ELSEVIER
Neuroscience Letters 188 (1995) 137-139
NEUROSCI[NC[ LETTERS
Alpha2-adrenoceptor-mediated inhibition of capsaicin-evoked release of glutamate from rat spinal dorsal horn slices Mutsuaki U e d a a, Tatsuya Oyama., Yasushi Kuraishi b, Akinori Akaike a, Masamichi Satoh o,* aDepartment of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-01, Japan bDepartment of Applied Biochemistry, Research Institutefor Wakan-yaku, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan CDepartment of Molecular Pharmacology, Facultyof Pharmaceutical Sciences, Kyoto University, Kyoto 606-01, Japan Received 5 January 1995; revised version received 13 February 1995; accepted 13 February 1995
Abstract
It is known that the descending noradrenergic system suppresses nociceptive transmission in the spinal dorsal horn. To determine whether noradrenaline--a-adrenoceptor systems exert inhibitory influence on the release of glutamate from the nociceptive primary afferents, the effects of a-adrenoceptor agonists on the capsaicin (3 ktM)-evoked release of glutamate from rat spinal dorsal horn slices were examined using an on-line continuous monitoring system for glutamate. The application of a2-agonists clonidine (0.1-10 /zM) and ST-91 (1 and l 0/~M), respectively, decreased the capsaicin-evoked release of glutamate in a concentration-dependent manner. The partial a2-agonist oxymetazoline (1 and 10/~M) produced a slight inhibition in the evoked release of glutamate. In contrast, the a 1agonist phenylephrine (1 and 10/tM) did not show any significant effects on the evoked release of glutamate. The inhibitory action of 10/~M clonidine on the evoked glutamate release was antagonized by the a2-antagonist yohimbine (1/zM) but not by the opioid antagonist naloxone (10/.tM). These findings suggest that noradrenaline, probably released from the descending inhibitory system, reduces the release of glutamate from the capsaicin-sensitive primary afferent terminals through a2-adrenoceptors.
Keywords:a2-Agonists; Glutamate; On-line monitoring; Capsaicin; Primary afferents; Analgesia It is experimentally and clinically demonstrated that the descending noradrenergic system modulates the transmission of nociceptive information in the spinal cord [5,8,11]. The studies based on the agonist and antagonist pharmacology suggest the involvement of the a 2- rather than aradrenergic receptor (for review, see Ref. [17]). Ligand binding studies using the autoradiographic technique have shown the existence of the high density of a zreceptor in the superficial laminae of the spinal dorsal horn [13]. Both pre- and postsynaptic actions of a2agonists to the primary afferent terminals have been postulated [4,12]. Presynaptically, noradrenaline inhibited the release of substance P elicited by noxious mechanical stimulation from the rabbit spinal dorsal horn [9]. However, whether noradrenaline regulates the release of glutamate from nociceptive primary afferent fibers remains to be examined because of the difficulties in specifying the origin of the release of glutamate [7]. Capsaicin is thought to act specifically on a subset of primary afferent * Corresponding author, Tel.: +81 75 7534526; Fax: +81 75 7534586.
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fibers [1,6] and it has been demonstrated that capsaicin evoked the release of glutamate from the spinal dorsal horn slices in a tetrodotoxin-resistant and dorsal rhizotomy-sensitive manner [15]. Therefore, in the present study, we investigated the effects of a-agonists on the capsaicin-evoked release of glutamate from the spinal dorsal horn slices, using an on-line continuous monitoring system for glutamate. Male adult Sprague-Dawley rats (200-250 g) were used in the present experiments. The dorsal horn slices of the lumbar spinal cord were prepared according to the method previously described [15]. The slices were preincubated at 37°C for 40 min in modified Krebs solution (pH 7.4) gassed with 95% 02 and 5% CO2. The composition of the solution (in mM) was as follows : NaCi, 124; KCI, 2; KH2PO4, 1.24; MgSO 4, 5; NaHCO3, 26; Dglucose, 10; CaC12, 2. After the pre-incubation, the slices were transferred into a superfusion chamber and superfused with the gassed Krebs solution containing 1 mM flNAD (Sigma), 10~M DL-threo-fl-hydroxyaspartic acid (Sigma), 0.3/~M tetrodotoxin and the cocktail of peptide
© 1995 Elsevier Science Ireland Ltd. All rights reserved
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M. Ueda et al. / Neuroscience Letters 188 (1995) 137-139
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and post hoc Dunnet's test. Statistical differences were considered significant at P < 0.05. Clonidine hydrochloride (RBI), oxymetazoline hydrochloride (RBI), ST-9I hydrochloride (Boehringer Ingelheim), yohimbine hydrochloride (RBI) and naloxone hydrochloride (Sigma) were freshly dissolved in H20, respectively. The application of 3 / t M capsaicin produced a prolonged and at most 20% increase in the release of glutamate from rat spinal dorsal horn slices (Fig. 1A). In the present study, 43.7 _+ 2.38 pmol.mg -l protein (n = 6) of glutamate was released by 3/~M capsaicin in the control group. Although the application of clonidine did not show any apparent effect on the basal release of glutamate at 10ktM (Fig. 1B) and at any other concentrations examined (data not shown), 10ktM clonidine inhibited the 3ktM capsaicin-evoked release of glutamate compared with control (Fig. 1). As shown in Fig. 2, clonidine at 0.1-10ktM decreased the capsaicin-evoked release of glutamate in a concentration-dependent manner. At 10ktM, clonidine significantly decreased the evoked release by 50% of the control group (Fig. 2). A similar degree of inhibition in the evoked release of glutamate was produced by ST-91, a selective Ctz-agonist, at concentrations of 1 and 10ktM, and oxymetazoline (1 and 10ktM), a partial az-agonist, showed a slight but non-significant inhibition (Fig. 2). On the other hand, the selective a j agonist phenylephrine (1 and 10ktM) did not produce apparent alterations in the capsaicin-evoked release of
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Fig. 1. Effects of capsaicin stimulation on the release of glutamate from the spinal dorsal horn slices in the absence (A) or presence (B) of 10/~M clonidine. Solid and hatched bars represent the application of 3 ~M capsaicin and 10/~M clonidine, respectively.
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inhibitors (leupeptin l b t M , captopril 5 ~ M , bestatin i 0 / ~ M ) at a flow rate of 0.5 ml/min. Capsaicin (Sigma) at 3/~M (containing 0.01% dimethyl sulfoxide) was applied for 2 min by adding to the medium. The drugs tested were applied from 6 min prior to capsaicin stimulation to the end of superfusion experiment (i.e. for 25 min). Glutamate released from the slices into the superfusate was oxidized to a-ketoglutarate by the immobilized glutamate dehydrogenase in the column with concomitant conversion of N A D to N A D H and the fluorescence of N A D H (E x 340 rim, E m 460 nm) was continuously determined by a fluorometric detector (Shimadzu, RF-535). Evoked release of glutamate was calculated with the peak area of the fluorescence by a data integrator (Shimadzu, CR-4A). The standard peak area was calibrated by the application of 2 0 - 2 0 0 pmol glutamate for 2 min. Protein contents were determined according to the method of Bradford [2], using bovine serum albumin as a standard. All values were expressed as mean _+ SEM. Statistical analyses were performed with one-way analysis of variance ( A N O V A )
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c~-agonists (!LLM) Fig. 2. Inhibitory effects of a-adrenoceptor agonists on the capsaicinevoked release of glutamate from the spinal dorsal horn slices. Clonidine (O), ST-91 (A), oxymetazoline (V), phenylephrine (I), combination (CMB) of 1 ~M yohimbine with 10~M clonidine (I), and combination of 10/~M naloxone with 10~M clonidine (\) were administered from 6 min prior to the start of capsaicin (3/~M) stimulation. Each point represents the mean and SEM of 4-7 experiments. *P<0.05 versus control (CTL), #P<0.05 versus 10~M clonidine alone.
M. Ueda et al. / Neuroscience Letters 188 (1995) 137-139
glutamate (Fig. 2). ST-91, oxymetazoline and phenylephrine at concentrations examined did not alter the basal release of glutamate (data not shown). To confirm that clonidine inhibited the evoked release of glutamate from dorsal horn slices through a2adrenergic receptor, the effect of yohimbine, a nonimidazoline a2-antagonist, was examined. The simultaneous application of 1 ~ M yohimbine with 10/tM clonidine significantly antagonized the inhibitory effect of clonidine (Fig. 2). Since the opioid agonists, such as morphine, inhibited the capsaicin-evoked release of glutamate in previous experiments [16], the effect of naloxone on the inhibitory action of clonidine was examined. However, 10/~M naloxone did not modify the inhibitory effect of 1 0 ~ M clonidine on the capsaicin-evoked release of glutamate (Fig. 2). The present study shows that the a2-agonists clonidine and ST-91 inhibited the capsaicin-evoked release of glutamate from the spinal dorsal horn slices in a yohimbinereversible manner. Because the capsaicin-evoked release of glutamate is likely to be released from the terminals directly activated by capsaicin [ 1,6,15], the present finding indicates the inhibition mediated by a2-adrenergic receptors on the release of glutamate from the capsaicinsensitive primary afferent fibers. This supports the hypothesis that noradrenaline presynaptically inhibited nociceptive transmission in the spinal dorsal horn [9]. In the previous experiments, /~-opioid agonists inhibited the capsaicin-evoked release of glutamate from the spinal dorsal horn slices in a naloxone (1/~M)-reversible manner [16]. However, naloxone at even 10/~M did not modify the inhibitory effect of clonidine in this experiment, suggesting that the opioidergic system did not contribute to this effect. a2-Adrenergic receptors are pharmacologically classified into a2A-, a2B- and a2c-subtypes. The a2A-Subtype exhibits a high affinity for oxymetazoline and a low affinity for prazosin, whereas the a2B- and a2c-subtypes exhibit the reverse order of affinities for these drugs [3]. In the present experiments, oxymetazoline (1 and 10/~M) did not significantly inhibit the capsaicin-evoked release of glutamate from the dorsal horn slices, implying that a2A-receptor may not be involved in presynaptic inhibition on primary afferents. In contrast, the evoked release was significantly decreased by the application of ST-91, which showed antinociceptive effects through a non-a2Areceptor [14]. Indeed, an in situ hybridization study demonstrates that a2c-receptor m R N A was predominantly expressed in the dorsal root ganglion cells [10]. Therefore, together with these findings, it may be suggested that a2-agonists inhibit the capsaicin-evoked release of glutamate through a2c- and/or a2B-subtypes, although it needs to be further examined using more selective ligands. In conclusion, the present results indicate the inhibition by a2-agonists of the release of glutamate from the
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capsaicin-sensitive primary afferent terminals and support the hypothesis that the descending noradrenergic system exerts an analgesia, at least partly, as a result of the inhibition of the release of the excitatory neurotransmitter glutamate from the nociceptive primary afferent fibers. [1] Bevan, S. and Szolcs~inyi,J., Sensory neuron-specific actions of capsaicin: mechanism and applications, Trends Pharmacol. Sci., 11 (1990) 330-333. [2] Bradford,M.M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dyebinding, Anal. Biochem., 72 (1976) 248-254. [3] Bylund,D.B., Ray-Prenger, C. and Murphy, T.J., Alpha-2A and alpha-2B adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one receptor subtype, J. Pharmacol. Exp. Ther., 245 (1988) 600-607. [4] Fleetwood-Walker,S.M., Mitchell, R., Hope, P.J., Molony, V. and Iggo, A., An a 2 receptor mediates the selective inhibition by noradrenaline of nociceptive responses of identified dorsal horn neurons, Brain Res., 334 (1985) 243-254. [5] Glynn, C.J., Jamous, M.A., Teddy, P.J., Moore, R.A. and Lloyd, J.W., Role of spinal noradrenergicsystem in transmissionof pain in patientswith spinal cord injury, Lancet, ii (1986) 1249-1250. [6] Holzer, P., Capsaicin: cellular targets, mechanism of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 43 (1991) 143-201. [7] Kamisaki,Y., Hamada, T., Maeda, K., Ishimura, M. and Itoh, T., Presynaptic a 2 adrenoceptors inhibit glutamate release from rat spinal cord synaptosomes,J. Neurochem., 60 (1993) 522-526. [8] Kuraishi,Y., Harada, Y. and Takagi, H., Noradrenergicregulation of pain-transmission of in the spinal cord mediated by aadrenoceptors, Brain Res., 174 (1979) 333-336. [9] Kuraishi, Y., Hirota, N., Sato, Y., Kaneko, S., Satoh, M. and Takagi, H., Noradrenergicinhibition of the release of substance P from the primary afferents in the rabbit spinal dorsal horn, Brain Res., 359 (1985) 177-182. [10] Nicholas, A.P., Pieribone, V. and Htikfelt, T., Distribution of mRNAs for alpha-2 adrenergic receptor subtypes in rat brain: an in situ hybridization study, J. Comp. Neurol., 328 (1993) 575594. [11] Reddy, S.V.R. and Yaksh, T.L., Spinal noradrenergic terminal system mediates antinociception, Brain Res., 189 (1980) 391401. [12] Satoh, M. Kawajiri, S., Ukai, Y. and Yamamoto, M., Selective and non-selectiveinhibition by enkephalins and noradrenalineof nociceptive response of lamina V type neurons in the spinal dorsal horn of the rabbit, Brain Res., 177 (1979) 384-387. [13] Sullivan, A.F., Dashwood, M.R. and Dickenson, A.H., a 2Adrenoreceptor modulation of nociception in rat spinal cord: location, effects and interactionswith morphine, Eur. J. Pharmacol., 138 (1987) 169-177. [14] Takano, Y., Takano, M. and Yaksh, T.L., The effect of intrathecally administered imiloxan and WB4101: possible role of a 2adrenoceptor subtypes in the spinal cord, Eur. J. Pharmacol., 219 (1992) 465-468. [15] Ueda, M., Kuraishi, Y., Sugimoto, K. and Satoh, M., Evidence that glutamate is released from capsaicin-sensitiveprimary afferent fibers in rats: study with on-line continuous monitoring of glutamate, Neurosci. Res., 20 (1994) 231-237. [16] Ueda, M., Oyama, T., Sugimoto, K., Kuraishi, Y. and Satoh, M., Opioidergic inhibition of capsaicin-evoked release of glutamate from rat spinal cord dorsal horn slices, Neuropharmacology, in press. [17] Yaksh, T.L. and Aimone, L.D., The central pharmacology of pain transmission. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, ChurchillLivingstone,London, 1989, pp. 181-205.