Research reportRole of spinal muscarinic and nicotinic receptors in clonidine-induced nitric oxide release in a rat model of neuropathic pain
Introduction
Spontaneous pain, hyperalgesia (increased pain intensity in response to noxious stimuli) and allodynia (normally innocuous stimuli become painful) are forms of neuropathic pain which are associated with peripheral nerve injury 2, 19. Neuropathic pain often is not relieved adequately by conventional treatments such as non-steroidal anti-inflammatory drugs and opioids [25]. Intrathecal injection of clonidine, an α2 adrenergic receptor agonist, has become an alternative treatment for neuropathic pain patients [28]. However, the underlying pharmacological mechanisms of the potent analgesic action of intrathecal clonidine in neuropathic pain are not fully understood.
Recent studies provide strong evidence that spinal nitric oxide (NO) is directly involved in the analgesic effect of morphine and clonidine. In this regard, we have demonstrated that clonidine causes NO release in spinal cords of rats and sheep 17, 37. We have also shown that intrathecal injection of nitric oxide synthase (NOS) inhibitors attenuates both the analgesic action of systemic morphine in normal rats using the heat as a stimulus and the antiallodynic effect of intrathecal clonidine in neuropathic rats 26, 33. Consistent with these findings, Kolesnikov et al. [16]have found that neuronal NOS-1, which is predominant at the spinal level, mediates the analgesic effect of morphine in mice. Furthermore, our previous studies indicate that acetylcholine is linked directly to clonidine-induced NO formation. For example, we have demonstrated that intrathecal clonidine causes acetylcholine release from the spinal cord 5, 15, which, in turn, evokes NO synthesis [37]. Thus, a cascade of activation of α2 adrenergic receptors followed by release of acetylcholine and NO has been proposed to be a likely mechanism of clonidine's analgesic action in the spinal cord. It is also known that spinal α2 adrenergic and muscarinic receptors are involved in the clonidine-elicited NO synthesis in spinal cords since atropine and idazoxan both attenuate NO release caused by clonidine [37]. Like many other tissues, both muscarinic and nicotinic cholinergic receptors are present in the spinal cord 4, 12. Nicotinic receptors have also been linked to NO production in the nervous system. In this regard, activation of nicotinic receptors is capable of evoking long-lasting NO release in the hippocampus [32]. However, it remains unclear whether nicotinic receptors are involved in clonidine-evoked NO release in the spinal cord. Therefore, in the present study, we tested a hypothesis that spinal nicotinic receptors are involved in clonidine-induced NO release.
Several behavioral studies have provided strong evidence that activation of muscarinic and nicotinic receptors in the spinal cord produces antinociception in animals 4, 13. These two types of cholinergic receptors are also involved in intrathecal clonidine-produced analgesia in a rat model of neuropathic pain [27]. But the spinal muscarinic and nicotinic receptors do not contribute equally to the antiallodynic effect of intrathecal clonidine in this model. We have shown that intrathecal muscarinic receptor antagonists nearly abolish the effect of clonidine, while nicotinic receptor antagonists only attenuate about 40% of the effect of clonidine [27]. Since spinal NO is ultimately involved in the analgesic effect of clonidine [26], we reasoned that the greater involvement of muscarinic receptors in clonidine-produced analgesia may reflect an increased coupling between muscarinic receptors and NO following activation of spinal α2 adrenergic receptors. We thus tested another hypothesis that spinal muscarinic receptors are more important than nicotinic receptors in clonidine-evoked NO release in neuropathic rats.
Section snippets
Surgical procedures
Male rats (Harlan Sprague–Dawley) weighing 150–180 g were used in this study. Ligation of L5 and L6 spinal nerves in rats was used in this study as an experimental model of neuropathic pain because it produces profound and sustained tactile allodynia, which resembles the condition observed in patients with neuropathic pain. Rats were anesthesized with halothane. The left L5 and L6 spinal nerves were isolated and ligated tightly with 4-0 silk suture, according to the method described by Kim and
Effect of clonidine on nitrite release in the spinal cord
Perfusion of clonidine significantly increased nitrite release in a dose-dependent manner from 10−11 to 10−9 M (Fig. 1) in spinal cords from neuropathic rats. The concentration of clonidine-producing maximal nitrite release was 10−9 M. Furthermore, the nitrite release induced by perfusion with 10−9 M of clonidine was stable for at least 1 h (Fig. 2). The dose-dependent effect and the time course of clonidine-induced NO release obtained from neuropathic rats were similar to what was observed in
Discussion
There are two important findings from the present study. We found that treatment with either spinal nicotinic or muscarinic receptor antagonists attenuated significantly NO release elicited by an α2 receptor agonist, clonidine. Our study provides the first evidence that, like muscarinic receptors, spinal nicotinic receptors mediate NO release caused by activation of α2 receptors in normal and neuropathic rats. Furthermore, our results indicate that muscarinic receptors play a more important
Acknowledgements
This study was supported by HL-60026 (to H.L. Pan) and GM-35523 (to J.C. Eisenach) from the National Institutes of Health. Dr. Pan currently holds an Independent Scientist Career Award from the National Heart, Lung, and Blood Institute.
References (38)
- et al.
Nicotinic antagonist, alpha-bungarotoxin, binding to rat hippocampal neurons containing nitric oxide synthase
Brain Res.
(1997) - et al.
Lack of analgesic effect of opioids on neuropathic and idiopathic forms of pain
Pain
(1988) - et al.
Autoradiographic distribution of []acetylcholine binding sites in the cervical spinal cord of man and some other species
Neurosci. Lett.
(1988) - et al.
Chronic neuropathic pain and its control by drugs
Pharmacol. Ther.
(1997) - et al.
Determination of nitric oxide using fluorescence spectroscopy
Methods Enzymol.
(1996) - et al.
Regional study of the colocalization of neuronal nitric oxide synthase with muscarinic receptors in the rat cerebral cortex
Neuroscience
(1995) - et al.
Muscarinic agonists as analgesics. Antinociceptive activity versus M1 activity: SAR of alkylthio-TZTP's and related 1,2,5-thiadiazole analogs
Life Sci.
(1995) - et al.
In vivo pharmacology of butylthio[2.2.2] (LY297802/NNC11-1053), an orally acting antinociceptive muscarinic agonist
Life Sci.
(1997) - et al.
Nicotine-evoked nitric oxide release in the rat hippocampal slice
Neurosci. Lett.
(1998) - et al.
Involvement of nitric oxide in development of tail-tremor induced by repeated nicotine administration in rats
Eur. J. Pharmacol.
(1997)