. In addition, glycine serves as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) acidic amino acid receptor subtype. The studies presented here characterize [3H]glycine binding to washed membranes prepared from rat spinal cord and cortex, areas enriched in glycine inhibitory and NMDA receptors, respectively, in an attempt to define the glycine recognition sites on the two classes of receptors. Specific binding for [3H]glycine was seen in both cortex and spinal cord. Saturation analyses in cortex were best fitted by a two-site model with respective equilibrium dissociation constants (KD values) of 0.24 and 5.6 microM and respective maximal binding constants (Bmax values) of 3.4 and 26.7 pmol/mg of protein. Similar analyses in spinal cord were best fitted by a one-site model with a KD of 5.8 microM and Bmax of 20.2 pmol/mg of protein. Na+ had no effect on [3H]glycine binding to cortical membranes but increased the binding to spinal cord membranes by greater than 15-fold. This Na+-dependent binding may reflect glycine binding to the recognition site of the high-affinity, Na+-dependent glycine uptake system. Several short-chain, neutral amino acids displaced [3H]glycine binding from both cortical and spinal cord membranes. The most potent displacers of [3H]glycine binding to cortical membranes were D-serine and D-alanine, followed by the L-isomers of serine and alanine and beta-alanine. In contrast, D-serine and D-alanine were similar in potency to L-serine in spinal cord membranes. Compounds active at receptors for the acidic amino acids had disparate effects on the binding of [3H]glycine. At 10 microM, NMDA resulted in a 25% increase, whereas D- and L-2-amino-5-phosphonovaleric acid at 100 microM resulted in a 30% decrease, in [3H]glycine binding to cortical membranes. Kynurenic acid was the most potent of the acidic amino acid-related compounds at displacing [3H]glycine binding. In cortical membranes, kynurenic acid displacement was resolved into a high- and a low-affinity component; the high-affinity component displaced the high-affinity component of [3H]glycine binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alterations in brain metabolism induced by chronic morphine treatment: NMR studies in rat CNS

High-resolution (500 MHz) multiresonance/multinuclear proton (¹H) nuclear magnetic resonance (NMR) spectroscopy was used to detect metabolic changes and cellular injury in the rat brain stem and spinal cord following chronic morphine treatment. Compensatory changes were observed in glycine, glutamate, and inositols in the brain stem, but not the spinal cord, of chronic morphine-treated rats. In spinal cord, increases were detected in lactate and N-acetyl-aspartate (NAA), suggesting that there is anaerobic glycolysis, plasma membrane damage, and altered pH preferentially in the spinal cord of chronic morphine-treated rats.