Tramadol [(1R, 2R) and (1S, 2S)-2-dimethylaminomethyl-1-(3-methoxyphenyl)-cyclohexanol hydrochloride] has been used as an analgesic for several decades. However, its mechanism of antinociception is unclear (1). Studies have shown that tramadol inhibits the uptake of [3H]5-HT into purified synaptosomes from the rat frontal cortex (2), which suggested that the analgesic effect of tramadol is related to serotonin as one of the mechanisms that modulate nociceptive perception. Recently, we found that tramadol inhibits the function of cholinergic receptors, such as muscarinic M1 and M3 receptors (3–5). These findings implicate G-protein coupled receptors (GPCR) as a target of tramadol action.
Serotonin (5-hydroxytryptamine, 5-HT) is a monoaminergic neurotransmitter that modulates numerous sensory, motor, and behavioral processes in the mammalian central and peripheral nervous systems (6). Seven 5-HT receptor families have been identified, and diverse responses are elicited via the activation of these receptor subtypes (7). The 5-HT type 2C receptor (5-HT2CR) is one of the major 5-HT receptor subtypes in the brain (8) and belongs to the GPCR family (9). Because the receptor is expressed in cortical and subcortical neurons, including hippocampal pyramidal neurons and neurons in thalamic sensory relay nuclei where nociceptive transmission is regulated (7), its message is widely distributed in the brain (9). 5-HT2CR is implicated in many important effects of 5-HT, including pain, feeding, and locomotion (8). Several reports have indicated that 5-HT2CR-deficient mice show abnormal control of feeding behavior, resulting in overweight mice (8) that are prone to spontaneous death from seizures (10).
We previously reported that volatile anesthetics like halothane inhibit 5-HT type 2A receptors expressed in Xenopus oocytes (11). Moreover, recent studies have shown that tramadol has analgesic properties that are mediated by serotonergic 5HT1 and 2/3 receptors (12,13). These implicate the effects of anesthetics on 5-HT2CR in one of the mechanisms modulating nociceptive perception. However, there is little information on the action of tramadol on 5-HT2C receptors.
The Xenopus oocyte expression system is a superior technique for studying a multiplicity of brain receptors with pharmacological properties that mimic those of native brain receptors (14). Stimulation of 5-HT2CR leads to G-protein-dependent activation of phospholipase C (PLC), producing myo-inositol-1, 4, 5-trisphosphate (IP3) and diacylglycerol (DG) (15). IP3 causes the release of Ca2+ from the endoplasmic reticulum, which in turn triggers the opening of Ca2+-activated Cl− channels (14). This system has been well characterized, and has proven useful for evaluating the effects of analgesics on GPCR (4,5,11,16). Therefore, we used this technique for this investigation.
This study examined the effects of tramadol on 5-HT-induced Ca2+-activated C− currents in Xenopus oocytes expressing 5-HT2CR. In addition, we investigated the mechanism of the effects of tramadol on 5-HT2CR function.
Adult female Xenopus laevis frogs were purchased from Seac Yoshitomi (Yoshitomi, Fukuoka, Japan), 5-HT was from Sigma (St. Louis, MO), tramadol hydrochloride was a kind gift from Nippon Shinyaku (Kyoto, Japan), bisindolylmaleimide I (GF109203×) was from Calbiochem (La Jolla, CA), and the Ultra-comp E. coli Transformation Kit was from Invitrogen (San Diego, CA). A Qiagen Kit (Chatworth, CA) was used to purify plasmid cDNA. 5-HT2CR cDNA from rats was kindly provided by Dr. Henry Lester (Caltech,Pasadena, CA). The 5-HT2CR cDNA was linearized with Xba I, and rat 5-HT2CR cRNA was prepared using an mCAP mRNA Capping Kit, and transcribed with a T7 RNA Polymerase in vitro Transcription Kit (Stratagene, La Jolla, CA).
Xenopus oocytes were isolated and microinjected, as described by Minami et al. (11,17). Briefly, Xenopus oocytes were injected with 50 ng of cRNA encoding 5-HT2CR. The oocytes were placed in a 100-μL recording chamber and perfused with MBS (modified Barth’s saline) containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, and 0.91 mM CaCl2, (pH 7.5) at a rate of 1.8 mL/min at room temperature. The recording and clamping electrodes (1–5 MΩ) were pulled from capillary tubing with a 1.2-mm outside diameter and filled with 3 M KCl. A Warner OC 725-B oocyte clamp (Hampden, CT) was used to voltage-clamp each oocyte at −70 mV. We analyzed the peak of the transient inward current component of the 5-HT2CR-induced currents because this component is dependent on 5-HT concentration and is quite reproducible, as described by Minami et al. (11). Tramadol was pre-applied for 2 min to allow complete equilibration in the bath. The solution of IV tramadol was freshly prepared immediately before use. The concentrations in the figures represent the bath concentrations.
To determine whether protein kinase C (PKC) activation plays a role in the modulatory effect of tramadol on 5-HT2CR-mediated events, oocytes were treated with the PKC inhibitor bisindolylmaleimide I (GF109203×) (200 nM) (18) in MBS for 240 min. 5-HT was applied at 60, 120, 180, and 240 min during the GF109203× treatment. As a control, 5-HT was similarly applied to oocytes that were not treated with GF109203X. We also investigated the effects of tramadol on 5-HT-induced Cl− currents in oocytes pretreated with GF109203× for 120 min.
The binding of [3H]5-HT to Xenopus oocytes was examined in the following manner. For groups expressing or not expressing 5-HT2CR, 3 Xenopus oocytes per tube were incubated for 60 min at 25°C with MBS (final volume 0.5 mL) containing [3H]5-HT (0.1–10 nM) in the presence or absence of tramadol. Three oocytes per tube were used for total binding and nonspecific binding at a tramadol concentration of 10−5M. After incubation, binding was terminated by rapidly washing the oocytes 4 times with 5 mL of ice-cold MBS buffer under vacuum through Whatman GF/C glass-fiber filters, and the oocytes were placed in counting vials containing scintillation cocktail. The radioactivity was counted in an Aloka LSC-3500E counter (Tokyo, Japan). Specific binding of [3H]5-HT was defined as the binding inhibited by 10−8M 5-HT.
Results are expressed as percentages of the control responses attributable to the variable 5-HT2CR expression in oocytes. The control responses were measured before and after applying each drug to take into account possible shifts in the control currents as recording proceeded. The “n” values refer to the number of oocytes studied. Each experiment was performed with oocytes from at least two different frogs. Statistical analyses were performed using either Student’s t-test or a one-way analysis of variance. Curve fitting and estimation of IC50 values for the concentration-response curves were performed using GraphPad In-plot Software (San Diego, CA).
With drugs such as anesthetics, the modulation of receptor function often depends on the degree of receptor activation (14). Therefore, it was necessary to determine the 5-HT concentration-response relationship under our experimental conditions before examining the effect of tramadol (Fig. 1). Nonlinear regression analysis of the curve yielded an EC50 for 5-HT of 26 nM, a Hill coefficient of 1.1, and the maximal current was observed at 10−5 M (Fig. 1). Based on the results in Figure 1, the effects of tramadol on 5-HT-induced currents were examined at a 5-HT concentration of 10−8M.
Compared with the Ca2+-activated Cl− currents induced by 10−8M 5-HT as a control, tramadol suppressed the Cl− currents (Fig. 2A). After 60 min, all the Cl− currents produced by 10−8M 5-HT recovered completely (Fig. 2A). However, using the Cl− currents treated with 10−8 M 5-HT as a control current, 10−7 and 10−6 M 5-HT compensated the inhibitory effects of 10−6 M tramadol, respectively (Fig. 3).
Several reports have shown that some anesthetics inhibit GPCR functions by modulating PKC pathways (11,16). Therefore, we also investigated whether the inhibitory effects of tramadol on 5-HT2CR-mediated events could be the result of modulation of PKC pathways. For this purpose, we used Xenopus oocytes pre-treated with the specific PKC inhibitor GF109203×(200 nM), which has a Ki value for inhibiting PKC activity of 20 nM (18). Treatment with GF109203× alone produced a 5.2-fold enhancement in the initial Cl− currents induced by 10−8 M 5-HT (Fig. 4A and B). Tramadol also inhibited the 5-HT-induced currents in oocytes pretreated with GF109203X (Fig. 4C and D).
We further examined the effects of tramadol on the binding of [3H]5-HT to 5-HT2CR. pecific binding of [3H]5-HT was saturable with increasing [3H]5-HT concentration (0.1–10 nM) (Fig. 5A). A Scatchard analysis showed a single population of binding sites, with an apparent dissociation constant (Kd) of 7 nM and maximal binding (Bmax) of 25.6 pmol/3 oocytes (Fig. 5B). The specific binding of [3H]5-HT was inhibited by 10−5 M tramadol and was not reversed by increasing the concentration of [3H]5-HT (Fig. 5A). From the Scatchard plot analysis, tramadol altered the Kd of [3H]5-HT binding (28 nM) without changing Bmax (26.0 pmol/3 oocytes) (Fig. 5B).
In this study, we showed that tramadol inhibited the 5-HT2CR expressed in Xenopus oocytes. According to Lintz et al. (19), the concentration of tramadol in human serum reaches 612.7 ± 221 ng/mL (approximately 2 μM) after IV injection of 100 mg tramadol, which is the clinical dosage. In this study, 1 and 10 μM tramadol inhibited the 5-HT-induced Cl− currents to 79.16% ± 3.27% and 69.09% ± 5.11% of the control, respectively. These findings suggest that tramadol suppresses the function of 5-HT2CR at clinically relevant concentrations.
Next, we examined how tramadol inhibits 5-HT2CR function. There is considerable evidence that PKC plays an important role in regulating several types of GPCR. Ethanol and volatile anesthetics such as isoflurane are reported to suppress the function of muscarinic (17) and substance P (16) receptors via the modulation of PKC pathways. Alcohols and volatile anesthetics also inhibit 5-HT2AR, and these actions are dependent on PKC (11). We showed that the selective PKC inhibitor GF109203× enhanced 5-HT2CR function, indicating that 5-HT2CR function can be modulated by changing the PKC activity. However, GF109203× did not abolish the inhibitory effects of tramadol on 5-HT2CR function, suggesting that tramadol inhibits 5-HT2CR function without modulating PKC pathways. Moreover, we previously reported that tramadol does not interfere with the pathway after G-protein-coupled signal transduction, such as PLC activation, intracellular Ca2+ release, and Ca2+-activated Cl− currents (4). It has also been reported that tramadol has no effects on the function of another Gq-coupled substance P receptor, although they share the same intracellular signaling pathways as 5-HT2CR (unpublished data). Moreover, tramadol did not inhibit the Cl− currents induced by large 5-HT concentration, suggesting that tramadol competitively inhibits 5-HT2CR function. From this and previous evidence, it is likely that the inhibitory effect of tramadol on the 5HT-induced Cl− current is attributable to the direct inhibition of 5HT2CR.
To confirm this hypothesis, we examined the effects of tramadol on [3H]5-HT binding to 5-HT2CR expressed in Xenopus oocytes. Scatchard plot analysis of [3H]5-HT binding revealed that tramadol altered the Kd without changing the Bmax, indicating competitive inhibition. These findings suggest that tramadol inhibits 5-HT2CR function by interfering with the binding of 5-HT to the receptor. We could not determine the dual binding site of tramadol on 5-HT2CR, although it is essential to reveal the effect of tramadol on 5-HT receptors in detail. Moreover, it is not clear where5-HT binds to 5-HT2C receptors. To answer these questions, it is necessary to investigate the region of 5-HT2C responsible for tramadol binding using chimera 5-HT2C or site-directed mutagenesis experiments, such as radio-ligand tramadol binding experiments.
The roles of 5-HT receptors in antinociception and analgesic actions have been investigated with a variety of approaches. 5-HT causes nociception (20,21). By contrast, several investigators have demonstrated that intrathecal serotonin induces antinociception in a variety of animal species (22,23). The role of serotonin signaling in pain sensation is still controversial. However, it has been reported that the peripheral nociceptive actions of IV administered 5-HT in the rat require dual activation of both 5-HT2 and 5-HT3 receptor subtypes (24), suggesting that 5-HT2CR participates in central pain procession. Inhibition of the function of 5-HT2CR by tramadol would modulate its antinociceptive effects. Conversely, some 5HT2 receptor antagonists have mood- and motivation-improving effects (25). Tramadol alters mood (26), and this effect is a result of inhibition of the function of 5-HT2CR. Moreover, 5-HT2CR mutant mice display both an epilepsy and obesity phenotype (27). Although these symptoms may be related to the inhibition of 5-HT2cR by tramadol (28), more animal studies are necessary to answer these questions.
In conclusion, our results suggest that tramadol inhibits 5-HT2CR function by competing for 5-HT binding sites. This suggests that the inhibition of 5-HT2CR function by tramadol explains one of the pharmacological properties of tramadol.
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