Journal of Undergraduate Research
Volume 5, Issue 5 - February 2004
The Influence of Morphine and GR89696 on NMDA receptor Phosphorylation
Jenny Liang
ABSTRACT
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 1mg/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.
INTRODUCTION
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
Sample Preparation
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 lysate buffer, boiled, and centrifuged. The supernatant
was collected.
Immunoprecipitation
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 40°C. Samples were incubated for 1hour with
20 ul of either anti-goat IgG or anti-rabbit IgG immobilized in agarose
beads (Sigma) at 40°C. Immunocomplexes were collected by centrifugation
and washed. The pellets were resuspended with 2X Tris-Glycine SDS
sample buffer (Invitrogen).
Western Blotting
Protein samples were subjected SDS-PAGE. The gel was transferred to
a PDVF membrane and blocked. The membranes were incubated overnight
at 40°C 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 x-ray film.
RESULTS
Immunoprecipitation
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 40°C. Samples were incubated for 1hour with 20 ul of either anti-goat IgG or anti-rabbit IgG immobilized in agarose beads (Sigma) at 40°C. Immunocomplexes were collected by centrifugation and washed. The pellets were resuspended with 2X Tris-Glycine SDS sample buffer (Invitrogen).
Western Blotting
Protein samples were subjected SDS-PAGE. The gel was transferred to
a PDVF membrane and blocked. The membranes were incubated overnight
at 40°C 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 x-ray film.
RESULTS
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).
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.
| Table 1 Density (Mean ±SE) of Phosphorylated NR1, NR2A and NR2B Subunits at Serine, Threonine and Tyrosine Residues. |
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| Subunits | MD | MV | GD | GV |
|---|---|---|---|---|
| NR1 Serine | 0.491 ± 0.306 | 0.191 ± 1.150 | 9.47e-4 ± 0.049* | 0.480 ± 0.546 |
| NR1 Threonine | 0.254 ± 0.327 | 0.060 ± 0.575* | 0.219 ± 0.369 | 0.154 ± 0.307 |
| NR1 Tyrosine | 0.145 ± 1.910 | 0.032 ± 0.371* | 0.287 ± 2.250 | 0.219 ± 0.349 |
| NR2A Serine | 0.0485 ± 0.200* | 0.003 ± 0.065* | 0.323 ± 0.459 | 0.299 ± 1.284 |
| NR2A Threonine | 0.075 ± 0.558 | 0.170 ± 0.791 | 0.138 ± 0.877 | 0.286 ± 0.465 |
| NR2A Tyrosine | 0.225 ± 0.315 | 0.174 ± 0.352 | 0.611 ± 0.272 | 0.404 ± 0.096 |
| NR2B Serine | 0.361 ± 0.400 | 0.218 ± 0.313 | 0.098 ± 7.44 | 0.483 ± 0.543 |
| NR2B Threonine | 0.266 ± 0.368 | 0.136 ± 0.317 | 0.272 ± 0.548 | 0.014 ± 0.122* |
| NR2B Tyrosine | 0.218 ± 0.810 | 0.433 ± 0.090 | 0.167 ± 0.542 | 0.214 ± 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.
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