2A-Adrenoceptor Stimulation Reduces Capsaicin-Induced Glutamate Release from Spinal Cord Synaptosomes

Glutamate (Glu) is involved in excitatory neurotransmission and nociception and plays an essential role in relaying noxious stimuli in the spinal cord. Intrathecal or epidural injection of 2-adrenergic agonists produces potent antinociceptive effects, alters spinal neurotransmitter release, and effectively treats acute nociceptive and chronic neuropathic pain. Although it is generally believed that 2-adrenergic receptor stimulation reduces excitatory neurotransmitter release from peripheral afferents, the subtype of receptor causing this effect and its specificity to nociceptive neurotransmission have been inadequately studied. We therefore examined the pharmacology of adrenergic agents to inhibit Glu release in spinal cord from stimulation with capsaicin, a specific agonist for receptors on nociceptive afferents. Capsaicin evoked Glu release in synaptosomes from normal rat dorsal spinal cord in a concentration-dependent manner. Glu release from 30 M capsaicin was inhibited by adrenergic agonists with a relative potency of clonidine dexmedetomidine norepinephrine ST91 phenylephrine 0, consistent with an action on 2A/D subtype receptors. Also consistent with this interpretation was the observation that inhibition of capsaicin-induced Glu release by clonidine or dexmedetomidine was blocked by the 2A/D antagonist BRL44408 but not by the 2B/C-preferring antagonist ARC239. Similar results were obtained in perfused spinal cord slices. These data suggest that capsaicin-evoked Glu release, likely reflecting stimulation of C fiber terminals, can be inhibited by activation of the 2A/D subtype, and this action of adrenergic agonists may reflect in part their efficacy in the treatment of acute pain. 2-Adrenergic agonists, like opioids, are powerful analgesics and are considered to act at multiple sites. Both classes of analgesics are more potent after intrathecal than systemic administration, indicating a site of action in the spinal cord, where the receptors on which they act are concentrated (Yaksh et al., 1984). The mechanisms by which opioids and 2-adrenergic agonists act remain an active topic of investigation. For 2adrenergic agonists, it has been suggested that they inhibit release of excitatory neurotransmitters from nociceptive afferents by a direct action on primary afferent terminals (Kuraishi et al., 1985). However, much of this work has been indirect, either examining inhibition of stimuli, such as depolarization with high concentrations of potassium, which excite all types of afferents (Kamisaki et al., 1993; Shinomura et al., 1999), or examining effects in complex systems, such as spinal cord slices, in which direct and indirect effects could occur (Ueda et al., 1995). One purpose of the current study was to examine the action of 2-adrenergic agonists using a specific activator or nociceptive afferents (capsaicin) and using a simplified system that primarily reflects direct actions on nerve terminals (synaptosomes). 2-Adrenoceptors can be divided by either pharmacologic or molecular approaches into three major subtypes: 2A (or the D homolog in the rat), 2B, and 2C. Rat spinal cord dorsal horn contains primarily 2A/D and 2C subtypes, as defined by immunohistochemistry (Stone et al., 1998). There is strong evidence that antinociception from intrathecally administered 2adrenergic agonists reflects actions on the 2A/D subtype in normal animals (Millan, 1992; Stone et al., 1997), although there is also some support for nonA subtypes causing antinociception in normal animals (Takano and Yaksh, 1993; Guo et al., 1999). We have previously demonstrated that the 2-adrenergic subtype mediating autoinhibition of norepinephrine release in the spinal cord was the 2A/D subtype (Li et al., 2000). A secondary purpose of the current study was to determine the 2-adrenergic subtype subserving inhibition of capsaicinevoked glutamate release in the spinal cord in normal animals. A combination of methods was used, including both complex and simple systems (spinal cord slices and synaptosomes), specific activation of afferents with capsaicin, and determination of a structure-activity relationship for 2-adrenergic agonists and antagonists. Experimental Procedures Materials. ST91 was provided by Boehringer Ingelheim Pharmaceuticals USA (Ridgefield, CT). Dexmedetomidine was provided by Orion Pharmaceuticals, Inc. (Turku, Finland). ARC239 dihydrochloSupported in part by National Institutes of Health Grant GM35523. ABBREVIATION: Glu, glutamate. 0022-3565/01/29903-939–944$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 299, No. 03 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 4200/944659 JPET 299:939–944, 2001 Printed in U.S.A. 939 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from ride and BRL44408 maleate were obtained from Tocris Cookson (St. Louis, MO). MgSO4, KCl, sodium bicarbonate, and glucose were obtained from Fisher Scientific (Fair Lawn, NJ). Capsaicin (8-methyl-N-vanillyl-6-nonenamide), glutamate, -nicotinamide adenine dinucleotide ( -NAD), glutamate dehydrogenase, clonidine, and remaining chemicals were obtained from Sigma (St. Louis, MO). Synaptosome Preparation. After obtaining Animal Care and Use Committee approval, male Sprague-Dawley rats (250 g) were used for all experiments. Animals were deeply anesthetized with 1.5 to 2.1% halothane and then decapitated. The spinal cord was quickly removed and placed in aerated (with 95%O2/5%CO2) ice-cold modified Krebs-Ringer buffer containing 135 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, 12.5 mM Hepes, and 10 mM glucose, at pH 7.4. The dorsal half of the lumbar spinal cord was dissected from two rats and homogenized in 14 ml of ice-cold 0.32 M sucrose, and a crude synaptosomal pellet (P2) was prepared by differential centrifugation with 1,000g for 5 min followed by 15,000g for 20 min as previously described (Lonart and