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Vol. 302, Issue 2, 568-576, August 2002
-D-glucuronide Suppresses Inhibitory
Synaptic Transmission in Rat Substantia Gelatinosa
University Centre for Neuroscience and Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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High doses of intrathecally applied morphine or
morphine-3
-D-glucuronide (M3G) produce allodynia and
hyperalgesia. Whole-cell patch-clamp recordings were made from
substantia gelatinosa neurons in transverse slices of adult rat lumbar
spinal cord to compare the actions of M3G with those of the µ-opioid
agonist, DAMGO
([D-Ala2,N-Met-Phe4,Gly-ol5]-enkephalin),
and the ORL1 agonist, nociceptin/orphanin FQ (N/OFQ). M3G
(1-100 µM) had little or no effect on evoked excitatory postsynaptic currents (EPSC) and no effect on postsynaptic membrane conductance. In
contrast, 1 µM DAMGO and 1 µM N/OFQ reduced the amplitude of evoked
EPSCs and activated an inwardly rectifying K+ conductance.
M3G did not attenuate the effect of DAMGO or N/OFQ on evoked EPSC
amplitude. However, 1 to 100 µM M3G reduced the amplitude of evoked
GABAergic and glycinergic inhibitory postsynaptic current (IPSC) by up
to 48%. This effect was naloxone-insensitive. The evoked IPSC was also
attenuated by DAMGO, but not by N/OFQ. Because M3G reduced the
frequency of tetrodotoxin-insensitive miniature IPSCs and
increased paired-pulse facilitation, it appeared to act presynaptically
to disinhibit substantia gelatinosa neurons. This effect, which does
not appear to involve µ-opioid or ORL1 receptors, may
contribute to the allodynia and hyperalgesia observed after intrathecal
application of high doses of morphine.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Morphine
is widely used for the management of moderate to severe pain. It is
converted by glucuronidation into two major metabolites, morphine-3-D-glucuronide (M3G) and
morphine-6-D-glucuronide (M6G) (Boerner et al., 1975
;
Christrup, 1997). M6G has high affinity for the µ-opioid receptor
(Pasternak et al., 1987; Paul et al., 1989; Löser et al., 1996)
and appears to be a more potent opioid agonist than morphine (Pasternak
et al., 1987; Paul et al., 1989; Frances et al., 1992; Osborne et al.,
2000). In contrast, M3G does not bind to µ-, 
-, or 
-opioid
receptors (Pasternak et al., 1987; Lambert et al., 1993; Löser et
al., 1996) and appears to be devoid of analgesic activity (Pasternak et
al., 1987; Yaksh and Harty, 1988). Furthermore, M3G does not interact
with N-methyl-D-aspartate, GABAA, or glycine receptors (Bartlett et al.,
1994) and has no effect on membrane conductance or action potential
discharge in locus coeruleus neurons (Osborne et al., 2000). M3G also
does not affect A- or C-fiber-evoked responses in dorsal horn
neurons (Sullivan et al., 1989; Hewett et al., 1993). It does however produce hyperalgesia and allodynia when administered intrathecally or
intracerebroventricularly (Woolf, 1981; Yaksh et al., 1986; Yaksh and
Harty, 1988) and progressively higher doses can cause seizures (Smith
et al., 1990; Halliday et al., 1999). These findings are consistent
with the suggestion that morphine metabolites may be responsible for
the development of hyperalgesia, allodynia, and myoclonus during
clinical opioid therapy (De Conno et al., 1991; Sjogren et al., 1998).
Therefore, the aim of the present study was to examine the cellular
effects of M3G on neurons in the rat substantia gelatinosa. Actions of
M3G were compared with those of the µ-opioid agonist, DAMGO
([D-Ala2,N-Met-Phe4,Gly-ol5]-enkephalin),
and the ORL1 agonist, nociceptin/orphanin FQ
(N/OFQ). Although it is established that M3G does not interact with
µ-, -, or -receptors, we sought to examine possible
interactions with other mechanisms within the dorsal horn, including
the more recently defined ORL1 receptor (Meunier
et al., 1995; Reinscheid et al., 1995). Some of these findings have
been communicated to the Society for Neuroscience (Moran and Smith,
2000).
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Spinal Cord Slice Preparation. All procedures were carried out in compliance with the guidelines of the Canadian Council for Animal Care, the University of Alberta Health Sciences Laboratory Animal Services Welfare Committee, and the Committee for Research and Ethical Issues of the International Association for the Study of Pain.
Sprague-Dawley rats (14-35 days old) were deeply anesthetized with urethane (1.5 g/kg, i.p.). A lumbosacral laminectomy was performed, and ~2 cm of spinal cord with attached ventral and dorsal rootlets was transferred into ice-cold oxygenated (95% O2-5% CO2) dissection solution containing 118 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.3 mM MgSO4, 1.2 mM NaH2PO4, 1.5 mM CaCl2, 5 mM MgCl2, 25 mM D-glucose, and 1 mM kynurenic acid. The dura mater was removed, and the spinal cord was glued to an agar block with cyanoacrylate glue. Transverse slices (300-500 µm) were cut using a Vibratome (Pelco International, Reading, CA) in ice-cold dissection solution and were then incubated at room temperature (22-24°C) in oxygenated dissection solution (see above, without 1 mM kynurenic acid) for 1 h before recording.Recording and Stimulation. Spinal cord slices were superfused (flow rate ~2-4 ml/min) at room temperature (22-24°C) with 95% O2-5% CO2 saturated artificial CSF (127 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 26 mM NaHCO3, 1.3 mM MgSO4, 2.5 mM CaCl2, and 25 mM D-glucose, pH 7.4). For recording excitatory postsynaptic currents (EPSCs), 10 µM bicuculline and 1 µM strychnine were included to block inhibitory synaptic inputs. For recording inhibitory postsynaptic currents (IPSCs), 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 µM DL-2-amino-5-phosphonovaleric acid (AP5) were included to block excitatory synaptic inputs. The GABAergic component of the IPSC was observed in the presence of CNQX and AP5, plus strychnine, whereas the glycinergic component was observed in the presence of CNQX and AP5, plus bicuculline. Tetrodotoxin (TTX; 1 µM) was included when recording miniature IPSCs (mIPSC).
The substantia gelatinosa was identifiable as a translucent band across the dorsal horn. Whole-cell recordings were made with an NPI SEC 05L amplifier (NPI Electronic GmbH, Tamm, Germany) in discontinuous single-electrode voltage-clamp or bridge-balance current-clamp mode using either the "blind" whole-cell patch-clamp technique or from visually identified substantia gelatinosa neurons using infrared-differential interference contrast video microscopy. Patch pipettes were pulled from thin-walled borosilicate glass (WPI, Sarasota, FL). These had resistances of 5 to 10 M
when filled with
an internal solution containing 130 mM potassium gluconate, 1 mM
MgCl2, 2 mM CaCl2, 10 mM
HEPES, 10 mM EGTA, 4 mM Mg-ATP, and 290-300 mOsm Na-GTP, pH 7.2. In
some experiments, the sodium channel blocker QX-314 (5 mM) was included
in the internal solution to prevent action potential discharge. Resting
membrane potential was typically between 
55 to 65 mV in
bridge-balance current-clamp mode. For voltage-clamp experiments,
membrane potential was clamped at 60 or 70 mV for recording EPSCs
and 0 mV for recording IPSCs. Switching frequencies were typically
between 30 and 40 kHz. Signals were filtered at 2 kHz and digitized
between 5 and 10 kHz. EPSCs were evoked at 0.05 Hz with a bipolar
concentric stimulating electrode (FHC Inc., Bowhoinham, ME) that was
placed on the dorsal root or near the dorsal root entry zone to
activate primary afferent fibers. Stimulus duration was 100 or 400 µs. IPSCs were evoked at 0.05 Hz by focal stimulation with a patch
pipette containing artificial CSF. For paired-pulse experiments, IPSCs
were evoked at 0.05 Hz with two paired stimuli (interstimulus interval
50-100 ms), and 10 consecutive responses were averaged for analysis. EPSCs and IPSCs were identified as being monosynaptic by their ability
to follow high frequency stimulation (10 or 20 Hz) with constant
latency and the absence of failures. An Axopatch 1D (Axon Instruments,
Inc., Foster City, CA) was used for recording mIPSCs, and data were
only used if the series resistance was below 25 M. Currents were
filtered at 1 kHz and digitized at 5 kHz, and data were stored on disk.
Data Analysis. Data were acquired and analyzed using pCLAMP 8.0 (Axon Instruments, Inc.). Statistical comparisons were made using GraphPad Instat 3.05 (GraphPad Software Inc., San Diego, CA). mIPSCs were analyzed using Mini Analysis Program (Synaptosoft, Decatur, GA) on the basis of amplitudes exceeding a threshold set above the baseline noise of the recording. Between 500 and 1000 individual mIPSC were analyzed for each cell. Detected events were reexamined visually and either accepted or rejected. The program was used to measure amplitudes and interevent intervals (frequency) and cumulative probability plots were constructed. Statistical analysis for each neuron was performed using the Kolmogorov-Smirnov nonparametric test. Distributions were considered statistically different if p < 0.05. Figures were produced with Origin 6.1 (OriginLab Corp., Northhampton, MA) or Igor Pro 3.1 (Wavemetrics, Lake Oswego, OR).
Drugs and Chemicals. Drugs were applied by bath superfusion for 5 to 6 min. This was long enough for equilibration with the tissue, as drug responses to 5- to 6-min applications were no larger with longer application times (up to 20 min). DAMGO, naloxone, and strychnine were obtained from Sigma-Aldrich (St. Louis, MO). Nociceptin, bicuculline, CNQX, and AP5 were from Tocris Cookson Inc. (Ballwin, MO). QX-314 was supplied by AstraZeneca Pharmaceuticals LP (Wilmington, DE) and TTX was from Alomone Labs (Jerusalem, Israel). Morphine sulfate was from British Drug Houses (Toronto, ON, Canada). M3G was from Lipomed (Arlesheim, Switzerland) and contained 0.28% morphine (HPLC analysis, Neurochemistry Research Unit, University of Alberta, Canada).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Morphine-3-D-glucuronide Does Not Affect Evoked
Excitatory Postsynaptic Currents.
Whole-cell patch-clamp
recordings were obtained from substantia gelatinosa neurons from slices
maintained in vitro for up to 10 h and stable recordings were made
from individual neurons for up to 3 h.
60
or 70 mV. Superfusion of 1 to 100 µM M3G had no significant effect
on EPSC amplitude in any of the 19 cells tested. By contrast, and
confirming previous reports (Glaum et al., 1994; Kohno et al., 1999),
the µ-opioid agonist DAMGO (1 µM) reduced EPSC amplitude by an
average of 46.9 ± 4.82% (n = 19/25 cells
tested). Similarly, the ORL1 agonist N/OFQ (1 µM) reduced EPSC amplitude by 39.6 ± 7.10% (n = 5/6 cells tested), which confirms the findings of Liebel et al.
(1997) and Lai et al. (1997). Sample data records are shown in Fig.
1. In Fig. 1A the EPSC is unaffected by
M3G but is suppressed by N/OFQ. In Fig. 1B, M3G is again ineffective
but the EPSC is suppressed by DAMGO. Time courses of the effects of
these drugs are shown in Fig. 1, C and D. The histogram in Fig. 1E
summarizes the effects of M3G, DAMGO, and N/OFQ on evoked EPSCs.
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Morphine-3-D-glucuronide Does Not Interact with
ORL1 or µ-Opioid Receptors in Substantia Gelatinosa
Neurons.
Because M3G had no noticeable effect on excitatory
transmission at synapses where N/OFQ and DAMGO suppressed transmission, it is unlikely to act as an ORL1 or µ-opioid
agonist. M3G was also devoid of antagonist activity at these receptors.
Superfusion of 1 or 10 µM M3G did not occlude the effects of N/OFQ.
In the presence of M3G, superfusion of 1 µM N/OFQ reduced EPSC
amplitude by 41.8 ± 7.84% (n = 3; Fig.
2A), which is similar to the actions of
N/OFQ by itself on EPSC amplitude (p > 0.85, t test; compare with Fig. 1A). In a similar series of
experiments, superfusion of 1 or 10 µM M3G did not occlude the
actions of DAMGO. In the presence of M3G, superfusion of DAMGO reduced
EPSC amplitude by 40.9 ± 4.30% (n = 3; Fig. 2B),
which is similar to the actions of DAMGO alone (p > 0.85, t test; compare with Fig. 1B). Figure 2, C and D,
shows time courses of these drug effects on EPSC amplitude. These
observations are consistent with binding studies that show that M3G
does not bind to µ-opioid receptors (Pasternak et al., 1987; Lambert
et al., 1993; Löser et al., 1996).
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Comparison of Postsynaptic Actions of
Morphine-3-D-glucuronide, Nociceptin/Orphanin FQ, and
DAMGO.
Membrane conductance measured from a voltage-ramp protocol
was unaffected by M3G (1 µM, n = 11 or 100 µM,
n = 5). Figure 3A shows
the lack of effect of 100 µM M3G on currents evoked by a voltage ramp
from 140 to 0 mV. In the same cell (Fig. 3B), 1 µM DAMGO increased
conductance at negative voltages, reflecting its activation of an
inwardly rectifying conductance (Grudt and Williams, 1994; Schneider et
al., 1998). M3G (1 µM) also had no effect on excitability
(n = 5), as evaluated by the frequency of action
potential discharge in response to depolarizing current pulses (data
not shown). These findings are similar to those of Osborne et al.
(2000) who found no effect of M3G on membrane conductance or action
potential firing in locus coeruleus neurons.
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140 mV (Fig. 3C). This was very similar to the findings in medullary
dorsal horn (Jennings, 2001). The reversal potential for the
N/OFQ-induced current of 97.0 ± 2.51 mV in 2.5 mM
[K+]o (n = 8) was shifted to 75.9 ± 4.28 mV in 6.5 mM
[K+]o (n = 3), consistent with the activation of a K+
conductance. DAMGO (1 µM) also activated an inwardly rectifying K+ current of 60.5 ± 10.0 pA
(n = 5/12 cells tested) at 140mV (Fig. 3D), which
confirms previous reports (Grudt and Williams, 1994; Schneider et al.,
1998).
Actions of Morphine-3-D-glucuronide on Evoked and
Miniature Inhibitory Postsynaptic Currents.
In the presence of the
glutamate receptor antagonists, 50 µM AP5 and 10 µM CNQX, focal
stimulation generated IPSCs in substantia gelatinosa neurons at a
holding potential of 0 mV. In contrast to its lack of effect on evoked
EPSCs, M3G produced a concentration-dependent decrease in the amplitude
of the evoked IPSC (Fig. 4A). Sample data
records are illustrated in Fig. 4B.
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). To test for this possibility, effects of 100 µM M3G on the IPSC were studied in the presence of 100 µM naloxone. Data records are shown in Fig. 4D and the time course of
the effect of M3G in the presence of naloxone is shown in Fig. 4E.
Because the effect of M3G was unchanged, the actions of M3G do not
reflect contamination of the sample by µ-agonists. Moreover, they
confirm that the action of M3G is not mediated via µ-opioid receptors
and exclude possible interactions with - and -receptors.
To further characterize the action of M3G on inhibitory synaptic
transmission, we examined the effect of 100 µM M3G on TTX-insensitive mIPSCs. M3G (100 µM) reduced the frequency (n = 4/4
cells tested, Kolmogorov-Smirnov test, p < 0.05; Fig.
5, A-C), but had no effect on the
amplitude of the mIPSCs (n = 4/4 cells tested;
p > 0.05, Kolmogorov-Smirnov test; Fig. 5C). This
preferential effect on mIPSC frequency rather than amplitude suggested
that M3G acted presynaptically. Additional evidence for a presynaptic
site of action of M3G was obtained from paired-pulse experiments. Two identical stimuli separated by an interstimulus interval (50-100 ms)
resulted in paired-pulse facilitation of the evoked IPSC. The mean
ratio of the amplitude of the paired IPSCs was 1.04 ± 0.09 (IPSC2/IPSC1,
n = 4). In four of six cells, superfusion of M3G (100 µM M3G) produced an increase in the mean ratio of
IPSC2/IPSC1 to 1.67 ± 0.45 (n = 4). This reflected suppression of the evoked IPSC and a 29.9 ± 8.95% increase in the paired-pulse ratio by p < 0.005 (paired t test; n = 4). Sample data records are shown in Fig.
6A. In Fig. 6B, the IPSCs have been
normalized to the amplitude of IPSC1 to better
illustrate the change in the paired-pulse ratio. Figure 6C is a summary
histogram of the effect of 100 µM M3G on the paired-pulse ratio.
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Actions of N/OFQ and DAMGO on Evoked Inhibitory Postsynaptic
Currents.
Recently, N/OFQ has been reported to selectively
suppress glutamatergic synaptic inputs in the spinal cord dorsal horn
(Zeilhofer et al., 2000; Ahmadi et al., 2001) but to have no effect on
inhibitory synaptic currents. We observed a similar lack of effect of
N/OFQ (1 µM) on evoked IPSCs. N/OFQ reduced the amplitude of evoked IPSCs by only 5.88 ± 0.75% (p > 0.05, n = 5/5 cells tested; Fig. 8A). The actions of N/OFQ on synaptic
transmission in the substantia gelatinosa are the reverse of M3G, which
inhibits IPSCs, but fails to affect EPSCs. Furthermore, 1 µM DAMGO
reduced IPSC amplitude (49.6 ± 10.8%, n = 6/10
cells tested, p < 0.05; Fig. 8B) confirming the
findings of Grudt and Henderson (1998) but contradicting those of Kohno
et al. (1999), who found that DAMGO did not inhibit IPSCs in rat lumbar
dorsal horn. Figure 8C shows the time course of the effect of DAMGO on
IPSC amplitude. Figure 8D is a summary histogram that compares the
effect of DAMGO, M3G, and N/OFQ on IPSC amplitude.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, the cellular effects of M3G were compared with those of the ORL1 agonist N/OFQ and the µ-opioid agonist DAMGO in substantia gelatinosa neurons of rat lumbar spinal cord. M3G had little or no effect on excitatory synaptic transmission at synapses where ORL1 or µ-opioid agonists were effective. M3G also failed to affect postsynaptic membrane conductance or excitability, whereas, both N/OFQ and DAMGO activated an inwardly rectifying K+ conductance. Moreover, suppression of excitatory synaptic responses by N/OFQ or DAMGO was not antagonized by M3G. M3G is therefore neither an agonist nor antagonist at ORL1 or µ-opioid receptors. However, M3G produced a naloxone-insensitive, concentration-dependent suppression of inhibitory synaptic transmission. The GABAergic and glycinergic components of the IPSC were similarly affected. Analysis of TTX-insensitive mIPSCs indicated that this action of M3G was presynaptic. This finding was supported by paired-pulse experiments where M3G produced an increase in paired-pulse facilitation.
The lack of effect of 1 to 100 µM M3G on EPSCs is consistent with
binding studies (Bartlett et al., 1994) and neurochemical assays that
showed M3G has no affinity for the
N-methyl-D-aspartate receptor and does
not affect the release of glutamic acid from synaptosomes (Bartlett and
Smith, 1996). Moreover, intrathecal M3G has no effect on C-fiber-evoked
responses in the superficial dorsal horn (Sullivan et al., 1989; Hewett
et al., 1993)
The failure of M3G to antagonize the effects of DAMGO and the lack of
effect of M3G on evoked EPSCs is consistent with receptor binding
studies, which show M3G has little or no affinity for the µ-opioid
receptor (Pasternak et al., 1987; Löser et al., 1996). In
addition, our findings agree with a previous electrophysiological study
(Hewett et al., 1993), which indicated that M3G does not antagonize the
antinociceptive actions of intrathecal morphine. Moreover, lack of
antagonism of the actions of N/OFQ suggests that M3G also does not
interact with the ORL1 receptor in rat substantia gelatinosa.
By contrast, with its lack of effect on excitatory synaptic transmission, M3G produced a concentration-dependent suppression on inhibitory synaptic transmission. At a concentration of 100 µM, M3G reduced the amplitude of evoked IPSCs by approximately 45%. In the presence of TTX, M3G reduced the mIPSC frequency without affecting the amplitude distribution in all cells tested, suggesting the effect of M3G involved a presynaptic mechanism. If M3G had exerted an effect on postsynaptic GABAA or glycine receptors, a change in mIPSC amplitude would likely have been observed. Similarly, a postsynaptic action of M3G would not account for the observed increase in the paired-pulse ratio. The effect on inhibitory transmission was not caused by morphine contamination of our sample of M3G because a concentration of morphine equivalent to the amount of contamination had no effect. Involvement of potential µ-agonist contaminants (morphine and M6G) was also ruled out by the lack of effect of naloxone on M3G-induced suppression of IPSCs.
Because intrathecally administered GABAA and
glycine receptor antagonists (Beyer et al., 1985; Yaksh et al., 1986;
Kaneko and Hammond, 1997; Zhang et al., 2001) have pro-nociceptive
actions similar to M3G, selective suppression of inhibitory synaptic
transmission by M3G may explain its allodynic and hyperalgesic effects.
It may also explain the allodynia, hyperalgesia, and myoclonus observed after administration of high-dose morphine in humans (De Conno et al.,
1991; Sjogren et al., 1993, 1994, 1998; Heger et al., 1999). Our
observed effects may be especially relevant to palliative care
situations, where heroic doses of morphine (up to 20 g/day) are
required to produce analgesia in tolerant individuals (Hagen and
Swanson, 1997; Sjogren et al., 1998). In humans, intrathecal injection
of 1100 mg of morphine results in an accumulation of ~3 µM M3G in
CSF (Goucke et al., 1994). By extrapolation, the concentration of M3G
in the CSF of a palliative care patient who had received 20 g of
morphine within a day would approach 60 µM (Hagen and Swanson, 1997;
Sjogren et al., 1998). This falls within the range of concentrations
tested in the present study. Thus, suppression of GABA- and
glycine-mediated synaptic transmission by M3G may explain the
development of allodynia, hyperalgesia, seizures, and myoclonus that
occur with high-dose opioid administration and may dictate the limiting
dose of morphine that can be administered.
Although it is well established that DAMGO suppresses EPSCs in
substantia gelatinosa neurons (Glaum et al., 1994; Kohno et al., 1999),
its effect on IPSCs is controversial. In the substantia gelatinosa of
the lumbar spinal cord, DAMGO reportedly did not affect inhibitory
synaptic transmission (Kohno et al., 1999), whereas in trigeminal
nucleus pars caudalis DAMGO, suppressed GABAergic and glycinergic IPSCs
(Grudt and Henderson, 1998). We found that DAMGO suppresses IPSCs in
lumbar substantia gelatinosa, which supports the findings of Grudt and
Henderson (1998). One reason for the disparate findings may be the
temperature at which the various studies were done. The work of Kohno
et al. (1999) was done at 37°C, whereas our work and that of Grudt
and Henderson (1998) were done at lower temperatures (24 or 30°C). If
there is an increased safety factor for inhibitory synaptic
transmission at higher temperatures, this may explain the insensitivity
of IPSCs to DAMGO that was noted by Kohno et al. (1999). We also corroborated previous findings that N/OFQ selectively suppresses EPSCs
in the substantia gelatinosa (Lai et al., 1997; Liebel et al., 1997;
Zeilhofer et al., 2000; Ahmadi et al., 2001).
Because actions of M3G at µ-, -, and -opioid and
ORL1 receptors (Pasternak et al., 1987; Sullivan
et al., 1989; Löser et al., 1996) have now been excluded, the
receptor through which M3G exerts its effect remains to be determined.
Interestingly, the selective presynaptic effect of M3G on IPSCs is
similar to that of the recently identified neuropeptide, nocistatin.
Nocistatin, like M3G, selectively suppresses IPSCs in the dorsal horn
via a presynaptic mechanism (Zeilhofer et al., 2000) and also has pro-nociceptive actions in behavioral tests (Xu et al., 1999; Zeilhofer
et al., 2000; Ahmadi et al., 2001). It is thus possible that M3G
interacts with the nocistatin receptor, but until this receptor is
better characterized and antagonists are developed, this possibility
remains to be investigated.
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Acknowledgments |
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We thank Dr. Glen Baker for HPLC analysis of M3G, Dr. Fuad Abdulla for useful discussions, and Patrick Stemkowski for technical assistance.
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Footnotes |
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Accepted for publication April 24, 2002.
Received for publication February 28, 2002.
The Canadian Institutes of Health Research (CIHR), the Rick Hansen Neurotrauma Initiative, and the Alberta Heritage Foundation for Medical Research supported this work. T.D.M. was supported by a studentship from the CIHR.
DOI: 10.1124/jpet.102.035626
Address correspondence to: Dr. Peter A. Smith, Department of Pharmacology, 9-75 Medical Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2H7. E-mail: peter.a.smith{at}ualberta.ca
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Abbreviations |
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M3G, morphine-3-D-glucuronide;
M6G, morphine-6-D-glucuronide;
GABA, 
-aminobutyric
acid;
AP5, DL-2-amino-5-phosphonovaleric acid;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;
DAMGO, [D-Ala2,N-Met-Phe4,Gly-ol5]-enkephalin;
EPSC, excitatory postsynaptic current;
IPSC, inhibitory postsynaptic
current;
mIPSC, miniature IPSC;
N/OFQ, nociceptin/orphanin FQ;
TTX, tetrodotoxin;
HPLC, high-performance liquid chromatography;
CSF, cerebrospinal fluid;
QX-314, N-(2,6-dimethylphenyl)acetamide-2-triethylammonium
bromide.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-glucuronide is a more potent opioid agonist than morphine.
J Pharmacol Exp Ther
262:
25-311-, 2-, and µ-opioid receptor agonists on excitatory transmission in lamina II neurons of adult rat spinal cord.
J Neurosci
14:
4965-4971[Abstract].-aminobutyric acidA receptors in formalin-induced nociception in the rat.
J Pharmacol Exp Ther
282:
928-938-D-glucuronide but not morphine-3-O--D-glucuronide binds to µ-, - and -specific opioid binding sites in cerebral membranes.
Naunyn-Schmiedeberg's Arch Pharmacol
354:
192-197[Medline].-glucuronide has a higher efficacy than morphine as a mu-opioid receptor agonist in the rat locus coeruleus.
Br J Pharmacol
131:
1422-1428[CrossRef][Medline].-glucuronide, a very potent morphine metabolite.
J Pharmacol Exp Ther
251:
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T. D. Moran, W. F. Colmers, and P. A. Smith Opioid-Like Actions of Neuropeptide Y in Rat Substantia Gelatinosa: Y1 Suppression of Inhibition and Y2 Suppression of Excitation J Neurophysiol, December 1, 2004; 92(6): 3266 - 3275. [Abstract] [Full Text] [PDF]
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K. Hemstapat, G. R. Monteith, D. Smith, and M. T. Smith Morphine-3-Glucuronide's Neuro-Excitatory Effects Are Mediated via Indirect Activation of N-Methyl-D-Aspartic Acid Receptors: Mechanistic Studies in Embryonic Cultured Hippocampal Neurones Anesth. Analg., August 1, 2003; 97(2): 494 - 505. [Abstract] [Full Text] [PDF]
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, p < 0.05).
