| Summary: | 博士 === 國立臺灣大學 === 藥理學研究所 === 93 === Gabapentin, a gamma-aminobutyric acid (GABA) analogue anticonvulsant, possesses antinociceptive effects in several animal pain models and clinical trials. Up to now, the action mechanism(s) of gabapentin remains unknown and a variety of hypotheses have been proposed. Gabapentin was initially shown to have little affinity at GABAA or GABAB receptors but was recently reported to be a selective agonist at the GABAB receptor consisting of GABAB1a-GABAB2 heterodimers. In addition, activation of NMDA receptors and ATP-sensitive K+ (KATP) channels were also reported to be involved in the cellular actions of gabapentin. A specific binding site of [3H]gabapentin in the brain has been reported to be the alpha2delta subunit of voltage-dependent Ca2+ channels, at which the binding of gabapentin was modulated by magnesium chloride, ruthenium red and spermine. In this study, we validated if these proposed cellular mechanisms contribute to the spinal analgesic effect of gabapentin using a rat model of postoperative pain. The alpha2delta subunit is present in several types (L-, P/Q-, N-, T-type) of Ca2+ channels, which are all distributed in the spinal cord. Among all types of Ca2+ channels, T-type Ca2+ channels have been implicated in the synaptic plasticity at synapses from nociceptive nerve fibers, which might be involved in the central sensitization of inflammatory pain. We have examined the following in the postoperative pain model: 1) if gabapentin acts as an GABAB receptor agonist to achieve it antiallodynic effect; 2) if activation of NMDA receptors or KATP channels are involved in the antiallodynic effect of intrathecal gabapentin; 3) if magnisium chloride, ruthenium red or spermine could affect the antiallodynic effect of intrathecal gabapentin; 4) if the expression of spinal alpha2delta-1 subunit of Ca2+ channels was altered; 5) which type of Ca2+ channel blockers would mimic the antiallodynic effects of intrathecal gabapentin. Finally, the possible antinociceptive effects of intrathecal T-type Ca2+ channel blockers were examined in the rat formalin test.
The postoperative pain model was conducted in male Sprague-Dawley rats (250-300 gm). Under isoflurane anesthesia, the rats with chronic intrathecal catheters received an incision over plantar surface of right hindpaw to produce punctate mechanical allodynia. Withdrawal thresholds to von Frey filament stimulation near the incision site were measured before, 2 h after incision and 15, 30, 45, 60, 90 and 120 min after intrathecal drug administrations. Receptor or channel (GABAA, GABAB and NMDA receptors and KATP channel) ligands were intrathecally pretreated 5 or 10 min before gabapentin was administered.
The formalin test was conducted by giving subcutaneous injection of 5% formalin 50 microL into rat (Sprague-Dawley, male, 250-300 gm) right hindpaw. T-type Ca2+ channel blockers were given intrathecally 10 min before formalin injection. Biphasic flinching or shaking of the injected paw were observed after formalin injection. Phase 1 and 2 were defined as 0–9 and 10–60 min after formalin injection, respectively. The number of flinches was counted for 1-min periods at 1 and 5 min and at 5-min intervals from 10 to 60 min. The biting and licking time was also monitored during 0–5 and 20–40 min after formalin injection.
Intrathecal injection of gabapentin (30-200 microg) dose-dependently reduced incision-induced allodynia. GABAB receptor antagonists, CGP 35348 and CGP 55845, did not affect gabapentin-induced antiallodynic effect at doses effective in antagonizing the antiallodynic effect of baclofen, a GABAB receptor agonist. Intrathecal pretreatment with NMDA receptor antagonists, APV (6 microg) and MK-801 (14 microg), GABAA receptor antagonist, bicuculline (0.3 microg), and KATP channel blocker, glibenclamide (300 microg), did not attenuate the antiallodynic effect of gabapentin. The GABAA receptor agonist, isoguvacine (20 microg), and KATP channel openers, pinacidil (100-300 microg) and diazoxide (600-1200 microg), per se, had little effect on the postincision allodynic response.
The antiallodynic effect of gabapentin was inhibited by the alpha2delta subunit binding modulators, MgCl2 (5-20 microg) and ruthenium red (0.2-20 ng), non-competitively, but not by spermine at doses without inducing motor weakness. On the other hand, the antiallodynic effect of intrathecal morphine was unaffected by either alpha2delta subunit binding modulators. In addition, an upregulation of the alpha2delta-1 subunit of Ca2+ channels in the L4-6 dorsal spinal cord was observed in this postoperative pain model, similar to the findings in other gabapentin-sensitive animal pain models.
Intrathecal omega-conotoxin GIVA (0.1-3 microg), an N-type Ca2+ channel blocker, like gabapentin, attenuated the allodynic response induced by paw incision. However, P/Q-type (omega-agatoxin IVA 0.1-0.3 microg), L-type (verapamil 300 microg, diltiazem 500 microg and nimodipine 500 microg) and T-type (mibefradil 600 microg) Ca2+ channel blockers had little effect on the postincisional allodynia. On the other hand, intrathecal injection of T-type Ca2+ channel blocker, mibefradil (50-500 microg) or nickel (1-10 microg), but not ethosuximide (100-1200 microg) dose-dependently inhibited the nociceptive behaviors in phase 1 and 2 of the formalin test.
In conclusion, our results provide behavioral evidences to support that the alpha2delta subunit of N-type Ca2+ channels, but not GABAB receptors, NMDA receptors and KATP channels, may be involved in the antiallodynic action of intrathecal gabapentin in the postoperative pain model. In addition, activation of spinal T-type Ca2+ channels might be involved in formalin-induced nociceptive behaviors.
|
|---|
