Tramadol is an analgesic that has been used clinically. Its mechanisms of action of tramadol have been reported by several investigators. Tramadol binds to μ-opioid receptors, and this has been considered the mechanism of antinociception by this compound, although its binding affinity is relatively low (1). Inhibition of the reuptake of norepinephrine and serotonin has been identified as a further mode of action of tramadol. This inhibitory effect may also contribute to the analgesic effects of tramadol by the inhibition of pain transmission in both the central nervous system and the spinal cord (2,3). We (4) have reported that tramadol inhibits the functions of nicotinic acetylcholine receptor ion channels. However, its mechanism of action is not yet completely understood.
Tramadol undergoes biotransformation in the liver by two metabolic pathways to form five N- or O-desmethylated metabolites. O-desmethyl tramadol (M1) is one of the five main metabolites of tramadol; the other four are mono-N-desmethyl tramadol (M2), di-N-desmethyl tramadol (M3), tri-N,O-desmethyl tramadol (M4), and di-N,O-desmethyl tramadol (M5) (5). Of the five, only O-desmethyl tramadol has been shown to have analgesic activity in mice and rats, as assessed by the tail-flick response, with two to four times the potency of tramadol (1). In biochemical receptor binding studies, O-desmethyl tramadol has more affinity for the μ-opioid receptor than does the parent compound tramadol (1). This evidence suggests that O-desmethyl tramadol might play an important role in the mechanism of action of tramadol.
Muscarinic receptor signaling in the central nervous system plays important roles in the level of consciousness, memory, and learning (6–8). Spinal muscarinic receptors mediate antinociception (9). It is reported that M1 and M3 receptors may play a major role in antinociception (10–12). Several investigators have shown that anesthetics inhibit muscarinic receptor function; ketamine, halothane, and isoflurane suppress muscarinic receptor function (13–15). These findings suggest that the inhibition of muscarinic function is one action of some anesthetics and analgesics (8). We (16,17) found that tramadol inhibits the function of muscarinic receptors. Shiraishi et al. (16) showed that tramadol inhibits the function of M1 receptors via binding at the acetylcholine (ACh)-binding site. Further, Shiga et al. (17) reported that tramadol inhibits the function of the M3 receptors. Our findings suggest that muscarinic M1 and M3 receptors are possible sites of action of tramadol. However, the effects of O-desmethyl tramadol have not been studied.
The Xenopus oocyte expression system has been widely used to study brain receptors with pharmacological properties that mimic those of native brain receptors (18). Stimulation of muscarinic M1 and M3 receptors expressed in oocytes activates Ca2+-activated Cl– currents (19,20); stimulation of M1 and M3 receptors leads to the Gq protein-mediated activation of phospholipase C, which causes the formation of inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum and triggers the opening of endogenous Ca2+-activated Cl– channels. This system has been well characterized and has proven useful for studying the effects of drugs acting on Gq protein-coupled receptors, such as M1 and M3 receptors.
In this study, we investigated the effects of the metabolite of tramadol, O-desmethyl tramadol, on M1 and M3 receptor functions in Xenopus oocytes expressing cloned muscarinic M1 or M3 receptors.
Adult female Xenopus laevis frogs were purchased from Seac Yoshitomi (Fukuoka, Japan). ACh was purchased from Sigma (St. Louis, MO). The Escherichia coli transformation kit was from Invitrogen (San Diego, CA). A kit from Qiagen (Chatworth, CA) was used to purify plasmid complimentary (c)DNA. Muscarinic M1 receptor cDNA was kindly provided by Dr. H. Lester (Caltech, Pasadena, CA), and cRNA for the M1 receptor was synthesized in vitro with T7 polymerase (Stratagene, La Jolla, CA) from cDNA linearized with Hind III. Muscarinic M3 receptor cDNA in pCD2 was a gift from Dr. T.I. Bonner (National Institutes of Health, Baltimore, MD). Within the M3 receptor coding region, the initiator codon and the BstPI site at 277 in the open reading frame were amplified by polymerase chain reaction and ligated with the remaining coding region, including the stop codon, using the BstPI/ApaI site. Then, the construct was subcloned into pCR3 vector (Invitrogen). cRNA for the M3 receptor was synthesized in vitro with T7 polymerase (Stratagene) from cDNA linearized with ApaI. Bisindolylmaleimide I (GF109203×) was from Calbiochem (La Jolla, CA); HEPES was from Nacalai Tesque (Kyoto, Japan); collagenase was from Nitta Zerachin (Osaka, Japan); O-desmethyl tramadol (M1) was a gift from Nippon Shinyaku (Kyoto, Japan).
This study conformed to the Guide for the Care and Use of Laboratory Animals adopted and promulgated by the Japanese National Institute of Health, and approval was granted by the Animal Research Committees of the University of Occupational and Environmental Health. Isolation and microinjection of Xenopus oocytes were performed as described by Minami et al. (15,21). Xenopus oocytes were injected with 50 ng of cRNA encoding the M1 or M3 receptor, and electrophysiological recording was performed at 2–5 days after injection. Oocytes were placed in a 100-μL recording chamber and perfused with modified Barth saline (MBS), containing 88 mM of NaCl, 1 mM of KCl, 2.4 mM of NaHCO3, 10 mM of HEPES, 0.82 mM of MgSO4, 0.33 mM of Ca(NO3)2, and 0.91 mM of CaCl2 (pH value of 7.5 adjusted with NaOH), at a rate of 1.8 mL/min at room temperature. Recording electrodes (1–5 ΜΩ) filled with 3 M of KCl were inserted into the animal pole. A Warner Oocyte-clamp OC 725-C (Warner, Hampden, CT) was used to voltage clamp each oocyte at −70 mV. The effects of O-desmethyl tramadol on ACh-induced currents were examined using an ACh concentration of 1 μM, according to methods previously described by Minami et al. (21), Shiraishi et al. (16), and Shiga et al. (17). We measured the peak of the transient inward current as the ACh-induced current because this component is dependent on ACh concentration and is quite reproducible, as performed by Minami et al. (21). O-desmethyl tramadol was preapplied for 2 min to allow for complete equilibration in the bath.
Protein kinase C (PKC) plays an important role in the regulation of M1 receptor function by anesthetics (15,21). To study whether the inhibitory effects of O-desmethyl tramadol on ACh-induced currents are modulated by PKC, oocytes expressing M1 receptors were exposed to the specific PKC inhibitor GF109203× (200 nM) (22) in MBS for 120 min. After exposure to GF109203×, oocytes were exposed to 1 μM of ACh, and the currents elicited were measured.
Xenopus oocytes were injected with 50 ng of cRNA encoding the M1 or M3 receptor, and the binding experiment was performed 2–5 days after injection. A single oocyte was incubated for 20 min at 25°C with MBS (final volume, 1 mL) containing [3H]quinuclidinyl benzilate ([3H]QNB; 0.5 nM) in the presence or absence of O-desmethyl tramadol (0.1 μM–1 mM). After incubation, binding was terminated and washed with 1 mL of MBS. The oocyte was placed in counting vials containing a scintillation cocktail. The radioactivity was counted in an Aloka LSC-3500E counter (Aloka, LSC-3500E, Tokyo, Japan). Specific binding of [3H]QNB was defined as the binding inhibited by 100 μM of atropine.
The results are expressed as percentages of control responses because of variability in oocyte expression. The control responses were measured before and after drug application. All values are presented as the mean ± se. The n values refer to the number of oocytes studied. Each experiment was conducted with oocytes from at least two different frogs. Statistical analyses were performed using either a t-test or a one-way analysis of variance. Half-maximal inhibitory concentration (IC50) values for concentration-response curves were estimated using Graph Pad Prism software (San Diego, CA).
Anesthetic modulation of receptor function often depends on the degree of receptor activation (18), and it was necessary to determine the ACh–concentration-response relationship under our experimental conditions before testing the effect of the O-desmethyl tramadol. Nonlinear regression analysis of these curves yielded an 50% effective concentration (EC50) for ACh of 1.0 ± 0.01 μM in the oocytes expressing M1 receptors. Maximal currents were observed at 100 μM (Fig. 1A). Nonlinear regression analysis of these curves yielded an EC50 for ACh of 1.0 ± 0.3 μM in the oocytes expressing M3 receptors. Maximal currents were observed at 100 μM (Fig. 1B). These results are consistent with our previous reports (16,17,21). Based on the results in Figure 1, the effects of O-desmethyl tramadol on ACh-induced currents were examined at an ACh concentration of 1.0 μM.
In the Xenopus oocytes expressing cloned M1 receptors, 1 μM of ACh induced robust Ca2+-activated Cl– currents (2207 ± 554 nA; n = 25). O-desmethyl tramadol inhibited the ACh-induced Ca2+-activated Cl– currents to 77.6% ± 7.5%, 72.9% ± 5%, and 58.7% ± 10.1% of control at 0.1 μM, 1 μM, and 10 μM, respectively (n = 22) (Figs. 2 and 3). The IC50 of O-desmethyl tramadol for the 1 μM of ACh-induced Cl– currents was 2 ± 0.6 μM (n = 22). In the Xenopus oocytes expressing cloned M3 receptors, 1 μM of ACh induced robust Ca2+-activated Cl– currents (790 ± 175 nA; n = 16). In contrast to the results with M1 receptors, O-desmethyl tramadol had little inhibitory effects on ACh-induced Ca2+-activated Cl– currents (Figs. 2 and 3).
PKC plays an important role in the regulation of M1 receptor function by anesthetics (15,21). Accordingly, we examined the effect of O-desmethyl tramadol on M1 receptor-stimulated currents in oocytes that had been pretreated with the PKC inhibitor GF109203×. The treatment of oocytes expressing the M1 receptor with GF109203× for 120 min at 200 nM, which is a concentration 10 times the Ki value for inhibiting PKC activity (22), enhanced the currents induced by 1 μM of ACh to 165% ± 16.5% of the control (Fig. 4). The inhibitory effects of O-desmethyl tramadol on ACh-induced currents in M1-expressing Xenopus oocytes were still observed after pretreatment with GF109203× (Fig. 4).
We next investigated whether O-desmethyl tramadol competes with ACh for M1 receptors. As shown in Figure 5, the inhibitory effect of O-desmethyl tramadol was overcome when the concentration of ACh was increased. The double-reciprocal plot analysis revealed that 10 μM of O-desmethyl tramadol competitively inhibited the ACh-induced currents (Fig. 5, A and B). From the double-reciprocal plot, the Emax values with O-desmethyl tramadol and without O-desmethyl tramadol were about equal. The Emax values were 1745 nA and 1660 nA with and without O-desmethyl tramadol, respectively. The KD values with O-desmethyl tramadol and without O-desmethyl tramadol were 0.3 μM and 0.5 μM, respectively.
Moreover, to confirm whether O-desmethyl tramadol binds to the muscarine binding site, we examined the effects of O-desmethyl tramadol on the binding of [3H]QNB to oocytes-expressed M1 receptors. The specific binding of [3H]QNB to oocytes-expressed M1 receptor was 3656 ± 354 cpm/oocyte. In Figure 5C, O-desmethyl tramadol concentration-dependently inhibited [3H]QNB binding to oocytes-expressing M1 receptors to 2694 ± 187 cpm/oocyte, 2545 ± 140 cpm/oocyte, 1685 ± 12 cpm/oocyte, and 12 ± 8 cpm/oocyte at 0.1, 10, 100, and 1000 μM, respectively. The IC50 of O-desmethyl tramadol for the binding of [3H]QNB to oocytes-expressing M1 receptor was 10.1 ± 0.1 μM (n = 8). However, O-desmethyl tramadol had little effect on [3H]QNB binding to oocytes-expressing M3 receptors (Fig. 5D).
The main finding of this study is that O-desmethyl tramadol inhibits the ACh-mediated response of M1 receptors expressed in Xenopus oocytes, which supports the concept that O-desmethyl tramadol, like tramadol, inhibits muscarinic receptors. Grond et al. (23) reported mean O-desmethyl tramadol concentrations of 200 mg after a bolus IV infusion and patient-controlled analgesia with demand doses of 20 mg for 24 h in 92 patients. In our study, the mean concentration of O-desmethyl tramadol was 84 ± 34 ng/mL (approximately 0.3 μM). Sindrup et al. (24) reported mean O-desmethyl tramadol concentrations of 5.0–122 ng/mL (approximately 0.4 μM) in patients who received 200–400 mg/d of tramadol. In our study, 0.1 μM of O-desmethyl tramadol inhibited ACh-induced Ca2+-activated Cl– currents to approximately 77.6% ± 7.5% of the control value (Fig. 3). Based on this evidence, O-desmethyl tramadol would inhibit M1 receptor functions clinically.
This study raises the question of how O-desmethyl tramadol inhibits M1 receptor-mediated responses. There is considerable evidence that PKC plays an important role in regulating the function of G-protein–coupled receptors (15,21). We reported that the PKC inhibitor GF109203× had no effect on the inhibition of M1 receptor function by tramadol (16). By contrast, several reports have shown inhibition by anesthetics via the PKC pathway (15,21). It was reported that halothane, F3 (1-chloro-1,2,2-trifluorocyclobutane), and ethanol inhibit the functions of 5-hydroxytryptamine 2A (5-HT2A) receptors (15). Halothane and ethanol inhibit M1 receptors (21). Halothane, isoflurane, enflurane, diethyl ether, and ethanol inhibit substance P receptors, which share the same signaling steps as the M1 receptor, in a PKC-dependent manner (25). In our present experiments, GF109203× did not alter the inhibitory effects of O-desmethyl tramadol on M1 receptor function, suggesting that PKC is not involved in the inhibitory effect of O-desmethyl tramadol on M1 receptor function, as was found for tramadol. Moreover, we tested the effect of O-desmethyl tramadol on substance P receptors, which share the same signaling steps as the M1 receptor, when expressed in Xenopus oocytes. O-desmethyl tramadol had no inhibitory effects on substance P receptor function (date not shown). From these findings, the inhibitory effect of O-desmethyl tramadol on the ACh-induced Cl– current is likely owing to direct inhibition of the M1 receptor.
To confirm our hypothesis, we examined the competition between O-desmethyl tramadol and ACh for M1 receptors. We found that O-desmethyl tramadol inhibited the specific binding of [3H]QNB to the oocyte-expressed M1 receptor, and O-desmethyl tramadol (100 μM) inhibited it completely. In this study, the IC50 of O-desmethyl tramadol for the Cl– currents induced by 1 μM of ACh was almost equal to that of tramadol (3.4 ± 2.3 μM) (16). According to Shiraishi et al. (16), tramadol also competitively inhibits the function of muscarinic receptors in adrenal medullary cells. From our results, a much larger concentration of O-desmethyl tramadol (1 mM) than that in clinical use is required for complete inhibition. Although O-desmethyl tramadol might inhibit the M1 receptor function competitively, the binding site might be near the site of QNB binding. It will be required to determine the binding site using a mutation study of M1 receptors.
Tramadol inhibited M3 receptor function. By contrast, O-desmethyl tramadol had little effect on M3 receptor function. The chemical structure of O-desmethyl tramadol is quite similar to that of tramadol (Fig. 2A). However, O-desmethyl tramadol had little effect on M3 receptor function. Previously, we suggested that tramadol binds to one of the ACh binding sites in M1 and M3 receptors (16,17). Based on our present findings, we speculate that O-desmethyl tramadol does not affect the ACh binding site on M3 receptors because of the small difference in its structure compared with that of tramadol.
The roles of brain muscarinic receptors in antinociception and analgesic action have been studied. Several lines of evidence have shown that muscarinic agonists enhance antinociceptive effects, which are blocked by pretreatment with M1, M2, or M3 muscarinic receptor antagonists (11). By contrast, inhibition of the muscarinic signaling pathway induced by reducing ACh levels, inhibiting ACh release, or administering scopolamine in rat brains decreases the minimum alveolar anesthetic concentration of inhaled anesthetics (26). Several anesthetics, such as isoflurane, depress M1 and M3 receptor function (13,20). Therefore, the actions of analgesics or anesthetics on muscarinic receptors may be more complex than currently believed (8), and further studies will be required to define the relationship between antinociception and muscarinic receptor functions. Clinically, tramadol sometimes causes a dry mouth and constipation (27), which may be caused by the inhibition of muscarinic receptors. Both glandular secretion and smooth muscle contraction are mediated primarily by stimulation of M3 receptors (28). O-desmethyl tramadol had little effect on M3 receptors, suggesting that it would not cause a dry mouth or constipation. Therefore, these anticholinergic effects of tramadol might be temporary.
In conclusion, O-desmethyl tramadol inhibited M1 muscarinic receptor function but had little effect on the function of M3 receptors. Our findings help to elucidate the pharmacological basis of tramadol and O-desmethyl tramadol and to better understand their neuronal action and anticholinergic effects.
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