Tramadol, (1RS; 2RS)-2-[(dimethylamino) methyl]- 1-(3-methoxyphenyl)-cyclohexanol hydrochloride, is an analgesic that is used clinically. 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, and this may also contribute to the analgesic effects of tramadol by the inhibition of pain transmission both in the central nervous system (CNS) and the spinal cord (2,3). However, we recently found that tramadol inhibits not only norepinephrine transporters, but also cholinergic receptors (4). The detailed study by Shiraishi et al. (5) showed that tramadol inhibits the function of type-1 muscarinic (M1) receptors via binding at the acetylcholine (ACh) binding site, suggesting that muscarinic receptors are the sites of action of tramadol.
Muscarinic receptors are involved in various neuronal functions in the CNS and autonomic nervous systems (6). Muscarinic signaling plays important roles in the level of consciousness, memory, and learning (7,8) in the CNS. Several investigators have shown that anesthetics inhibit muscarinic receptor function; the IV anesthetic ketamine inhibits M1 receptor function (9). Volatile anesthetics, such as halothane and isoflurane, also depress muscarinic receptor function (10–12). These findings suggest that the inhibition of muscarinic receptor function is one site of action for some anesthetics and analgesics (8).
Molecular cloning studies have revealed the existence of five subtypes of muscarinic receptors (M1–M5) (13). The M3 receptors belong to the G-protein-coupled receptor family, and their activation results in the production of inositol triphosphate and diacylglycerol, triggering Ca2+-mediated signaling pathways (14). It has been reported that the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide has antinociception by inhibiting presynaptic M3 receptor in the spinal cord in mice (15). Both glandular secretion and smooth muscle contraction are primarily mediated by M3 receptors (13). We recently reported that tramadol at clinically relevant concentrations inhibits muscarinic receptor function via modulation of the quinuclidinyl benzilate (QNB)-binding sites (5). It has been reported that isoflurane inhibits M3 receptor function through activation of protein kinase C (PKC) and that the relevant PKC phosphorylation sites are located outside the third intracellular loop (14).
The Xenopus oocyte expression system has been used to express a multiplicity of brain receptors from complimentary DNAs (cDNAs) or cRNAs with pharmacological properties that mimic those of native brain receptors (16). Stimulation of muscarinic M3 receptors expressed in oocytes activates Ca2+-activated Cl– currents (15); stimulation of M3 receptors leads to the Gq protein-mediated activation of phospholipase C, which causes the formation of inositol-1,4,5-trisphos- phate. Inositol-1,4,5-trisphosphate 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. In this study, we investigated the effects of tramadol on M3 receptor function using Xenopus oocytes expressing cloned M3 receptors.
Adult female Xenopus laevis frogs were purchased from Seac Yoshitomi (Fukuoka, Japan). ACh and atropine were purchased from Sigma (St Louis, MO). The Escherichiacoli transformation kit was from Invitrogen (San Diego, CA). A kit from Qiagen (Chatworth, CA) was used to purify plasmid cDNA. M3 receptor cDNA in pCD2 was a gift from Dr. T.I. Bonner (National Institutes of Health, MD) (17). The sequence between the initiation codon and Bst PI site of the oocyte cloned M3 receptor at 277 in the open reading frame were amplified by polymerase chain reaction. Sequences of forward and reverse primers were 5′-GTGGTACCGTCACAATGACCTTGC-3′ and 5′-CGATGATCAGGTCTGCACAG-3′, respectively. The resulting fragment was subcloned into pCR-Blunt II-TOPO vector and was verified by sequencing on both strands using an ABI 310 automated DNA sequencer (Perkin-Elmer, Wellesley, MA). Then, the fragment was subcloned into pCR3 vector and ligated with the remaining coding region of the M3 receptor using the Bst PI/ Apa I sites. Next, the construct was subcloned into pCR3 vector (Invitrogen, Carlsbad, CA). cRNA for the M3 receptor was synthesized in vitro with T7 polymerase (Stratagene, La Jolla, CA) from cDNA linearized with Apa I. Bisindolylmaleimide I (GF109203X) was from Calbiochem (La Jolla, CA); HEPES was from Nacalai Tesque (Kyoto, Japan); collagenase was from Nitta Zerachin, (Osaka, Japan); tramadol hydrochloride was a kind gift from Nippon Shinyaku (Kyoto, Japan); [3H]QNB (48 Ci/mmol) was from Amersham (Buckinghamshire, England).
Isolation and microinjection of Xenopus oocytes were performed as described by Minami et al. (12,18). Xenopus oocytes were injected with 50 ng of cRNA encoding the M3 receptor, and electrophysiological recording was performed 2–5 days after injection. Oocytes were placed in a 100-μL recording chamber and perfused with modified Barth’s 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 MΩ) filled with 3 M of KCl were inserted into the animal pole. A Warner Oocyte-clamp OC 725-C was used to voltage clamp each oocyte at −70 mV. 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. (18). Tramadol was preapplied for 2 min to allow for complete equilibration in the bath.
To study whether the inhibitory effects of tramadol on ACh-induced currents are modulated by PKC, oocytes expressing M3 receptors were exposed to the PKC inhibitor GF109203X (200 nM) (19) in MBS for 120 min. After exposure to GF109203X, 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 M3 receptor, and electrophysiological recording was performed 2–5 days after the injection. A single oocyte was incubated for 30 min at 25°C with MBS (final volume, 1 mL) containing [3H]QNB (0.5 nM) in the presence or absence of tramadol. Sixteen oocytes were used for total binding and nonspecific binding at each concentration of tramadol (0.1–100 μM), respectively. After incubation, binding was terminated, and the oocyte was rapidly washed four times with 5 mL of ice-cold Krebs-Ringer phosphate buffer under vacuum through Whatman GF/C glass-fiber filters and placed in counting vials containing a scintillation cocktail. The radioactivity was counted in an Aloka LSC-3500E counter. 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 ± sem. 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 a t-test or a one-way analysis of variance. The half-maximal inhibitory concentration values (IC50) for concentration-response curves were estimated using GraphPad Prism software (GraphPad, San Diego, CA).
Anesthetic modulation of receptor function often depends on the degree of receptor activation (16), and it was required to determine the ACh concentration-response relationship under our experimental conditions before testing the anesthetics (Fig. 1). Nonlinear regression analysis of these curves yielded an 50% effective concentration for ACh of 1.0 ± 0.1 μM and a Hill coefficient of 1.1 ± 0.1. Maximal currents were observed at 100 μM (Fig. 1). Based on the results in Figure 1, the effects of anesthetics and tramadol on ACh-induced currents were examined at an ACh concentration of 1.0 μM.
In the Xenopus oocytes expressing cloned M3 receptors, 1 μM of ACh induced robust Ca2+-activated Cl– currents (936 ± 448 nA, n = 10) (Fig. 2A). Tramadol inhibited ACh-induced Ca2+-activated Cl– currents to 94.4% ± 12.8%, 77.9% ± 10.3%, and 69.4% ± 7.4% of control at 100 nM, 1 μM, and 10 μM, respectively (Fig. 2B). The IC50 of tramadol for the 1 μM of ACh-induced Cl– currents was 1.0 ± 0.1 μM (n = 8).
Isoflurane inhibits M3 receptor function via stimulation of PKC activity (14). Accordingly, we examined the effect of tramadol on M3 receptor-stimulated currents with oocytes that had been pretreated with the PKC inhibitor GF109203X. Treatment of oocytes expressing the M3 receptor with GF109203X for 180 min at 200 nM, which is a concentration 10 times larger than the Ki value for inhibiting PKC activity (19), enhanced the currents induced by 1 μM of ACh to 163.0% ± 15.0% at 120 min and to 140.0% ± 14.0% at 180 min (Fig. 3), respectively. However, the inhibitory effects of tramadol on ACh-induced currents were still observed after 120-min pretreatment with GF109203X (Fig. 4).
We further examined the effects of tramadol on the binding of [3H]QNB to oocytes expressing M3 receptors. Tramadol concentration dependently inhibited [3H]QNB binding to oocytes to 81.1% ± 14.7%, 71.0% ± 5.5%, and 52.6% ± 14.5% of the control value at 0.1, 1, and 10 μM, respectively (Fig. 5).
Our main finding in this study is that tramadol inhibited the ACh-mediated response of M3 receptors expressed in Xenopus oocytes, and this supports the concept that tramadol has inhibitory effects on muscarinic receptors. The concentration of tramadol in human serum is reported to reach 612.7 ± 221 ng/mL (approximately 2 μM) after IV injection of 100 mg of tramadol, which is the clinical dosage (20). In our study, tramadol inhibited the ACh-induced Cl– currents with an IC50 of 1.0 ± 0.1 μM. From these findings, tramadol should suppress the function of M3 receptors at clinically relevant concentrations.
This study raised the question of how tramadol inhibits M3 receptor-mediated responses. There is considerable evidence that PKC plays an important role in regulating the function of G-protein-coupled receptors. We reported that halothane, F3 (1-chloro-1,2,2-trifluorocyclobutane), and ethanol inhibited the function of the 5-hydroxytryptamine 2A (13) and M1 receptors (18), in a PKC-dependent manner. In addition, more recently, Do et al. (14) reported that PKC activation with phorbol-12-myrisate-13-acetate significantly decreased the signaling of M3 receptors, suggesting that activation of PKC inhibits M3 receptor function. In our experiments, GF109203X enhanced the ACh-induced currents mediated by M3 receptors, suggesting that PKC activation suppresses M3 receptor function, which is consistent with the report by Do et al. (14). However, GF109203X did not alter the inhibitory effects of tramadol on M3 receptor function. These findings suggest that PKC is not involved in the inhibitory effect of tramadol on M3 receptor function. Moreover, we previously reported that tramadol had few effects on AlF4−-induced Ca2+-activated Cl– currents, which suggests that tramadol does not interfere with steps in the pathway after G-protein-coupled signal transduction, such as activation of phospholipase C, release of intracellular Ca2+, and activation of Ca2+-activated Cl– channels (5). From these results, it is likely that the inhibitory effect of tramadol on the ACh-induced Cl– current is caused by other inhibitory mechanisms.
We next examined the effects of tramadol on [3H]QNB binding to M3 receptors expressed in Xenopus oocytes. Our finding that tramadol inhibited the specific binding of [3H]QNB suggests that it inhibits muscarinic receptor function by interacting with the ACh binding sites. Mutational analysis of the rat M3 receptor has shown that two Thr (Thr231 and Thr234) and four Tyr (Tyr148, Tyr506, Tyr529, and Tyr533) residues are critical for high-affinity ACh binding (21,22). Based on these studies and our present findings, it is possible to speculate that the binding site of tramadol is at one of these six residues in the M3 receptor. It is of interest to determine whether radioactive tramadol binds to M3 receptors that are mutated at these residues. Such studies are required to clarify the site of tramadol binding to M3 receptors and are now underway in our lab.
The role of brain muscarinic receptors in antinociception and analgesic action has been investigated. 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 (23). In contrast, inhibition of the muscarinic signaling pathway induced by the reduction of ACh levels, inhibiting its release, or administrating scopolamine in rat brains, decreases the minimal alveolar anesthetic concentration of inhaled anesthetics (24). Isoflurane depresses M3 receptor function (10,14). Moreover, the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibits the second phase of nociception induced by subcutaneous injection of formalin into the paw of mice by inhibiting presynaptic M3 muscarinic receptors in the spinal cord (15). Thus, the actions of analgesics or anesthetics on muscarinic receptors may be more complex than currently considered (8), and further studies are required to define the relationship between antinociception and muscarinic receptor function. However, in a clinical situation, tramadol sometimes causes a dry mouth and constipation (25), which may be caused by the inhibition of muscarinic receptors. Both glandular secretion and smooth muscle contraction are primarily mediated by stimulation of M3 receptors (13). The inhibitory effects of tramadol on M3 receptors might explain these pharmacological effects on the autonomic nervous system.
In conclusion, tramadol at clinically relevant concentrations inhibits M3 muscarinic receptor function by interfering with the QNB binding sites on the receptor. Our findings help to unveil the pharmacological basis of tramadol to better understand its neuronal action and anticholinergic effects.
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