JOURNAL HOME CME HOME THIS MONTH PAST ISSUES ETOC COLLECTIONS
AUTHORS REVIEWERS EDITORIAL BOARD FEEDBACK RSS HELP
A&A International Anesthesia Research Society
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamakura, T.
Right arrow Articles by Shimoji, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamakura, T.
Right arrow Articles by Shimoji, K.
Anesth Analg 2000;91:225-229
© 2000 International Anesthesia Research Society

GENERAL ARTICLES

The Stereoselective Effects of Ketamine Isomers on Heteromeric N-Methyl-D-Aspartate Receptor Channels

Tomohiro Yamakura, MD*, Kenji Sakimura, PhD{dagger}, and Koki Shimoji, MD*

*Department of Anesthesiology, Niigata University School of Medicine; and {dagger}Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan

Address correspondence and reprint requests to Tomohiro Yamakura, MD, Department of Anesthesiology, Niigata University School of Medicine, 1-757 Asahimachi, Niigata 951-8510, Japan. Address e-mail to yamakura{at}med.niigata-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The effects of S(+)- and R(–)-ketamine on heteromeric N-methyl-D-aspartate receptor channels were investigated on the {epsilon}1/{zeta}1, {epsilon}2/{zeta}1, {epsilon}3/{zeta}1, and {epsilon}4/{zeta}1 channels expressed in Xenopus oocytes. S(+)-ketamine inhibited all four {epsilon}/{zeta} channels more effectively than R(–)-ketamine. The inhibitor concentrations for half-control response for S(+)-ketamine were quite similar among the four channels with 0.44–0.56 µM. However, the inhibitor concentrations for half-control response for R(–)-ketamine varied slightly among the four channels with 1.0 µM for {epsilon}2/{zeta}1 and {epsilon}3/{zeta}1 channels and 1.9–2.0 µM for {epsilon}1/{zeta}1 and {epsilon}4/{zeta}1 channels. Thus, the potency ratio of S(+)- and R(–)-ketamine for heteromeric channels was only slightly different among the {epsilon}/{zeta} channels.

Implications: The potency order and ratio of ketamine isomers for inhibition of N-methyl-D-aspartate receptor channels may not be so different between the brain region and the developmental stage.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Ketamine isomers differ in their analgesic and anesthetic properties, with S(+)-ketamine being more potent than R(–)-ketamine (1–3). However, more psychic emergence reactions are observed after R(–)-ketamine than S(+)-ketamine (2). Electrophysiological and receptor binding studies show that ketamine inhibits N-methyl-D-aspartate (NMDA) receptor channels in a stereoselective manner, with S(+)-ketamine being more potent than R(–)-ketamine (3–5).

The mouse NMDA receptor channel is composed of at least two families of subunits, the {epsilon} (rat NR2) and {zeta} (rat NR1) subfamilies of the glutamate receptor channel (6). The functional properties of {epsilon}/{zeta} heteromeric NMDA receptor channels are determined by the constituting {epsilon} subunit species ({epsilon}1–{epsilon}4). The heteromeric {epsilon}1/{zeta}1, {epsilon}2/{zeta}1, {epsilon}3/{zeta}1, and {epsilon}4/{zeta}1 channels exhibit different affinities for agonists and different sensitivities to Mg2+ block and competitive and noncompetitive antagonists (6). The {epsilon}1 and {zeta}1 subunit mRNAs are widely distributed in the brain, whereas the {epsilon}2 subunit mRNA is expressed abundantly in the forebrain (7). The {epsilon}3 subunit mRNA is predominantly found in the cerebellum, and the {epsilon}4 subunit mRNA is weakly expressed in the diencephalon and the brainstem (7). In the present investigation, we determined whether the potency order and ratio of optical isomers of ketamine are different among four kinds of heteromeric NMDA channels ({epsilon}1/{zeta}1, {epsilon}2/{zeta}1, {epsilon}3/{zeta}1, and {epsilon}4/{zeta}1 channels).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
This study was approved by the Committee for the Guidelines on Animal Experimentation of Niigata University. Subunit-specific mRNAs were synthesized in vitro with SP6 RNA polymerase (MEGAscript; Ambion, Inc., Austin, TX) in the presence of cap dinucleotides 7mGpppG. The {epsilon}1, {epsilon}2, {epsilon}3, {epsilon}4, and {zeta}1 subunit-specific mRNAs were synthesized by using pSPGR{epsilon}1, pSPGR{epsilon}2, pSPGR{epsilon}3, pSPGR{epsilon}4, and pSPGR{zeta}1, respectively (8). Xenopus laevis oocytes were injected with the {epsilon} and {zeta} subunit-specific mRNAs at a molar ratio of 1:1; the total amount of mRNAs injected per oocyte was ~0.6 ng for the {epsilon}1/{zeta}1 and {epsilon}2/{zeta}1 channels, and ~14 ng for the {epsilon}3/{zeta}1 and {epsilon}4/{zeta}1 channels.

After incubation at ~19°C for 2–3 days, whole-cell currents evoked by bath-application of agonists for ~15 s were recorded at -70 mV membrane potential with a conventional 2-micropipette voltage clamp. The current responses of {epsilon}/{zeta} channels to 10 µM L-glutamate plus 10 µM glycine were measured in Ba2+-Ringer’s solution to minimize the effects of secondarily activated Ca2+-dependent Cl- currents. For the measurement of ketamine effects on NMDA receptor channels, ketamine was continuously perfused during the experiment. Preapplication of ketamine in the absence of agonists produced no current response. Agonists were applied successively during perfusion of ketamine until no further inhibition by ketamine was observed. Three or four applications of agonists were necessary to fully establish inhibition, and the effects on the last application of agonists were evaluated. Ba2+-Ringer’s solution contained 115 mM NaCl, 2.5 mM KCl, 1.8 mM BaCl2, and 10 mM HEPES-NaOH (pH 7.2). The IC50 (inhibitor concentration for half-control response) and Hill coefficient values for ketamine of the {epsilon}/{zeta} channel were calculated according to the equation Rket = 1/[1 + (K/IC50)n], where Rket represents the relative response, K the concentration of ketamine isomers, and n the Hill coefficient. The EC50 (agonist concentration for half-maximum response) value for agonists of the {epsilon}/{zeta} channel was calculated according to the equation Rago = Fket/[1 + (EC50/A)n], where Rago represents the relative response, Fket the residual fraction of ketamine inhibition of responses to saturating concentrations of agonists, A the concentration of agonists, and n the Hill coefficient. The results obtained were statistically analyzed by using one-way analysis of variance (ANOVA) followed by Scheffé’s multiple comparison tests. P values < 0.05 were considered significant. Data were represented as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The sensitivities of four kinds of heteromeric NMDA receptor channels, {epsilon}1/{zeta}1, {epsilon}2/{zeta}1, {epsilon}3/{zeta}1, and {epsilon}4/{zeta}1 channels to ketamine isomers were examined by measuring current responses evoked successively by the application of 10 µM L-glutamate plus 10 µM glycine during continuous perfusion of ketamine isomers. Ketamine isomers inhibited the current responses of {epsilon}/{zeta} NMDA receptor channels in a use-dependent manner, i.e., three or four applications of agonists were necessary until no inhibition by ketamine was observed. After ketamine isomers were washed out, three to five applications of agonists almost fully recovered the current responses.

S(+)-ketamine (1 µM) inhibited the {epsilon}1/{zeta}1 channel more effectively than 1 µM R(–)-ketamine (Fig. 1A). The dose-inhibition relationships for ketamine isomers of four kinds of heteromeric channels were examined. Ketamine isomers inhibited four heteromeric channels in a concentration-dependent manner, and sensitivities to S(+)-ketamine were higher than R(–)-ketamine for all four {epsilon}/{zeta} channels (Fig. 1B). The IC50 values of the {epsilon}1/{zeta}1, {epsilon}2/{zeta}1, {epsilon}3/{zeta}1, and {epsilon}4/{zeta}1 channels for S(+)-ketamine were quite similar among the four channels with 0.44–0.56 µM, which were not significantly different (log[IC50] values were compared by using ANOVA, P > 0.34) (Table 1). However, the IC50 values for R(–)-ketamine varied slightly among the four channels (ANOVA followed by Scheffé’s multiple comparison tests, P < 0.001). The resulting ratios of IC50 values for R(–)/S(+)-ketamine were slightly different among {epsilon}/{zeta} channels (Table 1).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 1. Effects of S(+)- and R(–)-ketamine on heteromeric N-methyl-D-aspartate receptor channels. A, Representative tracings of inhibition of the {epsilon}1/{zeta}1 channel by 1 µM S(+)- and R(–)-ketamine. Inward current is downward. The duration of bath application of 10 µM L-glutamate plus 10 µM glycine is indicated by bars without taking into account the dead space time in the perfusion system (~2 s). Sensitivity to ketamine isomers was examined by measuring current responses during continuous perfusion of ketamine isomers (indicated by hatched column). After ketamine isomers were washed out, application of agonists three to five times fully recovered the current responses. B, The dose-inhibition relationships for S(+)- and R(–)-ketamine of four heteromeric {epsilon}/{zeta} channels. Each point represents the mean ± SEM of measurements on six to eight oocytes; SEM are indicated by bars when larger than the symbols. See Table 1 for inhibitor concentration for half-control response and Hill coefficient values calculated from these data. The control current responses (nA) of the {epsilon}1/{zeta}1, {epsilon}2/{zeta}1, {epsilon}3/{zeta}1, and {epsilon}4/{zeta}1 channels obtained before perfusion of ketamine isomers were 190–840, 190–900, 100–450, and 150–570, respectively.

 

View this table:
[in this window]
[in a new window]
  		Table 1. The IC50 and Hill Coefficient Values of Heteromeric NMDA Receptor Channels for S(+)- and R(-)-ketamine and Their IC50 Value Ratio (R/S).
 
We examined the effects of S(+)- and R(–)-ketamine on the dose-response relationships of NMDA receptor channels for agonists. S(+)- and R(–)-ketamine markedly suppressed the maximal current responses to saturating concentrations of L-glutamate (Fig. 2A). The EC50 values of the {epsilon}2/{zeta}1 channel for L-glutamate in the presence of 10 µM glycine during perfusion of 1 µM S(+)- and R(–)-ketamine were not significantly different from the control values (ANOVA, P > 0.20). Similarly, S(+)- and R(–)-ketamine inhibited the maximal current responses to glycine without affecting the EC50 values (Fig. 2B). These results suggest the noncompetitive inhibition of NMDA receptor channels by S(+)- and R(–)-ketamine.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Effects of S(+)- and R(–)-ketamine on the dose-response relationships of the {epsilon}2/{zeta}1 channel for L-glutamate and glycine. A, The dose-response relationships of the {epsilon}2/{zeta}1 channel for L-glutamate in the presence of 10 µM glycine before and during perfusion of 1 µM S(+)- and R(–)-ketamine. The measured current responses were normalized to the control current responses to 10 µM L-glutamate plus 10 µM glycine. The agonist concentrations for half-maximum response (µM) of the {epsilon}2/{zeta}1 channel for L-glutamate before and during perfusion of S(+)- and R(–)-ketamine were 0.98 ± 0.05, 0.92 ± 0.06, and 0.81 ± 0.07, respectively, and the Hill coefficient values for those were 1.41 ± 0.09, 1.34 ± 0.06, and 1.28 ± 0.09, respectively (n = 4). B, The dose-response relationships of the {epsilon}2/{zeta}1 channel for glycine in the presence of 10 µM L-glutamate before and during perfusion of 1 µM S(+)- and R(–)-ketamine. The agonist concentrations for half-maximum response (µM) of the {epsilon}2/{zeta}1 channel for glycine before and during perfusion of S(+)- and R(–)-ketamine were 0.30 ± 0.01, 0.27 ± 0.02, and 0.26 ± 0.02, respectively, and the Hill coefficient values for those were 1.49 ± 0.09, 1.46 ± 0.13, and 1.38 ± 0.05, respectively (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Cloning studies have revealed the molecular diversity of NMDA receptor channels. The identification of multiple subunits with distinct distribution, properties, and regulation implies that NMDA receptor channels are heterogeneous in their pharmacological properties, depending on the brain region and the developmental stage (6). However, we have demonstrated that S(+)-ketamine is more potent than R(–)-ketamine for four kinds of heteromeric NMDA receptor channels, and that the potency ratio varies only slightly among the channels (1.9–3.6). These results suggest that the potency order and ratio of ketamine isomers are not so different among the brain region and the developmental stage. Previous electrophysiological studies have shown that S(+)-ketamine is 1.9–3.5 times more potent than R(–)-ketamine at native NMDA receptor channels in hippocampal, cortical, and spinal cord neurons (4,5).

Both S(+)- and R(–)-ketamine inhibition of {epsilon}/{zeta} channels exhibited use-dependence for the onset and offset of block as described previously for racemic ketamine (9), which is consistent with the open channel block mechanism. The conserved asparagine residues in the channel-lining segment M2 of the {epsilon}2 and {zeta}1 subunits have been shown to constitute the block sites for Mg2+ and dissociative anesthetics (8,10). In our investigation, the IC50 values for S(+)-ketamine were quite similar among the four {epsilon}/{zeta} channels, whereas the IC50 values for R(–)-ketamine varied slightly among the channels. Because the asparagine residues in segment M2 are conserved for all {epsilon} and {zeta} subunits of NMDA receptor channels, additional residues might be involved in forming the action sites for R(–)-ketamine.

Clinical studies report that more psychic emergence reactions are observed after R(–)-ketamine than S(+)-ketamine (2). Because the potency order of ketamine isomers for inhibition of four {epsilon}/{zeta} channels is opposite of that for psychic emergence reactions, NMDA receptor channels are not likely to be the main targets for psychic emergence reactions. However, the analgesic and anesthetic effects of S(+)-ketamine are 3 to 4 times more potent than those of R(–)-ketamine (2,3). Furthermore, ketamine analgesia occurs at considerably smaller plasma levels (~0.5 µM) than those required for anesthesia (~10 µM) (11). The IC50 values of NMDA receptor channels for S(+)-ketamine were 0.44–0.56 µM, and those for R(–)-ketamine were 1.0–2.0 µM.

Thus, the high sensitivity of NMDA receptor channels to ketamine, together with the similar potency order and ratio of ketamine isomers, argues that NMDA receptor channels are crucial targets for the analgesic, rather than anesthetic, effects of ketamine. The relative potency of S(+)- and R(–)-ketamine for binding to opioid receptors also correlates with their relative analgesic potency (12,13). However, it is not likely that opioid receptors are involved in the mechanism of ketamine analgesia, because µ and {kappa} opioid receptors have low sensitivity to ketamine (affinity constant values of 42 and 28 µM, respectively) and ketamine antagonizes opioid receptor function (13).


   Acknowledgments
 
Supported by Grant-in-Aid (10671407) from the Japanese Ministry of Education, Science and Culture, Tokyo, Japan.

The authors thank Parke-Davis Pharmaceutical Research (Ann Arbor, MI) for kindly providing ketamine isomers.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Ryder S, Way WL, Trevor AJ. Comparative pharmacology of the optical isomers of ketamine in mice. Eur J Pharmacol 1978;49:15–23.[Web of Science][Medline]
  2. White PF, Ham J, Way WL, Trevor AJ. Pharmacology of ketamine isomers in surgical patients. Anesthesiology 1980;52:231–9.[Web of Science][Medline]
  3. Klepstad P, Maurset A, Moberg ER, Øye I. Evidence of a role for NMDA receptors in pain perception. Eur J Pharmacol 1990;187:513–8.[Web of Science][Medline]
  4. Ebert B, Mikkelsen S, Thorkildsen C, Borgbjerg FM. Norketamine, the main metabolite of ketamine, is a non-competitive NMDA receptor antagonist in the rat cortex and spinal cord. Eur J Pharmacol 1997;333:99–104.[Web of Science][Medline]
  5. Zeilhofer HU, Swandulla D, Geisslinger G, Brune K. Differential effects of ketamine enantiomers on NMDA receptor currents in cultured neurons. Eur J Pharmacol 1992;213:155–8.[Web of Science][Medline]
  6. Yamakura T, Shimoji K. Subunit- and site-specific pharmacology of the NMDA receptor channel. Prog Neurobiol 1999;59:279–98.[Web of Science][Medline]
  7. Watanabe M, Inoue Y, Sakimura K, Mishina M. Developmental changes in distribution of NMDA receptor channel subunit mRNAs. Neuro Report 1992;3:1138–40.[Web of Science][Medline]
  8. Yamakura T, Mori H, Masaki H, et al. Different sensitivities of NMDA receptor channel subtypes to non-competitive antagonists. Neuro Report 1993;4:687–90.[Web of Science][Medline]
  9. MacDonald JF, Miljkovic Z, Pennefather P. Use-dependent block of excitatory amino acid currents in cultured neurons by ketamine. J Neurophysiol 1987;58:251–66.[Abstract/Free Full Text]
  10. Mori H, Masaki H, Yamakura T, Mishina M. Identification by mutagenesis of a Mg2+-block site of the NMDA receptor channel. Nature 1992;358:673–5.[Medline]
  11. Reich DL, Silvay G. Ketamine: an update on the first twenty-five years of clinical experience. Can J Anaesth 1989;36:186–97.[Web of Science][Medline]
  12. Hustveit O, Maurset A, Øye I. Interaction of the chiral forms of ketamine with opioid, phencyclidine, {varsigma} and muscarinic receptors. Pharmacol Toxicol 1995;77:355–9.[Web of Science][Medline]
  13. Hirota K, Okawa H, Appadu BL, et al. Stereoselective interaction of ketamine with recombinant µ, {kappa}, and {delta} opioid receptors expressed in Chinese hamster ovary cells. Anesthesiology 1999;90:174–82.[Web of Science][Medline]
Accepted for publication March 17, 2000.




This article has been cited by other articles:


Home page
 J. Neurosci.Home page
X. Chen, S. Shu, and D. A. Bayliss
HCN1 Channel Subunits Are a Molecular Substrate for Hypnotic Actions of Ketamine
J. Neurosci., January 21, 2009; 29(3): 600 - 609.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
W. Nagels, R. Demeyere, J. Van Hemelrijck, E. Vandenbussche, K. Gijbels, and E. Vandermeersch
Evaluation of the Neuroprotective Effects of S(+)-Ketamine During Open-Heart Surgery
Anesth. Analg., June 1, 2004; 98(6): 1595 - 1603.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
R. J. Callahan, J. D. Au, M. Paul, C. Liu, and C. S. Yost
Functional Inhibition by Methadone of N-Methyl-D-Aspartate Receptors Expressed in Xenopus Oocytes: Stereospecific and Subunit Effects
Anesth. Analg., March 1, 2004; 98(3): 653 - 659.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
K.-M. Scheufler, C. Thees, J. Nadstawek, and J. Zentner
S(+)-Ketamine Attenuates Myogenic Motor-Evoked Potentials at or Distal to the Spinal {alpha}-Motoneuron
Anesth. Analg., January 1, 2003; 96(1): 238 - 244.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
M. Reeves, D. E. Lindholm, P. S. Myles, H. Fletcher, and J. O. Hunt
Adding Ketamine to Morphine for Patient-Controlled Analgesia After Major Abdominal Surgery: A Double-Blinded, Randomized Controlled Trial
Anesth. Analg., July 1, 2001; 93(1): 116 - 120.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamakura, T.
Right arrow Articles by Shimoji, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamakura, T.
Right arrow Articles by Shimoji, K.

Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press