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The Influence of Morphine and GR89696 on NMDA receptor Phosphorytation
Pain is suppressed by the opioids morphine and GR89696 in rodent models of inflammation, neuropathy and
neuritis. Electrophysiological data suggests that these agents can alter the function of N-methyl-D-aspartate (NMDA)
receptors while recent work has demonstrated that spinal cord NMDA receptors are phosphorylated in animals
demonstrating hyperalgesia and allodynia. Therefore in this study, we tested the hypothesis that morphine and
GR89696 reduced the level of phosphorylation of spinal cord NMDA receptors. Adult male Sprague Dawley rats were
injected with img/kg morphine, 1 mg/kg GR89696 or saline subcutaneously. Four hours following the injections the
animals were sacrificed and the lumbar spinal cords removed and homogenized. NMDA receptor subunits were
then immunoprecipitated from the homogenates, run on western blots and probed with anti-phosphoserine,
phosphothreonine or phosphotyrosine antibodies. NR1 and NR2A subunits of the NMDA receptor demonstrated a decrease
in phosphorylation at serine residues in response to GR89696 and an increase in phosphorylation at tyrosine resides in
response to morphine. GR89696 suppressed threonine phosphorylation on NR2B subunits. These data indicated that
opioid agonists alter phosphorylation of NMDA receptors in the spinal cord and suggest that the two opioids modulate pain
using different mechanisms.
The increase in activity of NMDA (N-methyl-D-Aspartate) receptors in the mammalian central nervous system is known to
be involved in chronic pain. The role of NMDA receptor in chronic pain is a target for pain therapies (Boris, et al.).
Hyperalgesia, the enhanced experience of pain and allodynia, the experience pain from harmless stimuli are two symptoms
of many chronic pain patients (Eliav, et.al.). Phosphorylation is one mechanism that can enhance the function of
NMDA receptors. Protein phosphorylation has been recognized as a major mechanism for the regulation of receptor
function (Gou, et. al.). Regulation of NMDA receptor phosphorylation appears to be controlled by protein kinases
and phosphatases (Zou, et al.). Activation of NMDA receptors by protein phosphorylation has demonstrated the development
of spinal hyperexcitability and persistent pain (Guo, et al) (Figure 1).
Figure 1. A Schematic representation of the activation of NMDA receptor in relation to pain.
B Schematic representation of the NR1 and NR2 NMDA receptors.
We suspect the reduced activity of NMDA receptors in opioid analgesics could be due to decreased phosphorylation of
the protein. Previous studies demonstrated the blockage of hyperalgesia and allodynia in models of peripheral neuropathy
and neuritis by GR89696 as well as morphine (Eliav, et al.; Ho, et al.). In this study changes in NMDA activity will be
observed by changes in protein phosphorylation status of splice variants of spinal cord NR1, NR2A and NR2B subunits in
non-inflamed rat lumbar spinal cord after morphine and GR89696 injections. We tested the hypothesis that morphine
and GR89696 reduced the level of phosphorylation of spinal cord NMDA receptors.
MATERIALS AND METHODS
Adult male rats were separated into three groups. One as a
control, one group was injected subcutaneously with 1mg/kg
of morphine and one group was injected subcutaneously with 1mg/
kg GR89696. The animals were sacrificed four hours after the
injection. The lumbar region of spinal cords were dissected
from euthanized rats and homogenized with cell tysate buffer,
boiled, and centrifuged. The supernatant was collected.
Immunoprecipitation and western blotting analysis of spinal cord
tissue were conducted under the following conditions. 500 mg
of protein was incubated for 1 hour with either 4-20 mg of
rabbit polyclonal Exon 21 (C1) Ab or goat polyclonal NR1 Ab (Santa
Cruz Biotechnology), NR2A Ab, or NR2B Ab at 40ï¿½ C. Samples
were incubated for 1hour with 20 ut of either anti-goat IgG or
anti-rabbit IgG immobilized in agarose beads (Sigma) at 40ï¿½
C. Immunocomplexes were collected by centrifugation and washed.
The pellets were resuspended with 2X Tris-Glycine SDS sample
Protein samples were subjected SDS-PAGE. The get was transferred to
a PDVF membrane and blocked. The membranes were
incubated overnight at 40ï¿½C with mouse polyclonal
Phosphoserine antibody (1:1000, Sigma), polyclonal
Phosphothreonine antibody (1:1000) or polyclonal
Phosphotyrosine antibody (1:1000). The membranes were
either incubated with secondary rabbit IgG HRP-linked Ab (1:4000,
Cell Signaling) or mouse IgG HRP-linked Ab (1:4000, Cell Signaling) for
1 hour. The membranes were exposed to a chemiluminescent
detection system by LumiGlo (Cell Signaling) and exposed to Kodak
The relative density of immunoblots phosphorylated NR1, NR2A
and NR2B subunit protein from rat spinal cord tissue after
morphine and GR89696 intrathecal injection was compared with
control spinal cord tissue (Figure 2). Data was normalized to
baseline measurements of the control group and graphed (Figure 3).
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Figure 2. Western analysis of the spinal cord lumbar
enlargement tissue in adult rats with Phosphoserine,
Phosphothreonine and Phosphotyrosine after being
immunoprecipated with NR1 Ab, NR2A Ab or NR2B Ab.
There was no significant change in the immunoblots for NR1 and
NR2 threonine subunits and NR2B serine and tyrosine subunits (p
> 0.05). However, immunoblots for phospho-NR1-serine dorsal
GR89696 injection, phospho-NR2A-serine dorsal and ventral
morphine injection and phospho-NR2B-threonine ventral
GR89696 injection showed a significant decrease in protein
between the groups in comparison to controls (p < 0.05).
However, immunoblots for phospho-NR2A-tyrosine ventral GR9696
and phospho-NR1-tyrosine ventral morphine injections showed
a significant increase between the two groups in comparison to
controls (p < 0.05). Results are summarized in Table 1.
Density (Mean ï¿½SE) of Phosphorylated NR1, NR2A and NR2B Subunits at Serine, Threonine and Tyrosine Residues.
Subunits MD Mv GD Gv
NR1 Serine 0.491 ï¿½ 0.306 0.191 ï¿½1.150 9.47e-4 ï¿½ 0.049* 0.480 ï¿½ 0.546
NR1 Threonine 0.254 ï¿½ 0.327 0.060 ï¿½ 0.575* 0.219 ï¿½ 0.369 0.154 ï¿½ 0.307
NR1 Tyrosine 0.145 ï¿½ 1.910 0.032 ï¿½ 0.371* 0.287 ï¿½ 2.250 0.219 ï¿½ 0.349
NR2A Serine 0.0485 ï¿½ 0.200* 0.003 ï¿½ 0.065* 0.323 ï¿½ 0.459 0.299 ï¿½ 1.284
NR2A Threonine 0.075 ï¿½ 0.558 0.170 ï¿½ 0.791 0.138 ï¿½ 0.877 0.286 ï¿½ 0.465
NR2A Tyrosine 0.225 ï¿½ 0.315 0.174 ï¿½0.352 0.611 ï¿½0.272 0.404 ï¿½ 0.096
NR2B Serine 0.361 ï¿½ 0.400 0.218 ï¿½ 0.313 0.098 ï¿½ 7.44 0.483 ï¿½ 0.543
NR2B Threonine 0.266 ï¿½ 0.368 0.136 ï¿½ 0.317 0.272 ï¿½ 0.548 0.014 ï¿½ 0.122*
NR2B Tyrosine 0.218 ï¿½ 0.810 0.433 ï¿½ 0.090 0.167 ï¿½ 0.542 0.214 ï¿½ 0.115
MD, Morphine Dorsal Spinal Cord Injection; Mv, Morphine Ventral Spinal Cord Injection; GD, GR9696 Dorsal Spinal Cord Injection; Gv, GR6996 Ventral Spinal Cord Injection.
* Significantly different from control (t-test), p < 0.05.
Previous studies have determined the effectiveness of NMDA receptors in the prevention of hyperalgesia and allodynia
(Ho, Mannes, Dubner, and Caudle, 1997; Gou, Zou, Guan, Ikeda, Tai, Dubner, and Ren). This study demonstrates GR89696
and Morphine to decrease as well as increase phosphorylation in NR1, NR2A and NR2B subunits. Our results complement
other studies that find GR89696 to inhibit hyperalgesia. For example, Guo et al. found tyrosine phosphorylation of the
NR2B subunit of NMDA receptor in the spinal cord to cause inflammatory hyperalgesia. Increases in tyrosine
phosphorylation from our data support their findings.
In addition, we find serine and threonine residues decrease phosphorylation of NMDA receptors. Because
protein phosphorylation is a major means for regulating receptor function, morphine and GR89696 can be used to control
and prevent hyperalgesia. Many previous studies have focused efforts in determining effectiveness of NMDA antagonists
on inflamed or injured spinal cords. Data from this study indicated that opioid agonists alter phosphorylation of NMDA
receptors in the spinal cord and suggest that the two opioids modulate pain using different mechanisms. Thus, data
gathered from this study can be used to help improve on currently used analgesics.
I would like to thank the UF Scholars program and the UF College of Dentistry for granting me the opportunity to complete
my research. I would also like to thank my mentor Dr. Robert Caudle and Federico Perez for their continued guidance
and support. It truly was an honor to work in their lab.
Boris A. Chizh, Harald Schlutz, Manuela Scheede and Werner Englberger. Grunenthal GmbH,
Research Centre, Zieglerstrasse 6, 52078 Aachen, Germany. October 2000.
Caudle, R., Mannes, A., ladarola, M. (1997). GR89696 is a kappa-2 opioid receptor agonist and
a kappa 1 opioid receptor antagonist in the guinea pig hippocampus. The Journal of Pharmacology and
Experimental Therapeutics, 283(3), 1342-1349.
Dingledin, R., Borges, K., Bowie, D., and Traynelis, S. (1999). The Glutamate Receptor Ion
Channels. Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia.
Eliav, E., Herzberg, U., Caudle, R. (1999). The kappa opioid agonist GR89696 blocks
hyperalgesia and allodynia in rat models of peripheral neuritis and neuropathy. Pain, 79, 255-264.
Guo, W., Zou, S., Guan, Y., Ikeda, T., Tai, M., Dubner, R., Ren, K. (2002, July) Tyrosine
Phosphorylation of the NR2B Subunit of the NMDA receptor in the spinal cord during
development and maintenance of inflammatory hyperalgesia. The Journal of Neuroscience, 22 (14), 6206-6217.
Ho, J., Mannes, A., Dubner, R., Caudle, R. (1997). Putative kappa-2 opioid agonists are
antihyperalgesic in a rat model of inflammation. The Journal of Pharmacology and Experimental Therapeutics, 281, 136-141.
Svendsen, F., Rygh, L., Hole, K., Tjolsen, A. (1999). Dorsal horn NMDA receptor function is
changed after peripheral inflammation. Pain, 83, 517-523.
Terayama, R., Dubner, R., Ren, K. (2002). The roles of NMDA receptor activation and nucleus
reticularis gigantocellularis in the time-dependent changes in descending inhibition after
inflammation. Pain, 97, 171-181.
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