Xinhui Li

Nicotinic acetylcholine receptor regulation of spinal norepinephrine release.

Background Neuronal nicotinic acetylcholine receptor (nAChR) agonists produce antinociception in animals. nAChRs exist almost exclusively on presynaptic terminals in the central nervous system and stimulate neurotransmitter release. This study tested whether nAChR agonists stimulate spinal release of the neurotransmitter norepinephrine either by direct actions on noradrenergic terminals or indirectly by stimulating release of other neurotransmitters to induce norepinephrine release. Methods Adult male rats were anesthetized and microdialysis probes inserted in the L2–L4 dermatomes of the spinal cord. Probes were perfused with artificial cerebrospinal fluid containing nicotine, the specific &agr;4&bgr;2* nAChR agonist metanicotine, or nicotine plus nAChR antagonists and norepinephrine measured in the microdialysates. The effects of specific glutamate receptor antagonists and nitric oxide synthase inhibitors were also examined. To determine direct effects on noradrenergic terminals, synaptosomes were prepared from spinal cord and incubated with nAChR agonists and antagonists. Results Both nicotine and metanicotine induced norepinephrine release in spinal microdialsyates, an effect reduced by nicotinic antagonists but not glutamate antagonists or nitric oxide synthase inhibitors. Both of the nicotinic agonists stimulated norepinephrine release in synaptosomes, and the effect of metanicotine was blocked at lower concentrations of &agr;4&bgr;2*- than &agr;7*-preferring nAChR antagonists. Conclusion These results suggest that one mechanism by which nAChR agonists act for analgesia is to stimulate spinal norepinephrine release. They do so by actions on &agr;4&bgr;2* nAChRs, and perhaps other subtypes, most likely located on noradrenergic terminals, rather than by indirectly stimulating norepinephrine release through glutamate release or nitric oxide synthesis.

Spinal noradrenergic activation mediates allodynia reduction from an allosteric adenosine modulator in a rat model of neuropathic pain

&NA; Activation of adenosine A1 receptors by endogenous adenosine or synthetic agonists produces anti‐nociception in animal models of acute pain and also reduces hypersensitivity in models of inflammatory and nerve‐injury pain. Allosteric adenosine modulators facilitate adenosine agonist binding to the A1 receptor. The purpose of the current study was to examine the effect, mechanisms of action, and interaction with noradrenergic systems of intrathecal (i.t.) or oral administration of the allosteric adenosine modulator T62 in a rat model of neuropathic pain. A spinal nerve ligation rat model (SNL; ligation of left L5 and L6 spinal nerve roots) was used. One week after SNL surgery, an i.t. catheter was inserted. Withdrawal threshold to mechanical stimulation of the left hind paw was determined before and after surgery, confirming mechanical hypersensitivity. Oral or i.t. T62 reduced hypersensitivity induced by SNL. The effects of i.t. T62 were inhibited by i.t. injection of an A1 receptor antagonist and by an &agr;2‐adrenergic antagonist but not by an A2 adenosine receptor antagonist. Anti‐dopamine &bgr; hydroxylase (D&bgr;H)‐saporin treatment reduce spinal norepinephrine content by 97%, accompanied by an almost complete loss of D&bgr;H immunoreactive axons in the spinal dorsal horn and neurons in the locus coeruleus. The effect of T62 was completely lost in animals treated with anti‐D&bgr;H‐saporin. These data support the hypothesis that activation of the A1 receptor by the allosteric modulator, T62, produces anti‐nociception via spinal noradrenergic activation.

α2-Adrenoceptor Activation by Clonidine Enhances Stimulation-evoked Acetylcholine Release from Spinal Cord Tissue after Nerve Ligation in Rats

Background:Spinally administered clonidine produces analgesia via &agr;2-adrenergic receptors. The analgesic potency of clonidine and its dependency on muscarinic acetylcholine receptors increase in rats after nerve injury. The authors hypothesized that these changes reflect greater acetylcholine release from the spinal cord by clonidine, either through direct or indirect effects. Methods:Male Sprague-Dawley rats were divided into two groups: no surgery or left L5 and L6 spinal nerve ligation (SNL). All experiments were performed 3 weeks after SNL. Crude synaptosomes were prepared from the spinal enlargement and loaded with [3H]choline. Samples were incubated with clonidine in the absence or presence of KCl depolarization. The authors also examined the effect of clonidine on KCl evoked acetylcholine release using perfusion of spinal cord slices, in which some spinal circuitry is maintained. Results:In synaptosomes, clonidine alone induced minimal acetylcholine release, which was actually greater in tissue from normal rats than in tissue from SNL rats. In the presence of KCl depolarization, however, clonidine enhanced acetylcholine release in tissue from SNL rats but inhibited release in tissue from normal rats. Similarly, in spinal cord slices, clonidine enhanced KCl evoked acetylcholine release in tissue from SNL animals but inhibited such release in tissue from normal animals. The &agr;2-adrenoceptor antagonist idazoxan inhibited the effects of clonidine in slices from SNL rats. Conclusion:These results suggest that clonidine enhances depolarization-induced acetylcholine release in neuropathic but not in normal spinal cord tissue. Interestingly, this enhanced acetylcholine release by clonidine occurs in a synaptosomal preparation, consistent with a direct effect on &agr;2 adrenoceptors on cholinergic terminals. Enhanced release of acetylcholine by clonidine could contribute to increased analgesia of clonidine in neuropathic pain.

Intrathecal Morphine Reduces Allodynia after Peripheral Nerve Injury in Rats via Activation of a Spinal A1 Adenosine Receptor

Background:The degree to which intrathecally administered morphine can alleviate hypersensitivity in animals after peripheral nerve injury is controversial, and the mechanisms by which morphine works in these circumstances are uncertain. In normal animals, morphine induces adenosine release, and in vitro data suggest that this link is disrupted after peripheral nerve injury. Therefore, using a controlled, blinded study design, the authors tested intrathecal morphine efficacy in rats with peripheral nerve injury and the role of spinal A1 adenosine receptors in the action of morphine. Methods:Male rats underwent intrathecal catheter implantation and lumbar spinal nerve ligation, resulting in hypersensitivity to tactile stimulation of the paw. Intrathecal morphine alone or with naloxone or the specific A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentyxanthine (DPCPX), was administered, and withdrawal threshold to von Frey filament application to the hind paw was determined. Results:Intrathecal morphine (0.25–30 &mgr;g) dose-dependently reversed mechanical hypersensitivity after spinal nerve ligation, with an ED50 of 0.79 &mgr;g. The effect of morphine was blocked by intrathecal naloxone. Intrathecal DPCPX alone had no effect on withdrawal threshold after spinal nerve ligation but completely reversed the effect of morphine, with an ID50 of 5.6 &mgr;g. Conclusions:This study is in accord with two recent reports that support short-term efficacy of intrathecal morphine to reverse hypersensitivity to mechanical stimuli in animal models of neuropathic pain. Despite previous studies demonstrating that morphine releases less adenosine after peripheral nerve injury, the current study suggests that the antihypersensitivity effect of morphine in these conditions is totally reliant on A1 adenosine receptor activation.

Morphine-induced spinal release of adenosine is reduced in neuropathic rats

Background Spinally administered opioids show decreased potency and efficacy in the treatment of neuropathic pain. As reported previously, morphine stimulates spinal opioid receptors to effect adenosine release, which acts at adenosine receptors to produce analgesia. The authors hypothesized that morphine induces less adenosine release in neuropathic compared with normal rats, explaining its reduced potency and efficacy. Methods Sprague-Dawley rats (200–250 g) were divided into three groups: no surgery (n = 52), sham surgery (n = 20), or left L5 and L6 spinal nerve ligation (n = 64). Two weeks after surgery, mechanical hypersensitivity of the left hind paw was verified. For each experiment, a crude synaptosomal P2 suspension was prepared by homogenizing cervical and lumbar dorsal spinal cord halves from four rats, followed by differential centrifugation, and aliquots incubated with morphine sulfate from 10−8 to 10−4 m alone or in presence of 10−5 m dipyridamole. Extrasynaptosomal concentrations of adenosine were analyzed by high-pressure liquid chromatography. Results Synaptosomal release of adenosine in the absence of morphine was similar between groups. Morphine produced a concentration-dependent adenosine release, which was less in synaptosomes from dorsal lumbar spinal cord in spinal nerve ligation compared with normal or sham animals. This reduction was removed by adding dipyridamole. Conclusion Morphine normally stimulates spinal release of adenosine, a potent antihypersensitivity compound. Because this effect of morphine is diminished in spinal nerve ligation animals, one explanation for decreased efficacy and potency of opioids in the treatment of neuropathic pain may be a dipyridamole-sensitive disruption in the opioid–adenosine link in the spinal cord.

Spinal adrenergic and cholinergic receptor interactions activated by clonidine in postincisional pain.

Background Previous pharmacologic and molecular studies suggest that the &agr;2-adrenoceptor subtype A is the target for spinally administered &agr;2-adrenergic agonists, i.e., clonidine, for pain relief. However, intrathecally administered &agr;2 C antisense oligodeoxynucleotide was recently reported to decrease antinociception induced by clonidine in the rat, suggesting non-A sites may be important as well. The current study sought to determine the subtype of &agr;2 adrenoceptors activated by clonidine in a rodent model for human postoperative pain, and to examine its interaction with spinal cholinergic receptors. Methods Postoperative hypersensitivity was produced in rats by plantar incision of the hind paw and punctuate mechanical stimuli were applied around the wound 24 h after surgery. Effects of intrathecal clonidine and 2-(2,6-diethylphenylamino)-2-imidazoline (ST91) on withdrawal thresholds to the stimulus were determined. To examine the adrenoceptor subtype and its interaction with spinal cholinergic receptors, animals were intrathecally pretreated with vehicles BRL44408 (an &agr;2 A subtype–preferring antagonist), ARC239 (an &agr;2 non-A subtype–preferring antagonist), atropine (a muscarinic antagonist), and mecamylamine (a nicotinic antagonist). Results Intrathecal ST91 showed a significantly greater efficacy when compared with clonidine. The analgesic effect of clonidine was diminished by pretreatment with either adrenoceptor antagonist, whereas the effect of ST91 was solely blocked by ARC239 pretreatment. Atropine and mecamylamine abolished the effect of clonidine effect but not the effect of ST91. Conclusions Both &agr;2 A and &agr;2 non-A adrenoceptors, as well as spinal cholinergic activation, are important to the antihypersensitivity effect of clonidine after surgery. ST91 is more efficacious in this model than clonidine and relies entirely on &agr;2 non-A adrenoceptors.

Allosteric modulation of adenosine A1 receptor coupling to G-proteins in brain

2‐Amino‐4,5,6,7‐tetrahydrobenzo(β)thiophen‐3‐yl 4‐chlorophenylmethanone (T62) is a member of a group of allosteric modulators of adenosine A1 receptors tested in animal models of neuropathic pain to increase the efficacy of adenosine. To determine its mechanisms at the level of receptor‐G‐protein activation, the present studies examined the effect of T62 on A1‐stimulated [35S]guanosine‐5′‐O‐(γ‐thio)‐triphosphate ([35S]GTPγS) binding in brain membranes, and by [35S]GTPγS autoradiography using the A1 agonist, phenylisopropyladenosine (PIA), to activate G‐proteins. In hippocampal membranes, T62 increased both basal and PIA‐stimulated [35S]GTPγS binding. The effect of T62 was non‐competitive in nature, since it increased the maximal effect of PIA, with no effect on agonist potency. GTPγS saturation analysis showed that T62 increased the number of G‐proteins activated by agonist but had no effect on the affinity of activated G‐proteins for GTPγS. [35S]GTPγS autoradiography showed that the neuroanatomical localization of T62‐stimulated [35S]GTPγS binding was identical to that of PIA‐stimulated activity. The increase in PIA‐stimulated activity by T62 varied between brain regions, with areas of lower A1 activation producing the largest percent modulation by T62. These results suggest a mechanism of allosteric modulators to increase the number of activated G‐proteins per receptor, and provide a neuroanatomical basis for understanding potential therapeutic effects of such drugs.

Adenosine reduces glutamate release in rat spinal synaptosomes.

Background:A1 adenosine receptor activation reduces hypersensitivity in animal models of chronic pain, but intrathecal adenosine does not produce analgesia to acute noxious stimuli. Here, the authors test whether increased inhibition by adenosine of glutamate release from afferents after injury accounts for this difference. Methods:Synaptosomes were prepared from the dorsal half of the lumbar spinal cord of normal rats or those with spinal nerve ligation. Glutamate release evoked by the TRPV-1 receptor agonist, capsaicin, was measured. Adenosine with or without adenosine A1 and A2 receptor antagonists was applied to determine the efficacy and mechanism of adenosine to reduce capsaicin-evoked glutamate release. Results:Capsaicin produced a concentration-dependent glutamate release similarly in normal and nerve-injured rats. Capsaicin-evoked glutamate release was inhibited by adenosine or R-PIA (R-N6-(2- phenylisopropyl)-adenosine) in a concentration-dependent manner, with a threshold of 10 nm in both normal and nerve-ligated synaptosomes. Blockade of capsaicin-evoked glutamate release by adenosine was reversed similarly in synaptosomes from normal and spinal nerve–ligated animals by an A1 adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) but not by an A2 adenosine receptor antagonist DMPX (3″7-dimethyl-1-proparaglyxanthine). Capsaicin-evoked glutamate release, as well as its inhibition by adenosine, did not differ between synaptosomes prepared from tissue ipsilateral and contralateral to spinal nerve ligation. Conclusion:These observations confirm previous neurophysiologic studies that presynaptic adenosine A1 receptor activation inhibits glutamate release from primary afferents. This effect is unaltered after peripheral nerve injury and thereby is unlikely to account for the enhanced analgesic efficacy of intrathecal adenosine in this setting.

Spinal adenosine receptor activation reduces hypersensitivity after surgery by a different mechanism than after nerve injury.

Background: Intrathecal adenosine has antinociceptive effects under conditions of hypersensitivity. T62 (2-amino-3-(4-chlorobenzoyl)-5,6,7,8-tetrahydrobenzothiophen) is an allosteric adenosine receptor modulator that enhances adenosine binding to the A1 receptor. Intrathecal T62 reduces hypersensitivity to mechanical stimuli in a rat model of neuropathic pain by a circuit that totally relies on activation of &agr;2 adrenoceptors. Here, the authors tested whether this same dependence was present in the acute setting of hypersensitivity after surgery. Methods: Intrathecal catheters were inserted in male Sprague-Dawley rats. An incision of the plantar aspect of the hind paw resulted 24 h later in hypersensitivity, as measured by applying von Frey filaments to the paw. At this time, rats received intrathecal T62, clonidine, or the combination in a blinded, isobolographic design. The effect of the &agr;2-adrenoceptor antagonist idazoxan on T62 was also tested. Results: Intrathecal T62 produced a dose-dependent antihypersensitivity effect, with no effect on ambulation or activity level. Clonidine also produced a dose-dependent antihypersensitivity effect. The ED40 (95% confidence interval) for T62 was 0.77 (0.63−0.91) μg, and that for clonidine was 1.23 (0.56−1.9) μg. Isobolographic analysis indicated synergism between T62 and clonidine. Intrathecal pretreatment with idazoxan only partially inhibited the antihypersensitivity effect of T62. Conclusions: Intrathecal T62 is effective for postoperative hypersensitivity. The synergy of T62 with clonidine and its only partial antagonism by idazoxan suggest that T62 does not rely entirely on activation of &agr;2adrenoceptors. These results indicate that, after surgery, T62 acts via a mechanism different from that of spinal nerve ligation, a model of chronic neuropathic pain.

Intrathecal adenosine following spinal nerve ligation in rat: Short residence time in cerebrospinal fluid and no change in A 1 receptor binding

Background Intrathecal adenosine produces a remarkably prolonged effect to relieve mechanical hypersensitivity after peripheral nerve injury in animals. The purpose of the current study was to investigate whether this reflected an alteration in kinetics of adenosine in cerebrospinal fluid or in the number of spinal A1 adenosine receptors after nerve injury. Methods Male rats were anesthetized, and the left L5 and L6 spinal nerves were ligated. Two weeks later, a lumbar intrathecal catheter and intrathecal space microdialysis catheter were inserted. Adenosine, 20 &mgr;g, was injected intrathecally in these and in normal rats, and microdialysates of the intrathecal space were obtained. Radioligand binding studies of adenosine A1 receptors were determined in spinal cord tissue from other normal and spinal nerve–ligated rats. Results Adenosine disappeared from rat cerebrospinal fluid within 30 min after intrathecal injection, with no difference between normal and spinal nerve–ligated animals. A1 adenosine receptor binding sites in the spinal cord were increased after spinal nerve ligation. This increase disappeared when adenosine deaminase was added to the membrane homogenates, suggestive of decreased endogenous adenosine in the membranes of nerve-ligated animals. Conclusion These data show that prolonged alleviation of hypersensitivity observed with intrathecal adenosine in this animal model of neuropathic pain is not due to prolonged residence in cerebrospinal fluid, although pharmacokinetics in tissues are unknown. Similarly, there is no evidence for up-regulation in spinal A1 adenosine receptors after spinal nerve ligation, and the adenosine deaminase experiment is consistent with a depletion of adenosine in spinal cord tissue after spinal nerve ligation.