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Small conductance calcium-activated K+ channels, SkCa, but not voltage-gated K+ (Kv) channels, are implicated in the antinociception induced by CGS21680, a A2A adenosine receptor agonist
Life Sciences 76 (2004) 367 – 377 www.elsevier.com/locate/lifescie
Small conductance calcium-activated K+ channels, SkCa, but not voltage-gated K+ (Kv) channels, are implicated in the antinociception induced by CGS21680, a A2A adenosine receptor agonist I. Regayaa,b, T. Phamc, N. Andreottid, N. Sauzea, L. Carregaa,d, M.F. Martin-Eauclairea, B. Jouiroua,b, J.C. Peragute, H. Vachera, H. Rochata,d,b, C. Devauxa, J.M. Sabatiera,b, R. Guieua,b,d,e,* a
UMR FRE CNRS 2738 Inge´nierie des Prote´ines, Faculte´ de Me´decine Nord, Bd P, Dramard 13015 Marseille, France b Laboratoire International Associe´ (LIA), Faculte´ de Me´decine Nord, Bd P Dramard 13015 Marseille, France c Service de Rhumatologie, Hoˆpital de la Conception, Marseille, France d Laboratoire de Biochimie, CHU Timone, Marseille France e Centre anti-douleur, Service de Neurochirurgie Fonctionnelle, CHU, Timone, Marseille, France. Received 20 May 2004; accepted 9 June 2004
Abstract It has been shown that A2A adenosine receptors are implicated in pain modulation. The precise mechanism by which activation of A2A receptors produces analgesic effects, however, remains unclear. The aim of this study was to investigate the possible involvement of apamin-sensitive calcium-activated potassium channels (SKCa) and voltage-gated potassium (Kv) channels in A2A receptor activationinduced analgesic effects. Using mice, we evaluated the influence of apamin, a non specific blocker of SKCa channels, Lei-Dab7 (an analog of scorpion Leiurotoxin), a selective blocker of SKCa2 channels, and kaliotoxin (KTX) a Kv channel blocker, on the CGS 21680 (A2A adenosine receptor agonist)induced increases in hot plate and tail pinch latencies. All drugs were injected in mice via the intracerebroventricular route. We found that apamin and Lei-Dab7, but not KTX, reduced antinociception
* Corresponding author. E-mail address: [email protected] (R. Guieu). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.06.023
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produced by CGS21680 on the hot plate and tail pinch tests in a dose dependent manner. Lei-Dab 7 was more potent than apamin in this regard. We conclude that SKCa but not Kv channels are implicated in CGS 21680-induced antinociception. D 2004 Elsevier Inc. All rights reserved. Keywords: SKCa channels; Kv channels; Adenosine; Antinociception
Introduction Antinociceptive effects of adenosine (ADO) or its analogs have been observed following peripheral administration in animals (Holmgreen et al., 1983; Ahlijanian and Takemori, 1985) and humans (Guieu et al., 1994; Belfrage et al., 1995; Sjolund et al., 1999), or following central administration in animals (Delander and Hopkins, 1987; Karlsten et al., 1990) or humans (Sollevi et al., 1995; Sollevi, 1997; Sjolund et al., 2001). ADO can act via adenosine A1, A2A, A2B or A3 receptors all of which belong to the G protein-coupled family (Ralevic and Burnstock, 1998). Activation of A1 (Sawynok et al., 1986) or A2A receptors (Delander and Hopkins, 1987) induced antinociceptive effects. Several mechanisms for antinociceptive effects of ADO receptors stimulation have been proposed, including pre- (Santicioli et al., 1992) and postsynaptic mechanisms (Li and Perl, 1994), but also receptor-receptor interactions (Sebastiao and Ribeiro, 2000). At the presynaptic level, activation of A1 receptors results in a decrease in cAMP production and an inhibition of voltage-gated calcium channels that leads to a decrease in the release of neurotransmitters such as acetylcholine, SP or CGRP implicated in the transmission of nociceptive stimuli (Sperlagh et al., 2001; Santicioli et al., 1992; for review see Ribeiro et al., 2002; Sawynok and Liu, 2003). At the postsynaptic level, the activation of A1 receptors induces the opening of several potassium channel subtypes including KATP (Ocana and Baeyens, 1994), inwardly rectified and calcium-activated potassium channels, resulting in the hyperpolarization of excitable membranes (Ralevic and Burnstock, 1998). It has been shown that A2A receptors are also involved in pain modulation at the spinal (Sawynok et al., 1986; Delander and Hopkins, 1987) and supraspinal levels (Pham et al., 2003). The mechanism by which A2A receptors are implicated in pain modulation remains unclear. At the supraspinal level, the activation of A2A receptors may facilitate or inhibit neurotransmitter release, depending on the brain area (Dunwiddie and Masino, 2001). It has been shown that A2A agonists inhibit transmitter release via an inhibition of voltage-dependent calcium channels (Latini et al., 1996; Edwards and Robertson, 1999). Nevertheless, it still remains possible that, as in the case of A1 receptors, activation of A2A receptors induces pain relief via modulation of potassium channel activity. Among the potassium channels, it has been shown that voltagegated (Kv) (Rasband et al., 2001) and apamin-sensitive small conductance Ca++-activated potassium channels (SKCa) (Boettger et al., 2002) are expressed on nociceptors and are involved in pain control. It has also been shown that SKCa are implicated in drug-induced antinociception (Galeotti et al., 2001; Granados-Soto et al., 2002). The aim of this study was thus to evaluate the possible involvement of Kv and SKCa potassium channels in the antinociception induced by activation of A2A receptors.
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Materials and methods Animals Male C57/BL6 mice (25–30g) bred in our laboratory were used at the age of 8 weeks. Animals were housed 10 per cage and had free access to food and water in a quiet environment. The room was maintained at 21–238C with a 12h light/dark cycle. Each animal underwent both tail pinch and hot plate tests at 30 min intervals in a random order. The experimenter was blind to the pharmacological treatment. Because latency variations of at least 10% were expected, 24 animals per group were included. Drugs and reagents CGS 21680 (2-[p-(carboxyethyl)-phenyl-ethylamine]-5V-N-ethylcarboxyamidoadenosine) as an A2A agonist and apamin (SKCa blocker) were purchased from Sigma (St Quentin Fallavier, France). N-Fmoc-L-amino acid derivatives, Fmoc-amide resin, and chemical reagents used for peptide synthesis were purchased from Perkin-Elmer Life Sciences (Shelton, CO), Novabiochem (Laufelfingen, Switzerland), and Laboratoire Neosystem (Strasbourg, France). Solvents were analytical grade products from SDS (Peypin, France). Peptide synthesis The chemical synthesis of Lei-Dab7, a peptide blocker of SKCa2 potassium channels, has been described elsewhere (Shakkottai et al., 2001). Briefly, the peptide was synthesized with the stepwise solid phase method (Merrifield, 1986) using an automated peptide synthesizer (Model 433A, Applied Biosystems Inc., Foster City, CA). The side-chain protecting groups used for trifunctional residues were: 2,2,5,7,8-pentamethylchroman-6-sulfonyl for Arg and homoarginine, tert-butyloxycarbonyl for Orn, Lys, and homolysine, and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-yliden)-3-methylbutyl for Dab. The reduced peptide was dissolved at 1 mM in 0.2 M Tris-HCl buffer, pH 8.3, and stirred under air to allow folding/oxidation (48 h, 258C). The folded/oxidized peptide was purified to homogeneity by reversedphase high-pressure liquid chromatography (HPLC) (C18 Aquapore ODS 20 Am, 250 10 mm) and lyophilized before injection. The homogeneity (N99%) and identity of the peptide were verified by: (i) analytical C18 reversed-phase HPLC, (ii) amino acid content determination after acid hydrolysis, and (iii) mass analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). Experimental procedure All injected drugs were dissolved in the vehicle (methanol/water, 3/7, V/V). Mice were injected intracerebroventriculary (ICV, 4 AL). Anesthesia was obtained with 1 ml of isofluorane (Forene (r), Abbott, France) on soaked cotton in a closed bottle for 30 s. Animals were injected with CGS 21680 at the doses of 0.1, 0. 5 and 1 nmol, alone or in combination with apamin (0.5, 2, 5 pmol), Lei-Dab7 (0.5, 2, 5 pmol) or KTX (0.5, 5 or 10 pmol). Controls were injected with the vehicle alone.
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Measurement of nociceptive response with the hot plate test Animals were placed on a 51F0.58C hot plate (Hot plate analgesia meter, Harvard apparatus, USA) and latencies of forepaw licking were measured 15, 30, and 60 min after ICV injection. Measurement of nociceptive response in the tail pinch test Nociceptive responses in the tail pinch test was measured as previously described (Takagi et al., 1996; Ochi et al., 1999). Briefly, mice were pretested by pinching their tail base with an artery clip (3 mm wide, 500 g constant pressure) and only mice that showed a nociceptive response (biting the clip or vocalizing) within 3 s were used for expriments. 20–25% of the mice pretested did not manifest any reaction and were excluded. To prevent tissue damage, a cut-off time of 10 s was selected. The nociceptive response to the tail pinch test was determined 15, 30, and 60 min after ICV injections, as for the hot plate test. Statistical analysis Data were analyzed by ANOVA with Fisher’s PLD post hoc test. A value of P lower than 0.05 was considered to be significant.
Results Effects of CGS 21680 alone At 0.1 nmol, CGS 21680 did not significantly modify latencies, regardless of the test (Fig. 1A and B). At 0.5 nmol, CGS 21680 increased hot plate test and tail pinch test latencies at 15, 30 and 60 min, respectively compared with 0.1 nmol (p b 0.05). At 1 nmol, it increased hot plate test and tail pinch test latencies at 15, 30 and 60 min, respectively compared with 0.5 nmol (p b 0.05). After 60 min, no increase in latencies was observed (data not shown). Effects of Lei-Dab7, apamin or KTX alone Lei-Dab7 (5 pmol), apamin (5 pmol), or KTX (10 pmol) did not significantly modify latencies in either test. At these concentrations, apamin or KTX do not deteriorate locomotor activity (Fournier et al., 2001). With 10 pmol of Lei-Dab 7, there were no observable manifestations of toxicity, including locomotor abnormalities. Effects of apamin, lei-Dab7 and KTX on CGS21680-induced latency increases in the hot plate and tail pinch tests Apamin decreased CGS21680-induced hot plate latency increases in a dose-dependent manner (Fig. 2A). Lei-Dab 7 (0.5 pmol) did not significantly decrease CGS 21680-induced latency increases in the hot plate test at any time point (Fig. 3). At 2 and 5 pmol, Lei-dab 7 decreased CGS 21680-
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Fig. 1. Effects of CGS21680 (A2A agonist) on latencies in the hot plate (A) and tail pinch tests (B). Mice (n = 24 per group) were injected (ICV) with CGS 21680 (4 AL in methanol/water 3v/7v) or vehicle (4 AL methanol/water, 3/7, V/V). *p b 0.05 compared with 0.1 nmol.
induced hot plate latency increases in a dose-dependent manner. At 30 and 60 min, Lei-dab 7 was more potent than apamin in reducing CGS 21680-induced latency increases (p b 0.05 in tail pinch and hot plate tests; Fig. 2B). In contrast to apamin and Lei-dab7, KTX did not significantly modify CGS21680-induced hot plate and tail pinch test latency increases, up to 10 pmol (data not shown).
Discussion There is no way of determining the specific loci of action of a substance in the brain after ICV injection and it cannot necessarily be assumed that CGS21680 or SKCA channel blockers act on the same brain structures. It is known, however, that both A2A receptors (Johansson and Fredholm, 1995; Rosin et al., 1998) and SKCa channels (Stocker and Pedarzani, 2000) are present in brain areas implicated in pain control (thalamus as example). It is well-established that a large number of potassium channel subtypes are involved in the modulation of noxious stimuli (Rasband et al., 2001),
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Fig. 2. Effects of apamin (a non specific SKCa blocker) on the latency increases in hot plate and tail pinch tests induced by CGS21680, a A2A receptor agonist. Mice (n = 24 per group) were injected ICV (4 AL) with CGS 21680 alone or combined with apamin. Error bars represent standard deviations. *p b 0.05 compared with CGS 21680 alone. **p b 0.05 compared with apamin 2pmol.
including voltage-gated K+ (Kv) channels (Clark and Tempel, 1998; Rasband et al., 2001), and KATP and Ca++-activated potassium channels (Galeotti et al., 2001; Boettger et al., 2002). The latter channels can be classified as BKCa, SKCa and IKCa (big, small and intermediate conductances, respectively (Latorre et al., 1989; Vergara et al., 1998). SKCa channels are in turn subdivided into SKCa1-3 which are largely distributed in the nervous system, including areas involved in pain control (Stocker and Pedarzani, 2000). Both subtypes play a major role in governing neuron excitability, and their presence and distribution in the nervous system contributes to pain perception. It was recently shown that the expression of SKCa1 and IKCa1 are modified in injured human
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Fig. 3. Effects of Lei-Dab7 (a specific SKCa2 blocker) on the latency increases in hot plate (A) and tail pinch tests (B) induced by CGS21680. Mice (n = 24 per group) were injected ICV (4 AL) with CGS 21680 alone or combined with Lei-Dab7. Error bars represent standard deviations. Error bars represent standard deviations. *p b 0.05 compared with CGS 21680 alone. **p b 0.05 compared with Lei-Dab7 (5 nmol).
sensory neurons of the dorsal root ganglion (Boettger et al., 2002), strongly suggesting that these channels are implicated in the control of noxious stimuli. SKCa channels modulate the firing pattern of neurons by generating slow membrane post-hyperpolarization (Xia et al., 1998; Rimini et al., 2000; Stocker and Pedarzani, 2000). The SKCa2 channel subtype is extensively expressed in the brain (Rimini et al., 2000; Stocker and Pedarzani, 2000), but until recently existing SKCa2 channel blockers were not selective enough to distinguish this channel from other SKCa subtypes.
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Our results suggests that SKCa1-3, but not Kv, are implicated in the analgesic effects induced by CGS21680. Most of these effects are due to the activation of A2A receptors. Indeed CGS21680 is 140 fold selective for the A2A versus A1 receptor (Hutchison et al., 1990). Thus it is difficult to conclude that most effects of CGS21680 are due to the activation of A1 rather than A2A receptors. Apamin and Lei-Dab 7 may act by eliminating the medium IAHP current, and causing "bursting electrical activity" and enhanced neurotransmitter release in nociceptors. Since Lei-Dab 7 is more effective than apamin in reducing CGS21680-induced antinociception, we postulate that the SKCa2 subtype is more probably implicated in CGS21680-induced antinociception. Apamin, a peptide from bee venom, in fact exhibits only a 10-fold selectivity for SKCa2 over SKCa1 or SKCa3 (Kohler et al., 1996; Desai et al., 2000; Shah and Haylett, 2000; Strobaek et al., 2000), while Lei-Dab7 is a leiurotoxin I analog that selectively blocks SKCa2 homotetramers with a low nanomolar affinity. This was shown in electrophysiological studies (Shakkottai et al., 2001). Finally, our results may be affected by the possibility that SKCa blocking can elicit profound effects on neuronal excitability and that the observed effects are unrelated to A2A modulation. This hypothesis can be discarded, however, since SKCa blockers alone have no effects on behavioral latencies to nociceptive stimuli. There is also no evidence that SKCa blockers are specifically coupled with A2A receptors since it has been shown that some drugs that which are not adenosine analogs possess analgesic effects via the modulation of SKCa channels (Galeotti et al., 2001; Granados-Soto et al., 2002). Interestingly, some of these drugs interact with neuronal adenosine uptake (Phillis, 1984). Other drugs act via BK but not SKCa channels (Santos et al., 2003). Adenosine or its analogs are involved in the control of a wide range of pain mechanisms including neuropathic pain (Guieu et al., 1994, 1996; Sjolund et al., 2001). Most of these effects are secondary to the activation of A1 receptors (Sawynok et al., 1986; Sawynok, 1998). On the other hand, some data suggest that the effectiveness of A2A agonists in alleviating pain is higher (Delander and Hopkins, 1987) or similar (Pham et al., 2003) to A1 receptor agonists. We used CGS21680 which possesses high affinity for A2A receptors (Ralevic and Burnstock, 1998) and long duration of action (Gao et al., 2001); thus we assume that the effects of this drug on behavioral responses are due primarly to the activation of A2A receptors, even though we cannot exclude the activation of A1 receptors, even minimal. It has been shown that CGS21680 alleviates inflammatory (Poon and Sawynok, 1998) and neuropathic pain (Lee and Yaksh, 1996). A2 receptors are thus implicated in the control of pain, but the mechanism by which central administration of A2A agonist induces antinociceptive effects remains unclear. It has been shown that A2A receptors are coupled with K ATP channels in smooth muscle (Dart and Standen, 1993; Prior et al., 1999; Haynes, 2000). In the present case, we found that SKCa channels are involved in the effects of CGS21680-induced antinociception. Kv channels are not implicated, since KTX had no effect on CGS21680 induced latency increases up to 10 pmol. Since SKCa blockers partially decreased CGS 21680-induced latency increases, however, other mechanisms may participate in the antinociceptive effects resulting from the activation of A2A receptors. It has been suggested that NMDA receptors, involved in the genesis of noxious stimuli (Bordi and Quataroli, 2000; Chizh et al., 2001) are targets for A2A receptors, through receptor-receptor interactions (Sebastiao and Ribeiro, 2000). This is because NMDA-receptor currents are suppressed by the activation of A2A receptors via the phospholipid C/IP3 cascade (Norenberg et al., 1997; Wirkner et al., 2000). The inhibition of NMDA receptors could thus participate in the analgesic effects induced by A2A receptor activation.
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In summary we found that SKCa, but not Kv participate in the analgesic effects of CGS21680. To our knowledge, there are no data on the interaction between A2A receptors and SKCa or Kv channels in an experimental acute pain model. Financial support provided by Fondation pour la Recherche Me´dicale and Centre National de la Recherche Scientifique.
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Sawynok, J., Sweeney, M.J., White, T.D., 1986. Classification of adenosine receptors mediating antinociception in the rat spinal cord. British Journal of Pharmacology 88, 923 – 930. Sebastiao, A.M., Ribeiro, J.A., 2000. Fine-tuning neuromodulation by adenosine. Trends in Pharmacological Science 21, 341 – 346. Shah, M., Haylett, D.G., 2000. The pharmacology of hSK1 Ca2+-activated K+ channels expressed in mammalian cell lines. British Journal of Pharmacology 129, 627 – 630. Shakkottai, V.G., Regaya, I., Wulff, H., Fajloun, Z., Tomita, H., Fathallah, M., Cahalan, M.D., Gargus, J.J., Sabatier, J.M., Chandy, K.G., 2001. Design and characterization of a highly selective peptide inhibitor of the small conductance calciumactivated K+ channel, SkCa2. Journal of Biological Chemistry 276, 43145 – 43151. Sjolund, K.F., Belfrage, M., Karlsten, R., Segerdahl, M., Arner, S., Gordh, T., Sollevi, A., 2001. Systemic adenosine infusion reduces the area of tactile allodynia in neuropathic pain following peripheral nerve injury: a multi-centre placebo-controlled study. European Journal of Pain 5, 199 – 207. Sjolund, K.F., Sedergahl, M., Sollevi, A., 1999. Adenosine reduces secondary hyperalgesia in two human models of cutaneous inflammatory pain. Anesthesia and Analgesia 88, 605 – 610. Sollevi, A., 1997. Adenosine for pain control. Acta Anaesthesiologica Scandinavica 110, 135 – 136. Sollevi, A., Belfrage, M., Lundeberg, T., Segerdahl, M., Hansson, P., 1995. Systemic adenosine infusion: a new treatment modality to alleviate neuropathic pain. Pain 61, 155 – 158. Sperlagh, B., Zsilla, G., Vizi, E.S., 2001. K(ATP) channel blockers selectively interact with A1-adenosine receptor mediated modulation of acetylcholine release in the rat hippocampus. Brain Research 889, 63 – 70. Stocker, M., Pedarzani, P., 2000. Differential distribution of three Ca(2+)-activated K(+) channel subunits, SK1, SK2, and SK3, in the adult rat central nervous system. Molecular Cell Neuroscience 15, 476 – 493. Strobaek, D., Joergensen, T.D., Christophersen, P., Ahring, P.K., Olesen, S.P., 2000. Pharmacological characterization of small-conductance Ca(2+)-activated K(+) channels stably expressed in HEK 293 cells. British Journal of Pharmacology 129, 991 – 999. Takagi, M., Imukai, T., Nalama, M., 1996. A modification of Haffner’s method for testing analgesics, Japanese. Journal of Pharmacology 16, 287 – 294. Vergara, C., Latorre, R., Marrion, N.V., Adelman, J.P., 1998. Calcium-activated potassium channels. Current Opinion in Neurobiology 8, 321 – 329. Wirkner, K., Assman, H., Koles, L., Gerevich, Z., Franke, H., Norenberg, W., Bohem, R., Illes, P., 2000. Inhibition by adenosine A2A receptors of NMDA but not AMPA currents in rat neostriatal neurons. British Journal of Pharmacology 130, 259 – 269. Xia, M., Fakler, B., Rivard, A., Wayman, G., Johnson-Pais, T., Keen, J.E., Ishii, T., Hirschberg, B., Bond, C.T., Lutsenko, S., Maylie, J., Adelman, J.P., 1998. Mechanism of calcium gating in small-conductance calcium-activated potassium channel. Nature 395, 503 – 507.
Small conductance calcium-activated K+ channels, SkCa, but not voltage-gated K+ (Kv) channels, are implicated in the antinociception induced by CGS21680, a A2A adenosine receptor agonist I. Regayaa,b, T. Phamc, N. Andreottid, N. Sauzea, L. Carregaa,d, M.F. Martin-Eauclairea, B. Jouiroua,b, J.C. Peragute, H. Vachera, H. Rochata,d,b, C. Devauxa, J.M. Sabatiera,b, R. Guieua,b,d,e,* a
UMR FRE CNRS 2738 Inge´nierie des Prote´ines, Faculte´ de Me´decine Nord, Bd P, Dramard 13015 Marseille, France b Laboratoire International Associe´ (LIA), Faculte´ de Me´decine Nord, Bd P Dramard 13015 Marseille, France c Service de Rhumatologie, Hoˆpital de la Conception, Marseille, France d Laboratoire de Biochimie, CHU Timone, Marseille France e Centre anti-douleur, Service de Neurochirurgie Fonctionnelle, CHU, Timone, Marseille, France. Received 20 May 2004; accepted 9 June 2004
Abstract It has been shown that A2A adenosine receptors are implicated in pain modulation. The precise mechanism by which activation of A2A receptors produces analgesic effects, however, remains unclear. The aim of this study was to investigate the possible involvement of apamin-sensitive calcium-activated potassium channels (SKCa) and voltage-gated potassium (Kv) channels in A2A receptor activationinduced analgesic effects. Using mice, we evaluated the influence of apamin, a non specific blocker of SKCa channels, Lei-Dab7 (an analog of scorpion Leiurotoxin), a selective blocker of SKCa2 channels, and kaliotoxin (KTX) a Kv channel blocker, on the CGS 21680 (A2A adenosine receptor agonist)induced increases in hot plate and tail pinch latencies. All drugs were injected in mice via the intracerebroventricular route. We found that apamin and Lei-Dab7, but not KTX, reduced antinociception
* Corresponding author. E-mail address: [email protected] (R. Guieu). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.06.023
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produced by CGS21680 on the hot plate and tail pinch tests in a dose dependent manner. Lei-Dab 7 was more potent than apamin in this regard. We conclude that SKCa but not Kv channels are implicated in CGS 21680-induced antinociception. D 2004 Elsevier Inc. All rights reserved. Keywords: SKCa channels; Kv channels; Adenosine; Antinociception
Introduction Antinociceptive effects of adenosine (ADO) or its analogs have been observed following peripheral administration in animals (Holmgreen et al., 1983; Ahlijanian and Takemori, 1985) and humans (Guieu et al., 1994; Belfrage et al., 1995; Sjolund et al., 1999), or following central administration in animals (Delander and Hopkins, 1987; Karlsten et al., 1990) or humans (Sollevi et al., 1995; Sollevi, 1997; Sjolund et al., 2001). ADO can act via adenosine A1, A2A, A2B or A3 receptors all of which belong to the G protein-coupled family (Ralevic and Burnstock, 1998). Activation of A1 (Sawynok et al., 1986) or A2A receptors (Delander and Hopkins, 1987) induced antinociceptive effects. Several mechanisms for antinociceptive effects of ADO receptors stimulation have been proposed, including pre- (Santicioli et al., 1992) and postsynaptic mechanisms (Li and Perl, 1994), but also receptor-receptor interactions (Sebastiao and Ribeiro, 2000). At the presynaptic level, activation of A1 receptors results in a decrease in cAMP production and an inhibition of voltage-gated calcium channels that leads to a decrease in the release of neurotransmitters such as acetylcholine, SP or CGRP implicated in the transmission of nociceptive stimuli (Sperlagh et al., 2001; Santicioli et al., 1992; for review see Ribeiro et al., 2002; Sawynok and Liu, 2003). At the postsynaptic level, the activation of A1 receptors induces the opening of several potassium channel subtypes including KATP (Ocana and Baeyens, 1994), inwardly rectified and calcium-activated potassium channels, resulting in the hyperpolarization of excitable membranes (Ralevic and Burnstock, 1998). It has been shown that A2A receptors are also involved in pain modulation at the spinal (Sawynok et al., 1986; Delander and Hopkins, 1987) and supraspinal levels (Pham et al., 2003). The mechanism by which A2A receptors are implicated in pain modulation remains unclear. At the supraspinal level, the activation of A2A receptors may facilitate or inhibit neurotransmitter release, depending on the brain area (Dunwiddie and Masino, 2001). It has been shown that A2A agonists inhibit transmitter release via an inhibition of voltage-dependent calcium channels (Latini et al., 1996; Edwards and Robertson, 1999). Nevertheless, it still remains possible that, as in the case of A1 receptors, activation of A2A receptors induces pain relief via modulation of potassium channel activity. Among the potassium channels, it has been shown that voltagegated (Kv) (Rasband et al., 2001) and apamin-sensitive small conductance Ca++-activated potassium channels (SKCa) (Boettger et al., 2002) are expressed on nociceptors and are involved in pain control. It has also been shown that SKCa are implicated in drug-induced antinociception (Galeotti et al., 2001; Granados-Soto et al., 2002). The aim of this study was thus to evaluate the possible involvement of Kv and SKCa potassium channels in the antinociception induced by activation of A2A receptors.
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Materials and methods Animals Male C57/BL6 mice (25–30g) bred in our laboratory were used at the age of 8 weeks. Animals were housed 10 per cage and had free access to food and water in a quiet environment. The room was maintained at 21–238C with a 12h light/dark cycle. Each animal underwent both tail pinch and hot plate tests at 30 min intervals in a random order. The experimenter was blind to the pharmacological treatment. Because latency variations of at least 10% were expected, 24 animals per group were included. Drugs and reagents CGS 21680 (2-[p-(carboxyethyl)-phenyl-ethylamine]-5V-N-ethylcarboxyamidoadenosine) as an A2A agonist and apamin (SKCa blocker) were purchased from Sigma (St Quentin Fallavier, France). N-Fmoc-L-amino acid derivatives, Fmoc-amide resin, and chemical reagents used for peptide synthesis were purchased from Perkin-Elmer Life Sciences (Shelton, CO), Novabiochem (Laufelfingen, Switzerland), and Laboratoire Neosystem (Strasbourg, France). Solvents were analytical grade products from SDS (Peypin, France). Peptide synthesis The chemical synthesis of Lei-Dab7, a peptide blocker of SKCa2 potassium channels, has been described elsewhere (Shakkottai et al., 2001). Briefly, the peptide was synthesized with the stepwise solid phase method (Merrifield, 1986) using an automated peptide synthesizer (Model 433A, Applied Biosystems Inc., Foster City, CA). The side-chain protecting groups used for trifunctional residues were: 2,2,5,7,8-pentamethylchroman-6-sulfonyl for Arg and homoarginine, tert-butyloxycarbonyl for Orn, Lys, and homolysine, and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-yliden)-3-methylbutyl for Dab. The reduced peptide was dissolved at 1 mM in 0.2 M Tris-HCl buffer, pH 8.3, and stirred under air to allow folding/oxidation (48 h, 258C). The folded/oxidized peptide was purified to homogeneity by reversedphase high-pressure liquid chromatography (HPLC) (C18 Aquapore ODS 20 Am, 250 10 mm) and lyophilized before injection. The homogeneity (N99%) and identity of the peptide were verified by: (i) analytical C18 reversed-phase HPLC, (ii) amino acid content determination after acid hydrolysis, and (iii) mass analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). Experimental procedure All injected drugs were dissolved in the vehicle (methanol/water, 3/7, V/V). Mice were injected intracerebroventriculary (ICV, 4 AL). Anesthesia was obtained with 1 ml of isofluorane (Forene (r), Abbott, France) on soaked cotton in a closed bottle for 30 s. Animals were injected with CGS 21680 at the doses of 0.1, 0. 5 and 1 nmol, alone or in combination with apamin (0.5, 2, 5 pmol), Lei-Dab7 (0.5, 2, 5 pmol) or KTX (0.5, 5 or 10 pmol). Controls were injected with the vehicle alone.
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Measurement of nociceptive response with the hot plate test Animals were placed on a 51F0.58C hot plate (Hot plate analgesia meter, Harvard apparatus, USA) and latencies of forepaw licking were measured 15, 30, and 60 min after ICV injection. Measurement of nociceptive response in the tail pinch test Nociceptive responses in the tail pinch test was measured as previously described (Takagi et al., 1996; Ochi et al., 1999). Briefly, mice were pretested by pinching their tail base with an artery clip (3 mm wide, 500 g constant pressure) and only mice that showed a nociceptive response (biting the clip or vocalizing) within 3 s were used for expriments. 20–25% of the mice pretested did not manifest any reaction and were excluded. To prevent tissue damage, a cut-off time of 10 s was selected. The nociceptive response to the tail pinch test was determined 15, 30, and 60 min after ICV injections, as for the hot plate test. Statistical analysis Data were analyzed by ANOVA with Fisher’s PLD post hoc test. A value of P lower than 0.05 was considered to be significant.
Results Effects of CGS 21680 alone At 0.1 nmol, CGS 21680 did not significantly modify latencies, regardless of the test (Fig. 1A and B). At 0.5 nmol, CGS 21680 increased hot plate test and tail pinch test latencies at 15, 30 and 60 min, respectively compared with 0.1 nmol (p b 0.05). At 1 nmol, it increased hot plate test and tail pinch test latencies at 15, 30 and 60 min, respectively compared with 0.5 nmol (p b 0.05). After 60 min, no increase in latencies was observed (data not shown). Effects of Lei-Dab7, apamin or KTX alone Lei-Dab7 (5 pmol), apamin (5 pmol), or KTX (10 pmol) did not significantly modify latencies in either test. At these concentrations, apamin or KTX do not deteriorate locomotor activity (Fournier et al., 2001). With 10 pmol of Lei-Dab 7, there were no observable manifestations of toxicity, including locomotor abnormalities. Effects of apamin, lei-Dab7 and KTX on CGS21680-induced latency increases in the hot plate and tail pinch tests Apamin decreased CGS21680-induced hot plate latency increases in a dose-dependent manner (Fig. 2A). Lei-Dab 7 (0.5 pmol) did not significantly decrease CGS 21680-induced latency increases in the hot plate test at any time point (Fig. 3). At 2 and 5 pmol, Lei-dab 7 decreased CGS 21680-
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Fig. 1. Effects of CGS21680 (A2A agonist) on latencies in the hot plate (A) and tail pinch tests (B). Mice (n = 24 per group) were injected (ICV) with CGS 21680 (4 AL in methanol/water 3v/7v) or vehicle (4 AL methanol/water, 3/7, V/V). *p b 0.05 compared with 0.1 nmol.
induced hot plate latency increases in a dose-dependent manner. At 30 and 60 min, Lei-dab 7 was more potent than apamin in reducing CGS 21680-induced latency increases (p b 0.05 in tail pinch and hot plate tests; Fig. 2B). In contrast to apamin and Lei-dab7, KTX did not significantly modify CGS21680-induced hot plate and tail pinch test latency increases, up to 10 pmol (data not shown).
Discussion There is no way of determining the specific loci of action of a substance in the brain after ICV injection and it cannot necessarily be assumed that CGS21680 or SKCA channel blockers act on the same brain structures. It is known, however, that both A2A receptors (Johansson and Fredholm, 1995; Rosin et al., 1998) and SKCa channels (Stocker and Pedarzani, 2000) are present in brain areas implicated in pain control (thalamus as example). It is well-established that a large number of potassium channel subtypes are involved in the modulation of noxious stimuli (Rasband et al., 2001),
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Fig. 2. Effects of apamin (a non specific SKCa blocker) on the latency increases in hot plate and tail pinch tests induced by CGS21680, a A2A receptor agonist. Mice (n = 24 per group) were injected ICV (4 AL) with CGS 21680 alone or combined with apamin. Error bars represent standard deviations. *p b 0.05 compared with CGS 21680 alone. **p b 0.05 compared with apamin 2pmol.
including voltage-gated K+ (Kv) channels (Clark and Tempel, 1998; Rasband et al., 2001), and KATP and Ca++-activated potassium channels (Galeotti et al., 2001; Boettger et al., 2002). The latter channels can be classified as BKCa, SKCa and IKCa (big, small and intermediate conductances, respectively (Latorre et al., 1989; Vergara et al., 1998). SKCa channels are in turn subdivided into SKCa1-3 which are largely distributed in the nervous system, including areas involved in pain control (Stocker and Pedarzani, 2000). Both subtypes play a major role in governing neuron excitability, and their presence and distribution in the nervous system contributes to pain perception. It was recently shown that the expression of SKCa1 and IKCa1 are modified in injured human
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Fig. 3. Effects of Lei-Dab7 (a specific SKCa2 blocker) on the latency increases in hot plate (A) and tail pinch tests (B) induced by CGS21680. Mice (n = 24 per group) were injected ICV (4 AL) with CGS 21680 alone or combined with Lei-Dab7. Error bars represent standard deviations. Error bars represent standard deviations. *p b 0.05 compared with CGS 21680 alone. **p b 0.05 compared with Lei-Dab7 (5 nmol).
sensory neurons of the dorsal root ganglion (Boettger et al., 2002), strongly suggesting that these channels are implicated in the control of noxious stimuli. SKCa channels modulate the firing pattern of neurons by generating slow membrane post-hyperpolarization (Xia et al., 1998; Rimini et al., 2000; Stocker and Pedarzani, 2000). The SKCa2 channel subtype is extensively expressed in the brain (Rimini et al., 2000; Stocker and Pedarzani, 2000), but until recently existing SKCa2 channel blockers were not selective enough to distinguish this channel from other SKCa subtypes.
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Our results suggests that SKCa1-3, but not Kv, are implicated in the analgesic effects induced by CGS21680. Most of these effects are due to the activation of A2A receptors. Indeed CGS21680 is 140 fold selective for the A2A versus A1 receptor (Hutchison et al., 1990). Thus it is difficult to conclude that most effects of CGS21680 are due to the activation of A1 rather than A2A receptors. Apamin and Lei-Dab 7 may act by eliminating the medium IAHP current, and causing "bursting electrical activity" and enhanced neurotransmitter release in nociceptors. Since Lei-Dab 7 is more effective than apamin in reducing CGS21680-induced antinociception, we postulate that the SKCa2 subtype is more probably implicated in CGS21680-induced antinociception. Apamin, a peptide from bee venom, in fact exhibits only a 10-fold selectivity for SKCa2 over SKCa1 or SKCa3 (Kohler et al., 1996; Desai et al., 2000; Shah and Haylett, 2000; Strobaek et al., 2000), while Lei-Dab7 is a leiurotoxin I analog that selectively blocks SKCa2 homotetramers with a low nanomolar affinity. This was shown in electrophysiological studies (Shakkottai et al., 2001). Finally, our results may be affected by the possibility that SKCa blocking can elicit profound effects on neuronal excitability and that the observed effects are unrelated to A2A modulation. This hypothesis can be discarded, however, since SKCa blockers alone have no effects on behavioral latencies to nociceptive stimuli. There is also no evidence that SKCa blockers are specifically coupled with A2A receptors since it has been shown that some drugs that which are not adenosine analogs possess analgesic effects via the modulation of SKCa channels (Galeotti et al., 2001; Granados-Soto et al., 2002). Interestingly, some of these drugs interact with neuronal adenosine uptake (Phillis, 1984). Other drugs act via BK but not SKCa channels (Santos et al., 2003). Adenosine or its analogs are involved in the control of a wide range of pain mechanisms including neuropathic pain (Guieu et al., 1994, 1996; Sjolund et al., 2001). Most of these effects are secondary to the activation of A1 receptors (Sawynok et al., 1986; Sawynok, 1998). On the other hand, some data suggest that the effectiveness of A2A agonists in alleviating pain is higher (Delander and Hopkins, 1987) or similar (Pham et al., 2003) to A1 receptor agonists. We used CGS21680 which possesses high affinity for A2A receptors (Ralevic and Burnstock, 1998) and long duration of action (Gao et al., 2001); thus we assume that the effects of this drug on behavioral responses are due primarly to the activation of A2A receptors, even though we cannot exclude the activation of A1 receptors, even minimal. It has been shown that CGS21680 alleviates inflammatory (Poon and Sawynok, 1998) and neuropathic pain (Lee and Yaksh, 1996). A2 receptors are thus implicated in the control of pain, but the mechanism by which central administration of A2A agonist induces antinociceptive effects remains unclear. It has been shown that A2A receptors are coupled with K ATP channels in smooth muscle (Dart and Standen, 1993; Prior et al., 1999; Haynes, 2000). In the present case, we found that SKCa channels are involved in the effects of CGS21680-induced antinociception. Kv channels are not implicated, since KTX had no effect on CGS21680 induced latency increases up to 10 pmol. Since SKCa blockers partially decreased CGS 21680-induced latency increases, however, other mechanisms may participate in the antinociceptive effects resulting from the activation of A2A receptors. It has been suggested that NMDA receptors, involved in the genesis of noxious stimuli (Bordi and Quataroli, 2000; Chizh et al., 2001) are targets for A2A receptors, through receptor-receptor interactions (Sebastiao and Ribeiro, 2000). This is because NMDA-receptor currents are suppressed by the activation of A2A receptors via the phospholipid C/IP3 cascade (Norenberg et al., 1997; Wirkner et al., 2000). The inhibition of NMDA receptors could thus participate in the analgesic effects induced by A2A receptor activation.
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In summary we found that SKCa, but not Kv participate in the analgesic effects of CGS21680. To our knowledge, there are no data on the interaction between A2A receptors and SKCa or Kv channels in an experimental acute pain model. Financial support provided by Fondation pour la Recherche Me´dicale and Centre National de la Recherche Scientifique.
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