1053 Anesthesiology 1999; 91:1053–63 © 1999 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Direct Inhibition of the N-methyl-D-aspartate Receptor Channel by High Concentrations of Opioids Tomohiro Yamakura, M.D.,* Kenji Sakimura, Ph.D.,† Koki Shimoji, M.D.‡ Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/91/4/1053/397882/0000542-199910000-00026.pdf by guest on 24 September 2021 Background: Electrophysiologic and receptor binding studies HIGH concentrations of various opioid agonists and an- showed that some opioids have noncompetitive N-methyl-D- tagonists protect neurons against central nervous system aspartate (NMDA) receptor antagonist properties. ischemia and injury and neurotoxicity of exogenously Methods: The effects and mechanisms of action of various 1–3 opioid compounds were examined on four kinds of hetero- applied N-methyl-D-aspartate (NMDA). More recent meric NMDA receptor channels, namely the e1/z1, e2/z1, e3/z1, electrophysiologic and receptor binding studies have and e4/z1 channels, expressed in Xenopus oocytes. Further- found that some opioid agonists, such as meperidine, more, the action sites of opioids on NMDA receptor channels methadone, and ketobemidone, reduce NMDA-induced were investigated by site-directed mutagenesis. depolarization in rat-brain slice preparations and inhibit Results: Meperidine inhibited four kinds of channels to a 3 similar extent with inhibitor concentrations for half-control [ H]MK-801 (dizocilpine) binding in rat cortical and fore- m 4–6 response (IC50s) of 210–270 M. Morphine, fentanyl, codeine, brain membranes. These studies suggest that some and naloxone also inhibited NMDA receptor channels with opioids have NMDA receptor antagonist properties. affinities comparable to meperidine. Opioid inhibition ex- However, it is uncertain whether the NMDA receptor hibited voltage dependence and was quite effective at nega- antagonist activity is a property common to various opi- tive potentials. Opioids also shifted the inhibition curve of Mg21 to the right. Furthermore, replacement of the con- oid compounds. In addition, mechanisms of NMDA re- served asparagine residue with glutamine in the channel- ceptor channel inhibition by opioids remain to be char- lining segment M2 of the z1 subunit, which constitutes the acterized. block sites of Mg21 and ketamine, reduced the sensitivity to Cloning and expression studies have revealed the mo- e opioids, whereas that of the 2 subunit barely affected the lecular heterogeneity of the NMDA receptor channel.7 opioid sensitivity. Conclusions: These results, together with previous findings, The mouse NMDA receptor channel is composed of at e z suggest that the low-affinity NMDA receptor antagonist activity least two families of subunits, the (rat NR2) and (rat is a common characteristic of various opioid compounds, and NR1) subfamilies of the glutamate receptor channel, that the inhibition is a result of channel-block mechanisms at which share amino acid sequence homology with the 21 the site, which partially overlaps with those of Mg and ket- subunits of the a-amino-3-hydroxy-5-methyl-4-isoxazole amine. This antagonist property of opioids may be clinically propionic acid (AMPA)– or kainate-selective glutamate significant in the spinal cord following epidural or intrathecal e z administration, after which the cerebrospinal fluid concentra- receptor channels. The functional properties of the / tions of some opioids reach the high micromolar level. (Key heteromeric NMDA receptor channels are determined words: Channel blocker; dissociative anesthetics; glutamate re- by the constituting e subunit species (e1–e4). The het- ceptor; subunit.) eromeric e1/z1, e2/z1, e3/z1, and e4/z1 channels exhibit different affinities for agonists and different sensitivities 21 * Instructor, Department of Anesthesiology. to Mg block and competitive and noncompetitive an- † Professor, Department of Cellular Neurobiology, Brain Research tagonists. The e1 and z1 subunit mRNAs are widely Institute. distributed in the brain, whereas the e2 subunit mRNA is ‡ Professor and Chairman, Department of Anesthesiology. expressed abundantly in the forebrain. The e3 subunit Received from the Departments of Anesthesiology and Cellular Neu- mRNA is predominantly found in the cerebellum, but the robiology, Niigata University School of Medicine, Niigata, Japan. Sub- e4 subunit mRNA is weakly expressed in the dienceph- mitted for publication January 19, 1999. Accepted for publication May alon and the brainstem. Reports on expression patterns 14, 1999. Supported by a grant-in-aid (no. 10671407) from the Japa- in the adult spinal cord are controversial. The z1, e1, and nese Ministry of Education, Science and Culture, Tokyo, Japan. e2 subunit mRNAs are the predominant transcripts de- Address reprint requests to Dr. Yamakura: Department of Anesthe- 8 z siology, Niigata University School of Medicine, 1-757 Asahimachi, Ni- tected in the mouse cervical cord, whereas the NR1- 1, igata 951-8510, Japan. Address electronic mail to: NR2C-e3, and NR2D-e4 subunit mRNAs are found in the [email protected] rat lumbar spinal cord.9 Anesthesiology, V 91, No 4, Oct 1999 1054 YAMAKURA ET AL. Fig. 1. Schematic representations of the proposed structure of glutamate receptor channel subunits. (A) Three transmem- brane segment topology model. (B) Amino acid sequences of segment M2 of the glutamate receptor channel subunits Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/91/4/1053/397882/0000542-199910000-00026.pdf by guest on 24 September 2021 selective for NMDA (e1, e2, e3, e4, and z1) and those selective for AMPA (a1, a2, a3, and a4). The box indicates the Q (glu- tamine)/R (arginine)/N (asparagine) site of the glutamate receptor channel sub- units, which determines the permeability and block by divalent cations. NMDA receptor channel subunits have four hydrophobic the e1/z1, e2/z1, e3/z1, and e4/z1 channels) were synthe- segments (M1–M4) within their central regions. According sized using pSPGRe1,13 pSPGRe2,14 pSPGRe3,15 to the three transmembrane segment model, segment M2 pSPGRe4,16 and pSPGRz1,15 respectively. The e2, e2- forms a reentrant membrane loop with both ends facing N589Q, z1, and z1-N598Q subunit-specific mRNAs (for the the cytoplasm, and the carboxyl-terminal region resides in expression of the e2-N589Q/z1, e2/z1-N598Q, and e2- the cytoplasm (fig. 1A).7 Site-directed mutagenesis has re- N589Q/z1-N598Q channels) were synthesized using vealed that the conserved asparagine (N) residues in seg- pBKSAe2,17 pBKSAe2-N589Q,11 pBKSAz1,18 and pBKSAz1- ment M2 of the e and z subunits govern both Mg21 block N598Q,11 respectively. The a1 and a2 subunit-specific mR- and Ca21 permeability of NMDA receptor channels, thus NAs for the a1/a2 AMPA-selective glutamate receptor indicating that segment M2 constitutes the ion-channel channel were synthesized using pSPGR1 and pSPGR2, re- pore of NMDA receptor channels.10,11 The position of this spectively.19 asparagine residue corresponds to that of glutamine (Q) or Xenopus laevis oocytes were injected with the wild- arginine (R) of the a subunits, which determines the Ca21 type or mutant e subunit–specific mRNA and the wild- permeability of the AMPA-selective glutamate receptor type or mutant z subunit–specific mRNA at a molar ratio channel 12 (fig. 1B). of 1:1, or with the a1 and a2 subunit–specific mRNAs at In the present investigation, we examined the effects a molar ratio of 10:1; the total amount of mRNAs injected and mechanisms of action of various opioid compounds per oocyte was approximately 0.6 ng for the e1/z1 and (phenylpiperidine derivatives meperidine and fentanyl, e2/z1 channels; 14 ng for the e3/z1 and e4/z1 channels; naturally occurring opioids morphine and codeine, and 4 ng for the e2-N589Q/z1, e2/z1-N598Q, and e2-N589Q/ opioid antagonist naloxone) on the e1/z1, e2/z1, e3/z1 z1-N598Q channels; and 10 ng for the a1/a2 channel. and e4/z1 heteromeric NMDA channels expressed in Xenopus oocytes. Furthermore, the action sites of opi- Electrophysiological Analyses oids on NMDA receptor channels were investigated by After incubation at approximately 19°C for 2 or 3 days, site-directed mutagenesis. whole-cell currents evoked by bath application of ago- nists for approximately 15 s were recorded at 270 mV membrane potential with a conventional two-micropi- Materials and Methods pette voltage clamp.19 The current responses of the wild-type and mutant e/z channels to 10 mML-glutamate Subunit-specific mRNA Preparation and Expression plus 10 mM glycine (almost saturating concentrations for in Xenopus Oocytes all e/z channels) were measured in Ba21-Ringer’s solu- Subunit-specific mRNAs were synthesized in vitro with tion to minimize the effects of secondarily activated SP6 or T3 RNA polymerase (Ambion MEGAscript) in the Ca21-dependent Cl- currents.20 The current responses of 7 presence of cap dinucleotides mGpppG. The e1, e2, e3, the a1/a2 channel to 100 mM kainate were measured in e4, and z1 subunit-specific mRNAs (for the expression of normal frog Ringer’s solution. For measurement of the Anesthesiology, V 91, No 4, Oct 1999 1055 CHANNEL BLOCK OF NMDA RECEPTORS BY OPIOIDS effects of opioids on NMDA receptor channels, opioids opioids, Kd(0) the equilibrium dissociation constant of were continuously perfused during the experiment. Pre- opioids at a membrane potential of 0 mV, z the charge of application of opioids in the absence of agonists did not opioids, d the portion of the membrane electric field produce any current response in either wild-type or sensed at the blocking site, E the membrane potential, F mutant channels. Agonists were applied three times suc- the Faraday constant, R the gas constant, and T the cessively during perfusion of opioids, and the effects on absolute temperature. The results obtained were statis- the second and third applications of agonists were aver- tically analyzed by the Student t test or one-way analysis Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/91/4/1053/397882/0000542-199910000-00026.pdf by guest on 24 September 2021 aged.