Editorial Manager(tm) for Anesthesiology Manuscript Draft
Manuscript Number: ALN201003055R2
Title: Purinergic P2X Receptor Regulates N-methyl-D-aspartate Receptor Expression and Synaptic Excitatory Amino Acid Concentration in Morphine-tolerant Rats
Article Type: Pain Medicine
Corresponding Author: Dr. Chih-Shung Wong, M.D., Ph.D.
Corresponding Author's Institution: Tri-Service General Hospital First Author: Yueh-Hua Tai, Ph.D.
Order of Authors: Yueh-Hua Tai, Ph.D.; Pao-Yun Cheng, Ph.D.; Ru-Yin Tsai, Ph.D.; Yuh-Fung Chen, Ph.D.; Chih-Shung Wong, M.D., Ph.D.
Abstract: Background: The present study examined the effect of P2X receptor antagonist 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) on morphine tolerance in rats.
Methods: Male Wistar rats were implanted with two intrathecal catheters with or without a
microdialysis probe, then received a continuous intrathecal infusion of saline (control) or morphine (tolerance induction) for 5 days.
Results: Long-term morphine infusion induced antinociceptive tolerance and upregulated N-methyl-D-aspartate receptor subunits NR1 and NR2B expression in both total lysate and synaptosome fraction of the spinal cord dorsal horn. TNP-ATP (50μg) treatment potentiated the antinociceptive effect of morphine, with a 5.5-fold leftward shift of the morphine dose-response curve in morphine-tolerant rats, and this was associated with reversal of the upregulated NR1 and NR2B subunits in the
synaptosome fraction. NR1/NR2B specific antagonist ifenprodil treatment produced similar effect as TNP-ATP; it also potentiated the antinociceptive effect of morphine. On day 5, morphine challenge resulted in a significant increase in aspartate and glutamate concentration in the cerebrospinal fluid dialysates of morphine-tolerant rats and this effect was reversed by TNP-ATP treatment. Moreover, the amount of immunoprecipitated postsynaptic density-95/NR1/NR2B complex was increased in
morphine-tolerant rats and this was prevented by the TNP-ATP treatment.
Conclusions: The findings suggest that attenuation of morphine tolerance by TNP-ATP is attributed to downregulation of N-methyl-D-aspartate receptor subunits NR1 and NR2B expression in the
synaptosomal membrane and inhibition of excitatory amino acids release in morphine-tolerant rats. The TNP-ATP regulation on the N-methyl-D-aspartate receptor expression may be involved in a loss of scaffolding proteins postsynaptic density-95.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Reviewers' Comments: Reviewer #1:
The authors present a revision of a manuscript that reports on interaction between P2X receptor activation and morphine tolerance implicating a mechanism that involves both EAA release and post synaptic NMDA receptor expression. The revisions are very helpful and make the manuscript clearer. I have a few relatively minor suggestions.
1. Suggestion for wording of summary statement:
Summary Statement: Purinoceptor P2X receptor antagonist TNP-ATP restores the antinociceptive effect of morphine in morphine tolerant rats, possibly via down regulation of NMDA receptor subunits NR1 and NR2B in the synaptosomal membrane and inhibition of excitatory amino acids release.
Answer: Thanks for your suggestion; we had revised the summary statement “Purinergic P2X receptor antagonist downregulated N-methyl-D-aspartate receptor subunits NR1 and NR2B in the synaptosomal membrane and inhibited excitatory amino acids release in morphine tolerant-rats” on pages 2, line 1-3.
2. The following piece of the discussion is a bit confusing. "26Although spinal infusion of morphine for 4 days has little effect on the concentration of EAAs, naloxone challenge evokes a dramatic increase in the release of L-glutamate and taurine, but not of other amino acids, in morphine-infused, but not saline-infused, rats.27 Similarly, in our previous study, acute morphine treatment increases the levels of DOPAC and glutamate in the striatum, nucleus accumbens, and locus coeruleus neurons in naloxone-precipitated morphine-tolerant rats.28" You cite reference 17 that did not show an increase in EAAs but response to spinal morphine for 4 days but in these experiments you did find an increase. Instead of (or maybe before) talking about your previous work why not just speculate on why the two spinal protocols were different. Maybe 5 was the magic day. Was there any other difference?
Answer: Thanks for your comments. We had changed the statement into “In previous and our recent studies, the results failed to demonstrate an increase in CSF EAA levels during induction of morphine tolerance.7,27,28 However, post-treatment with naloxone evoked a significant and time-dependent increase in the CSF dialysate glutamate and taurine concentration, but not other amino acids in chronic morphine-infused rats.27 Similarly, we demonstrated that morphine challenge induced an increase of glutamate and aspartate in the CSF dialysates of morphine-tolerant rats;
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it was also accompanied by a loss of morphine’s analgesic effect,7,28 and co-administration of morphine with the NMDA antagonist not only attenuated morphine tolerance development, but also blocked the morphine challenge induced spinal EAAs release.28” on pages 23, lines 4-13.
3. The idea of cross-talk between mu-opioid activation, P2X activation and protein kinase C is intriguing. Can you develop this more in your discussion? Might there be a role for GRK 2 or 3?
Answer: Thanks for your comments; we had added a statement of “Studies have indicates that P2X and μ-opioid receptors are functionally coupled in sensory neuron.50 Extracellular ATP-evoked P2X receptor inward current inhibited opioid sensitivity in neurons co-cultured with fibrosarcoma cells.51 Translocation and activation of protein kinase C enhance postsynaptic neuron excitability in morphine-tolerant rats.10,52,53 Moreover, activation of protein kinase C showed significantly potentiation of Ca2+ signal and inward cation current (predominately Na+) as well through P2X3 receptor in DT-40 3KO and HEK-293 cells.54”on pages 27, lines 7-14.
4. Your findings in the rat model are very intriguing but we all know that things do not always translate as hoped into humans. I would temper your last statement "We suggest that TNP-ATP can provide an alternative analgesic adjuvant for the treatment of patients who need long-term opioid administration for pain relief." with, "If these findings are validated in humans?"
Answer: Thanks for your comments; we had deleted the statement from pages 29.
************
Reviewer #2:
The revised paper is acceptable. The authors have satisfactorily addressed the issues I raised.
************
Reviewer #3:
1. Please define the measures of central tendency and variability used prior to the statistical methods section the first time they are reported. For example, is this mean +- SD, "The tail-flick latency was measured using the hot water immersion test (52 ?
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 0.5 C)?"
Answer: Thanks for your comments. The hot water tail immersion test unit serves to assess the tail flick reaction of rats when their tail is immersed in a constant temperature bath with the temperature range between 52 ±0.5℃.
2. Please simply define the factors and their nature when introducing the two-way ANOVA. For example stating that a between groups factor (dose) and repeated measures factor (time) were specified would be very helpful.
Answer: Thanks for your comments; we added the statement on page 15, line 3-6. “Tail-flick latencies and EAA concentration were analyzed using two-way (time and treatment) ANOVA followed by subsequent one-way ANOVA (at each time of the experiment) with a post hoc Student-Newman-Keuls test.”
3. Please report the nature of the inferences (e.g., two-tailed).
Answer: Thanks for your comments; we had added the statement on page 15, line 1-3;
P value on pages 16~21.
4. Please ensure that exact sample sizes can be discerned in the Figure Captions.
Answer: Thanks for your comments; we had addedsample size in the figure captions.
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Purinergic P2X Receptor Regulates N-methyl-D-aspartate Receptor Expression
and Synaptic Excitatory Amino Acid Concentration in Morphine-tolerant Rats
Yueh-Hua Tai, Ph.D.,* Pao-Yun Cheng, Ph.D.,+ Ru-Yin Tsai, Ph.D.,** Yuh-Fung Chen,
Ph.D.,++ Chih-Shung Wong M.D., Ph.D.+++
*
Postdoctoral Fellow, Department of Anesthesiology, Tri-service General Hospital and
National Defense Medical Center, Taipei, Taiwan.
**
Postdoctoral Fellow, Department of Medical Research, China Medical University
Hospital, Taichung, Taiwan
+
Assistant Professor, ++ Associate Professor, Graduate Institute of Chinese
Pharmaceutical Science, China Medical University, Taichung, Taiwan
+++
Professor, Department of Anesthesiology, Cathay General Hospital, Taipei, Taiwan
Acknowledgements
This study was supported by a grant from the National Science Council, Taipei,
Taiwan (NSC 97-2321-B-016-003-MY2). The study was performed at the
Nociception Signal Transduction Laboratory, Department of Anesthesiology,
Tri-service General Hospital and National Defense Medical Center, Taipei, Taiwan. *Manuscript (Title Page, Abstract, Body, References)
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Summary Statement: Purinergic P2X receptor antagonist downregulated
N-methyl-D-aspartate receptor subunits NR1 and NR2B in the synaptosomal
membrane and inhibited excitatory amino acids release in morphine tolerant-rats.
Corresponding author and author address: Chih-Shung Wong, MD, PhD,
Department of Anesthesiology, Cathay General Hospital, 280 Renai Rd. Sec.4, Taipei,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Abstract
Background: The present study examined the effect of P2X receptor antagonist
2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) on morphine tolerance in rats.
Methods: Male Wistar rats were implanted with two intrathecal catheters with or
without a microdialysis probe, then received a continuous intrathecal infusion of
saline (control) or morphine (tolerance induction) for 5 days.
Results: Long-term morphine infusion induced antinociceptive tolerance and
upregulated N-methyl-D-aspartate receptor subunits NR1 and NR2B expression in
both total lysate and synaptosome fraction of the spinal cord dorsal horn. TNP-ATP (50μg) treatment potentiated the antinociceptive effect of morphine, with a 5.5-fold leftward shift of the morphine dose-response curve in morphine-tolerant rats, and this
was associated with reversal of the upregulated NR1 and NR2B subunits in the
synaptosome fraction. NR1/NR2B specific antagonist ifenprodil treatment produced
similar effect as TNP-ATP; it also potentiated the antinociceptive effect of morphine.
On day 5, morphine challenge resulted in a significant increase in aspartate and
glutamate concentration in the cerebrospinal fluid dialysates of morphine-tolerant rats
and this effect was reversed by TNP-ATP treatment. Moreover, the amount of
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morphine-tolerant rats and this was prevented by the TNP-ATP treatment.
Conclusions: The findings suggest that attenuation of morphine tolerance by
TNP-ATP is attributed to downregulation of N-methyl-D-aspartate receptor subunits
NR1 and NR2B expression in the synaptosomal membrane and inhibition of
excitatory amino acids release in morphine-tolerant rats. The TNP-ATP regulation on
the N-methyl-D-aspartate receptor expression may be involved in a loss of scaffolding
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Introduction
Opioids, such as morphine, are a class of powerful analgesics used for treating
moderate to severe pain in the clinic. However, long-term administration induces
tolerance, which hampers their clinical use.1 Morphine tolerance is a complex
physiological response; in addition to opioid receptor uncoupling and
endocytosis/desensitization,2,3 glutamatergic receptor activation and
neuroinflammation had been demonstrated by ourselves and others.4-7
The excitatory amino acids (EAAs), glutamate and aspartate, are the principal
excitatory neurotransmitters in the central nervous system and have a variety of
functions, including nociceptive transmission and modification.8 The glutamatergic
receptor system, especially the N-methyl-D-aspartate (NMDA) receptor, plays an
important role in synaptic plasticity and chronic pain formation.9 NMDA receptors are
tetrameric hetero-oligomers consisting of the essential NR1 subunit and one or more
modulatory NR2A-D and NR3 subunits. Activation of spinal NMDA receptors plays a
crucial role in the development of morphine tolerance.4,10 Pharmacological blockade
of NMDA receptors or disruption of the NR1 subunit gene significantly attenuates
morphine tolerance,11,12 suggesting an involvement of NMDA receptors in morphine
tolerance.
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extracellular adenosine 5'-triphosphate (ATP) that are involved in pain mechanisms.13
The P2X3 and P2X2/3 receptors located on primary afferent nerve terminals in the
inner lamina II of the spinal cord play a significant role in neuropathic and
inflammatory pain.14,15 A number of studies have demonstrated the therapeutic
potential of modulating P2X receptors in treating neuropathic pain.16 Intrathecal
administration of ATP produces long lasting allodynia, probably through P2X2/3
receptors.17 Studies using gene knockout, antisense oligonucleotides, or the selective
P2X3 antagonist A-317491 indicate that ATP and P2X3 receptors are involved in
chronic pain, particularly chronic inflammatory and neuropathic pain.15,18-20
McGaraughty et al.21 reported that antagonism of P2X3 and P2X2/3 receptors reduces
inflammatory hyperalgesia and chemogenic nociception, possibly through the spinal
opioid receptor system. Mao et al.22 suggested that neuropathic pain and morphine
tolerance share common mechanisms of nociception sensitization and morphine
resistance. The present study examined the effect of the P2X receptor antagonist 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) on morphine tolerance and its possible mechanism.
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Materials and Methods
Animal preparation and intrathecal drug delivery
All experiments conformed to the Guiding Principles in the Care and Use of
Animals of the American Physiology Society and were approved by the National
Defense Medical Center Animal Care and Use Committee (National Defense Medical
Center, Taipei, Taiwan). Intrathecal catheters and microdialysis probe implantation
were performed as described previously.7 In brief, male Wistar rats (350-400 g) were
anaesthetized with phenobarbital (60 mg/kg, intraperitoneally) and implanted with
two intrathecal catheters (8.5 cm) with or without a microdialysis loop probe via the
atlanto-occipital membrane down to the lumbar enlargement L1–L2 of the spinal
bony structure. The levels of L1–L2 spinal bony structure correspond to the spinal
cord segments of L5, L6, and S1–S3, which are responsible for the tail-flick reflex.23
One intrathecal catheter was connected to a mini-osmotic pump for infusion of saline (1 μl/h) (Sal rats) or morphine (15 μg/h) (MO rats) for 5 days, while the other was used for the subsequent injection of saline (Sal/Sal or MO/Sal rats) or TNP-ATP
(Sal/TNP-ATP or MO/TNP-ATP rats) or ifenprodil (Sal/IFE or MO/IFE rats). On day
5, after development of morphine tolerance, the rats were injected with either
TNP-ATP (50 g or 12.5-50 g as indicated) or saline (as control) or ifenprodil (10 μg/5 μl, intrathecally), then, 30 min later, a single dose of morphine (15 μg/5 μl,
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intrathecally) was injected and its antinociceptive effect measured. All rats were
maintained on a 12-hr light/dark cycle with food and water freely available. Rats with
neurological deficits were excluded from the study. All drugswere purchasedfrom
Sigma (St. Louis, MO). Preliminary results did not show any abnormal motor
function after intrathecal injection of test drugs (data not shown).
Construction of the spinal cord microdialysis probe
The technique for spinal microdialysis probe construction was modified from
that in a previous study.24 The probe was constructed using two 7 cm PE5 tubes
(0.008 inch inner diameter, 0.014 inch outer diameter; Spectranetics, Colorado
Springs, CO, USA) and a 4.2 cm cuprophan hollow fiber (Hospal Co, Lyon, France).
A nichrome-formvar wire (0.0026 inch diameter; A-M System, Everret Inc., WA) was
passed through a polycarbonate tube (194 μm outer diameter, 102 μm inner diameter;
0.7 cm in length) and the cuprophan hollow fiber (active dialysis region), which was
then connected to a PE5 catheter using epoxy glue. The middle portion of the
cuprophan hollow fiber was bent to form a U-shaped loop, and both ends of the
dialysis loop, which consisted of silastic tubes, were sealed with silicone. The dead space of the dialysis probe was 8 μl. During in vitro measurements, the recovery rates of the probes were around 40% at an infusion rate of 5 μl/min.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Behavioral tests
The tail-flick latency was measured using the hot water immersion test (52 ± 0.5
o
C) with the rats placed in plastic restrainers. The average baseline tail-flick latency
was 2 ± 0.5 sec in naïve rats and the cut-off time was 10 sec. The percentage of the
maximal possible antinociceptive effect was calculated as (maximum latency-baseline
latency) / (cut off latency – baseline latency) × 100. Antinociceptive dose-response
curves were constructed for each study group.
Cerebrospinal fluid sample collection and measurement of excitatory amino
acids
One of the externalized silastic tubes was connected to a syringe pump
(CMA-100, Acton, MA) and perfused with Ringer’s solution (8.6 mg/ml of NaCl,
0.33 mg/ml of CaCl2, and 0.3 mg/ml of KCl). The cerebrospinal fluid (CSF)
dialysates were collected from the other externalized silastic tube of the microdialysis
probe using a standard procedure of a 50 min washout period, followed by a 30 min
sample collection period at a flow rate of 5 μl/min in a polypropylene tube on ice, and
were frozen at -80 oC until assayed. The concentrations of EAAs were measured by
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chromatography (Agilent 1100, Agilent Technologies, Santa Clara, CA) with a
reverse-phase ZORBAX Eclipse amino acid analysis column (4.6×150 mm2, 3.5 μm)
and fluorescent detector (Gilson model 121, set at 428 nm) as described previously.25
External standards (authentic amino acids at concentrations of 156.25, 312.5, 625,
1250, and 2500 μM ) were run at the beginning and end of each sample group. Peak
heights were normalized to the standard peaks and quantified based on the linear
relationship between peak height and the amount of the corresponding standard.
Preparation of spinal cord total lysate and synaptosomal membrane and
cytosolic fractions and Western blot analysis
After drug treatment, as described in animal preparation and intrathecal drug
delivery section, rats were sacrificed by exsanguination under isoflurane (ABBOTT,
Abbott Laboratories Ltd, Queenborough, Kent, United Kingdom) anesthesia,
laminectomy was performed at the lower edge of the 12th thoracic vertebra (L1-L2
spinal bony structure) and the lumbar enlargement of the spinal cord immediately
removed and stored at -80 oC until used for Western blotting. To prepare a total lysate,
the dorsal portion of the lumbar spinal cord enlargement was homogenized in ice-cold
lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2% Triton X-100, 100 μg/ml of
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lysate centrifuged at 12,000 g for 30 min at 4 ºC, and the supernatant used for Western
blotting. To prepare cellular fractions, the dorsal portion of the lumbar spinal cord
enlargement was fractionated into cytosolic, membrane,and nuclear fractions using a
Cytoplasmic, Nuclear, and Membrane compartment protein extraction kit as
recommended by the manufacturer (Biochain Institute, Inc., Hayward, Calif). The
membrane and cytosolicfractions were checked for specificity by Western blotting
with mouse anti-rat epidermal growth factor receptor (1:2000; MBL, Naka-ku Nagoya,
Japan) and anti-rat α-tubulin antibodies (1:5000; Laboratory Frontier, Seodaemun-gu,
Seoul, Korea), respectively. The protein concentrations of the samples were
determined by the bicinchoninic acid assay(Pierce, Thermo Fisher Scientific Inc,
Waltham, MA) using bovine serum albumin as the standard. Samples containing 20
µg of protein were adjusted to a similar volumewith loading buffer (10% sodium
dodecyl sulfate, 20% glycerin, 125 mM Tris, 1 mMEDTA, 0.002% bromophenol blue,
10% β-mercaptoethanol) and the proteins denaturedby heating at 95 °C for 5 min, separatedon 10 % sodium dodecyl sulfate-polyacrylamide gels, and transferred onto
polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes
were blocked with 5% non-fat milk in Tris-Tween buffer saline (50 mM Tris-HCl, 154
mM NaCl, 0.05% Tween 20, pH 7.4), then incubated overnight at 4°C with polyclonal
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dilution in 5% non-fat milk in Tris-Tween buffer saline) or monoclonal mouse anti-rat
PSD-95 antibodies (1:5000 dilution in 5% non-fat milk in Tris-Tween buffer saline)
(allfrom Millipore, Billerica, MA), then incubated for 1 h at roomtemperature with
horseradish peroxidase-conjugateddonkey anti-rabbit or anti-mouse IgG antibodies,
as appropriate (1:2000 in 5% non-fat milk in Tris-Tween buffer saline) (Jackson
ImmunoResearch, West Grove, PA). Membrane-bound secondary antibodies were
detectedusing Chemiluminescenceplus reagent (PerkinElmer LAS, Boston, MA) and
visualized using a chemiluminescence imaging system (Syngene, Cambridge, United
Kingdom). Finally, the blotswere incubated for 18 min at 56 °C in stripping buffer
(62.6 mM Tris-HCl, pH: 6.7, 2% sodium dodecyl sulfate, 100 mM mercaptoethanol)
and reprobed with monoclonal mouse anti-β-actin antibody (1:5000; Sigma) as a
loadingcontrol. The Western blot analysis was repeated three times. The density of
each specificband was measured using a computer-assisted imaging analysis system
(Gene Tools Match software, Syngene, Cambridge, United Kingdom).
Immunoprecipitation of post-synaptic density- 95/NR1 and NR2B subunits
complex
To determine the co-assembly of PSD-95, NR1 and NR2B subunits, the
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anti-PSD-95 antibody. Anti-PSD-95 antibody (1:50; Cell Signaling, Danvers, MA)
was covalently cross-linked to Dynabeads® protein A (invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The PSD-95/NR1 and NR2B complexes were isolated by incubating 200 μg of spinal cord dorsal horn membrane proteins
solubilized in Cytoplasmic, Nuclear, and Membrane compartment protein extraction
kit extraction buffer with 50 μl of Dynabeads® protein A for 1 h at room temperature.
The incubation performed with normal mouse serum was used as negative control.
Dynabeads were precipitated using a magnet, and then the beads were extensively
washed with phosphate-buffered saline. Precipitated proteins were eluted with 50 μl
sodium dodecyl sulfate-containing sample buffer, and 20 μl of the samples were used
for Western blots as described inWestern blot analysis.
Fluorescence immunocytochemistry and image analysis
For fluorescence immunocytochemistry, the lumbar spinal cord was post-fixed
overnight at 4 ℃ in 4% paraformaldehyde prepared in 0.1 M phosphate buffer (pH
7.4), then cryoprotected in 30% sucrose for 2 days. It was confirmed as lumbar spinal
cord by the cross anatomy, which showed nearly a circular shape with very large
anterior and posterior gray horns and relatively little white matter. Sections (5 μm)
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preincubated for 1 h with 4% normal goat serum in phosphate-buffered saline
containing 0.01% Triton X-100. After three washes times in ice-cold
phosphate-buffered saline, the sections were incubated overnight at 4 oC with
unlabeled mouse monoclonal anti-rat beta-III tubulin (Santa Cruz, CA, USA; 1:100
dilution in phosphate buffered saline with Triton X-100 containing 2% normal goat
serum) and rabbit polyclonal antibodies anti-rat NR1 or NR2B (both from Millipore;
1:500 dilution in phosphate buffered saline with Triton X-100 containing 2% normal
goat serum). The sections were then reacted for 1 h at room temperature with
rhodamine-labeled goat anti-rabbit IgG antibodies (red fluorescence) and fluorescein
isothiocyanate-labeled donkey anti-mouse IgG antibodies (green fluorescence) (both
from Jackson ImmunoResearch) and images were captured using an Olympus BX 50
fluorescence microscope (Olympus, Optical, Tokyo, Japan) and a Delta Vision
disconsolation microscopic system operated by SPOT software (Diagnostic
Instruments Inc. Sterling Heights, MI). The laser wavelength was set at 488 nm for
fluorescein isothiocyanate fluorescence and 568 nm for rhodamine fluorescence.
Controls without primary antibody were run to confirm that the staining was specific.
Data and statistical analysis
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using SigmaStat 3.0 software (SYSTAT Software Inc., San Jose, CA). The
appropriate paired t-test (two-tailed) or analysis of variance (ANOVA) was used to
determine the statistical significance with a criterion of p< 0.05. Tail-flick latencies
and EAA concentration were analyzed using two-way (time and treatment) ANOVA
followed by subsequent one-way ANOVA (at each time of the experiment) with a
post hoc Student-Newman-Keuls test. Values for the analgesic dose of 50% of the
maximal possible antinociceptive effect (AD50) were analyzed using a
computer-assisted linear regression program SigmaPlot 10.0 (SYSTAT Software
Inc.). The 95% confidence interval (CI) was calculated using the pharmacologic
calculations system PHARM/PCS version 4.2 (MicroComputer Specialists,
Philadelphia, PA). For immunoreactivity data, the intensity of each test band was
expressed as the optical density relative to that of the average optical density for the
corresponding control band. For statistical analysis, immunoreactivity was analyzed
by one-way ANOVA, followed by multiple comparisons with the
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Results
Treatment with the P2X receptor antagonist TNP-ATP restores the
antinociceptive effect of morphine in morphine-tolerant rats
As in our previous study, morphine challenge (15 μg / 5μl, intrathecally) on day
5, at 3 h after discontinuation of drug infusion, produced a significant antinociceptive
effect in saline-infused rats (Sal/Sal) (p<0.001), but not in morphine-tolerant rats
(MO/Sal) (p=0.017) (Fig. 1A). TNP-ATP alone did not produce an antinociceptive
effect in either saline-infused controls (p=0.502) or morphine-tolerant rats (p=0.962).
However, treatment with TNP-ATP (12.5, 25, 50 μg / 5μl, intrathecally) 30 min before
morphine challenge (MO/TNP-ATP) dose-dependent restored the antinociceptive
effect in morphine tolerant rats (p<0.001).The two-way ANOVA of these time-course
curves showed significant different in tail-flick latency by treatments, by time, and for
the interactions (P < 0.001). High dose of TNP-ATP (100μg/5μl) treatment produced
similar antinociceptive effect as TNP-ATP 50μg treatment in morphine-tolerant rats
(data not shown). As shown in Fig. 1B, TNP-ATP treatment 30 min before morphine
injection had no effect on the morphine dose-response curve in saline-infused rats
(Sal/TNP-ATP), the AD50 being 1.12 μg in Sal/Sal rats and 1.19 μg in Sal/TNP-ATP
rats. In morphine-tolerant rats, the morphine dose-response curve was shifted to the
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(50 μg) treatment restored the antinociceptive effect of morphine in morphine-tolerant rats, shifting the AD50 from 90.51 μg (MO/Sal) to 16.35 μg (MO/TNP-ATP).
Treatment with lower doses of TNP-ATP either 12.5 or 25 μg showed slightly restored
morphine’s antinociceptive effect in morphine-tolerant rats, with AD50 of 46.54 and
35.19 μg, respectively.
Effect of TNP-ATP on levels of NMDA receptor subtypes in the total lysate and
the synaptosomal membrane of morphine-tolerant rats
As shown in Fig. 2, immunoblot analysis showed that levels of NR1, NR2A and
NR2B in the spinal cord dorsal horn lysate from saline-infused rats (Sal/Sal) were
unaffected by TNP-ATP treatment (Sal/TNP-ATP) (NR1, p=0.057; NR2A, p=0.126
and NR2B, p=0.957, respectively). On day 5, long-term morphine infusion
upregulated levels of NR1 and NR2B subunits in the total lysate by approximately
50-100 % (MO/Sal) and this effect was not prevented by TNP-ATP treatment
(MO/TNP-ATP) (p<0.001). As shown in Fig. 3, in morphine-tolerant rats (MO/Sal),
cytosolic levels of NR1 and NR2B were no different from those in saline-infused
(Sal/Sal) or saline-infused TNP-ATP-treated (Sal/TNP-ATP) rats. However, TNP-ATP
treatment significantly increased cytosolic levels of NR1 and NR2B subunits in
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contrast, as shown in the right and bottom panels of Fig. 3, increased levels of NR1
and NR2B subunits were seen in the synaptosomal membrane in morphine-tolerant
rats (compare MO/Sal with Sal/Sal) and this effect was prevented by TNP-ATP
treatment (MO/TNP-ATP) (p<0.001). Expression of the
α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor GluR1 and GluR2 subunits in the cytosolic and synaptosomal membrane fractions was not affected by
any of the treatments (data no shown) (p=0.672 and 0.624, respectively). Epidermal
growth factor receptor and α-tubulin markers were used to confirm the identity of the
membrane and cytosolic fractions (Fig. 3). Fluorescence microscopy localization of
the NR1 and NR2B subunits is shown in Fig. 4 and 5, respectively. In
morphine-tolerant rats, a robust and extensive NR1 and NR2B subunit labeling was
evenly distributed throughout the entire neuron (MO/Sal), whereas labeling was
cytosolic after TNP-ATP treatment (MO/TNP-ATP).
NR1/NR2B antagonist ifenprodil treatment attenuated the antinociceptive
tolerance of morphine
As shown in Fig.6, on day 5 three hours after discontinuation of morphine
infusion, morphine challenge (15 μg) did not produce antinociceptive effect in
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was observed in saline-infused rats (Sal/Sal) (p<0.001). However, pretreatment with
ifenprodil (10 μg, intrathecally) 30 min before morphine challenge preserved its
antinociceptive effect in morphine tolerant rats (MO/IFE) (p<0.001). Ifenprodil alone
had no antinociceptive effect in either saline-infused control rats (p=0.543) or
morphine-tolerant rats (p=0.1). As shown in Fig. 6B, the dose-response showed that
the AD50 for morphine was 1.12 μg in Sal/Sal rats and 1.13 μg in Sal/IFE rats. In
morphine-tolerant rats, morphine’s dose-response curve was shifted to the right by
80-fold (MO/Sal, AD50=89.88 μg) compared to saline-infused rats (Sal/Sal,
AD50=1.12 μg), and ifenprodil treatment potentiated the antinociceptive effect of
morphine of morphine-tolerant rats, the AD50 were from 89.88 μg (MO/Sal) to 25.28
μg (MO/IFE).
TNP-ATP treatment suppresses the morphine challenge-evoked EAA release in
morphine-tolerant rats
In the CSF microdialysis experiment, TNP-ATP treatment 30 min before
morphine challenge had no significant effect on CSF EAA levels in either
saline-infused controls (aspartate, p=0.68; glutamate, p=0.338) or morphine-tolerant
rats (aspartate, p=0.635; glutamate, p=0.074). As shown in Figure 7, morphine
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(aspartate, p=0.658; glutamate, p=0.868) or saline-infused plus TNP-ATP-treated
(Sal/TNP-ATP) rats (aspartate, p=0.949; glutamate, p=0.814). As in our previous
study 6,7, morphine challenge resulted in a significant increase in aspartate and
glutamate release in morphine-tolerant rats (MO/Sal), and TNP-ATP treatment 30 min
before morphine challenge completely blocked this morphine-evoked EAAs release in
morphine-tolerant rats (MO/TNP-ATP) (p<0.001). Two-way ANOVA of these
time-course curves showed significant different in EAA concentrations by treatments,
by time, and for the interactions (P < 0.001).
TNP-ATP treatment downregulates synaptosomal membrane post-synaptic
density-95 expression in morphine-tolerant rats
In Fig. 8, the density of the PSD-95 band on immunoblots of the synaptosomal
membrane fraction from the saline-infused rat spinal cord dorsal horn (Sal/Sal) is
expressed as 1. TNP-ATP treatment alone had no effect on PSD-95 expression in
saline-infused rats (compare Sal/TNP-ATP and Sal/Sal). Long-term
morphine-infusion increased (by approximately 100%) synaptosomal membrane
PSD-95 expression (MO/Sal) and this effect were not only prevented by TNP-ATP
treatment (MO/TNP-ATP), but PSD-95 expression was lower than in the saline
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Effect of TNP-ATP treatment on the co-assembly of post-synaptic density-95 and
NR1 and NR2B subunits
PSD-95 provides a physical means for anchoring of NMDA receptor at the
postsynaptic site, and the co-assembly of PSD-95 with NR1 and NR2B in
morphine-tolerant rats was examined. As shown in Fig. 9, an increasing of the
co-assembly of three proteins was noted in the morphine-tolerant rat lumbar spinal
cord. TNP-ATP treatment dose-dependently reverses the increasing of PSD-95, NR1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Discussions
In the present study, TNP-ATP treatment restored the antinociceptive effect of
morphine and prevented the morphine-induced increase in aspartate and glutamate in
the spinal CSF of morphine-tolerant rats. Moreover, we found that long-term
morphine infusion upregulated expression of the NMDA receptor NR1 and NR2B
subunits in the total lysate of the lumbar enlargement of the spinal cord, and this was
unaffected by TNP-ATP treatment. However, TNP-ATP treatment significantly
increased the amount of cytosolic NR1 and NR2B, in contrast, reversed the increase
in NR1 and NR2B expression in the synaptosomal fraction of morphine-tolerant rat
spinal cords. Moreover, treatment with NMDA receptor NR1/NR2B antagonist
ifenprodil produced similar effect as TNP-ATP; it also potentiated the antinociceptive
effect of morphine. Therefore, the 5.5-fold left-ward shift in the AD50 of morphine in
tolerant rats by TNP-ATP treatment might via regulation of NMDA expression and
synaptic excitatory amino acid concentration in morphine-tolerant rats. In addition,
the upregulation of PSD-95 in the synaptosomal fraction was also observed in the
morphine-tolerant rat spinal cords, and this effect was reversed by TNP-ATP
treatment. Quantification of the co-precipitated complex revealed that treatment of
TNP-ATP dose-dependently downregulates PSD-95, NR1 and NR2B expression in
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NMDA receptor NR1 and NR2B subunits expression on the postsynaptic membrane
may be involved, at least in part, in the loss of PSD-95 expression.
Glutamate and aspartate have been shown to be involved in nociception
transmission in the spinal cord.26In previous and our recent studies, the results failed
to demonstrate an increase in CSF EAA levels during induction of morphine
tolerance.7,27,28 However, post-treatment with naloxone evoked a significant and
time-dependent increase in the CSF dialysate glutamate and taurine concentration, but
not other amino acids in chronic morphine-infused rats.27 Similarly, we demonstrated
that morphine challenge induced an increase of glutamate and aspartate in the CSF
dialysates of morphine-tolerant rats; it was also accompanied by a loss of morphine’s
analgesic effect,7,28and co-administration of morphine with the NMDA antagonist not
only attenuated morphine tolerance development, but also blocked morphine-induced
spinal EAAs release.28 The sustained potentiation of NMDA receptor-mediated
responses may be through μ-opioid receptor mediated protein kinase C activation.29
These evidence suggests a positive feedback control between opioid and
glutamatergic receptors, particularly the NMDA receptors. As known, chronic
morphine infusion induced tolerance and Gi-protein uncoupling, and the morphine
challenge in our present study may act via Gs-protein signal transduction, and result
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EAA concentration by morphine challenge in the present study might be reflecting a
direct action of morphine on NMDA receptor sensitization after chronic morphine
exposure. Co-administration of morphine with various drugs, such as the NMDA
antagonist MK-801, gabagentin, or amitriptyline, preserves the antinociceptive effect
of morphine by lowering CSF EAA levels.7,28,32 In the present study, we also found
that acute intrathecal morphine challenge induced an increase in glutamate and
aspartate levels in tolerant rat spinal CSF dialysates and loss of the antinociceptive
effect of morphine, and that TNP-ATP treatment prevented the morphine-evoked EAA
increase in the CSF. These findings suggest that the restoration of the antinociceptive
effect of morphine by TNP-ATP might partly result from a reduction in spinal EAA
release.
Activation of NMDA receptors has been shown to play a crucial role in the
development of tolerance to the analgesic effect of morphine.4 Pharmacological
analysis has demonstrated that blockade of NMDA receptor hyperfunction effectively
prevents the development of morphine tolerance.33,34 The competitive NMDA
receptor antagonist LY274614 prevents antinociceptive tolerance to the highly selective μ-opioid agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin.35 In the present
study, we also demonstrated that posttreatment with NMDA receptor specific
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morphine-tolerant rats. Studies involving alterations in synaptic NMDA receptor
expression, including antisense and transgenic knockdown of NMDA receptors,
support the idea that NMDA receptor activation is important for morphine-induced
plasticity and provide strong evidence that a unique pharmacological state is required
for inhibition of behavioral adaptations.12,36 Yang et al.37 demonstrated that the
amount of NMDA receptors at the synapse regulates synaptic responses and pain
sensitivity. The present study showed that long-term morphine infusion increased
NR1 and NR2B expression in the synapse and that this correlated with development
of morphine tolerance, in agreement with a previous report that morphine tolerance is
associated with time-dependentupregulation of the NR1 subunit in thespinal cord
dorsal horn compared to the saline control group.38 Presumably, enhancement of NR1
expression at the synapse strengthens NMDA receptor-mediated synaptic
transmission and thus increases NMDA receptor-evoked intracellular signals, leading
to central sensitization and behavioral manifestations.12,39 In morphine-tolerant rats,
treatment with the P2X receptor antagonist TNP-ATP significantly decreased synaptic
NR1 and NR2B subunit expression and decreased the morphine-evoked EAA release
and restored the antinociceptive effect. The rapid dynamic change in synaptic
NR1/NR2B in neurons was associated with decreased PSD-95 expression.
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receptor NR2 subunits in the post-synaptic membrane and mediates the triggering of
many physiological and pathophysiological functions via NMDA receptor
activation.40,41 Previous studies have demonstrated a critical role for the interaction of
PSD-95 with NMDA receptors in receptor trafficking to the neuron surface, synaptic
localization, and intracellular signaling.42-44 Co-transfection with PSD-95 and
NR1/NR2A or NR1/NR2B subunit clones results in increased NR2A and NR2B
subunit expression via interaction of the C-terminal threonine/serine/valine/valine
motif of the NR2 subunit with PSD-95, and results in increased cell-surface
expression of the assembled NR1/NR2A and NR1/NR2B subtypes.45-47 In addition,
binding of PSD-95 to the NR2B C-terminal serine/threonine-X-valine motif reduces
receptor endocytosis from the neuron surface and stabilizes NR2B-containing NMDA
receptors in the synapse,42,43 thereby increasing the residence time of receptors at the
cell surface. These studies suggest that PSD-95 plays a crucial role in the trafficking,
membrane targeting, and internalization of NMDA receptor complexes. In our present
study, PSD-95 expression was increased after long-term morphine infusion and this
effect was inhibited by acute TNP-ATP treatment before morphine challenge.
Quantification of the immunoprecipitated complex densities of PSD-95/NR1/NR2B
revealed a significant increase in morphine-tolerant rats; this phenomenon was
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lower level of PSD-95 results in loss of stability of NR1 and NR2B subunits in the
synapse, which reduces the communication/coupling of NMDA receptors with
intracellular signaling cascades. The underlying mechanisms between P2X receptor
and PSD-95 interaction need further investigation.
P2X receptors play a crucial role in facilitating pain transmission at peripheral
and spinal sites, as both peripheral sensory neurons and spinal cord dorsal horn
neurons can be depolarized by ATP.48,49 Studies have indicates that P2X and μ-opioid
receptors are functionally coupled in sensory neuron.50 Extracellular ATP-evoked P2X
receptor inward current inhibited opioid sensitivity in neurons co-cultured with
fibrosarcoma cells.51 Translocation and activation of protein kinase C enhance
postsynaptic neuron excitability in morphine-tolerant rats.10,52,53 Moreover, activation
of protein kinase C showed significantly potentiation of Ca2+ signal and inward cation
current (predominately Na+) as well through P2X3 receptor in DT-40 3KO and
HEK-293 cells.54 Upregulation of P2X3 receptor expression is seen following chronic
constriction injury of the sciatic nerve and provokes ectopic sensitivity to ATP.55,56
Recent reports using gene knockout, antisense oligonucleotides, or the selective P2X3
antagonist A-317491 all point to a crucial role of P2X3 receptors in chronic
inflammatory and neuropathic pain.20,57,58 Interestingly, P2X receptor agonist-induced
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antagonists.59 A direct interaction between the purinergic and glutamatergic receptor
systems in mediating nociceptive processing in the spinal cord is further supported by
evidence that P2X receptor activation can stimulate glutamate release in spinal dorsal
horn neurons.60 In the present study, we found that treatment with the P2X receptor
antagonist TNP-ATP preserves morphine’s antinociceptive effect in morphine tolerant
rats; the mechanisms might be involved a significant reduction of synaptosomal NR1
and NR2B expression and morphine-evoked EAA release from presynaptic nerve
terminals in morphine-tolerant rats. The above results provide direct evidence for an
interaction between the purinergic and NMDA receptor systems.
TNP-ATP is one of the potent P2X receptor antagonists and is selective for P2X1,
P2X3, and P2X2/3 receptors.61 Intrathecal administration of TNP-ATP attenuates
α,β-meATP-induced hyperalgesia in mice and the pronociceptive effect of formalin and capsaicin.59,62 In present study, intrathecal treatment with TNP-ATP (63 nmol)
alone did not produce any antinociceptive effect. Although previous studies indicated
that intrathecal administration of low doses of TNP-ATP (1-10 nmol) produces a
partial, but significant, antinociceptive effect in mice62 and intradermal administration
of larger doses (100-300 nmol) produces significant attenuation (approx. 50%) of
acute formalin-induced paw flinching.63 Intraperitoneal administration of sufficient
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abdominal constriction assay.64 These diverse results might be due to differences in
the doses of TNP-ATP, animal models and relevant site of action. The different needs
further investigation.
In conclusion, our present study demonstrates that TNP-ATP treatment restores
the antinociceptive effect of morphine in morphine tolerant rats possibly by inducing
internalization of NR1 and NR2B from the synaptosomal membrane into the neuron
cytosol, thus reducing NMDA receptor-mediated intracellular signaling and EAA
release in the CSF following morphine challenge. The synaptic trafficking of
glutamate receptor subunit NR1 and NR2B may be modulated by the synaptic
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 References
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