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Anesth Analg 2002;94:901-906
© 2002 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Tramadol Inhibits Norepinephrine Transporter Function at Desipramine-Binding Sites in Cultured Bovine Adrenal Medullary Cells

Kenichiro Sagata, MD*, Kouichiro Minami, MD, PhD*, Nobuyuki Yanagihara, PhD{dagger}, Munehiro Shiraishi, MD*, Yumiko Toyohira, PhD{dagger}, Susumu Ueno, MD, PhD{dagger}, and Akio Shigematsu, MD, PhD*

Departments of *Anesthesiology and {dagger}Pharmacology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan

Address correspondence and reprint requests to Kouichiro Minami, Department of Anesthesiology, University of Occupational and Environmental Health, School of Medicine, 1-1, Iseigaoka, Yahatanishiku, Kitakyushu, 807-8555, Japan. Address e-mail to kminami{at}med.uoeh-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Tramadol is a widely used analgesic, but its mode of action is not well understood. To study the effects of tramadol on norepinephrine transporter (NET) function, we assayed the effect of tramadol on [3H]-norepinephrine ([3H]-NE) uptake and [3H]-desipramine binding to plasma membranes isolated from bovine adrenal medulla. We then characterized [14C]-tramadol binding in cultured bovine adrenal medullary cells. Tramadol inhibited the desipramine-sensitive uptake of [3H]-NE by the cells in a concentration-dependent manner (50% inhibitory concentration = 21.5 ± 6.0 µM). Saturation analysis revealed that tramadol increased the apparent Michaelis constant of [3H]-NE uptake without changing the maximal velocity, indicating that inhibition occurred via competition for the NET (inhibition constant, Ki = 13.7 µM). Tramadol inhibited the specific binding of [3H]-desipramine to plasma membranes. Scatchard analysis of [3H]-desipramine binding revealed that tramadol increased the apparent dissociation constant (Kd) for binding without altering maximal binding, indicating competitive inhibition (Ki = 11.2 µM). The binding of [14C]-tramadol to the cells was specific and saturable, with a Kd of 18.1 ± 2.4 µM. These findings indicate that tramadol competitively inhibits NET function at desipramine-binding sites.

IMPLICATIONS: Tramadol competitively inhibits norepinephrine transporter function at desipramine-binding sites in the adrenal medullary cells and probably the noradrenergic neurons of the descending inhibitory system.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Tramadol [(1R, 2R) and (1S, 2S)-2-dimethylamino- methyl-1-(3-methoxyphenyl)-cyclohexanol hydro chloride] is an analgesic used for the management of intraoperative pain, postoperative pain, acute pain syndromes, and chronic pain (1). It binds to µ-opioid receptors with approximately 100 times less affinity than morphine (2), and this suggests that the antinociceptive action of tramadol may be due to mechanisms other than opioid receptor binding.

Pain perception is modulated by a variety of neurotransmitters, including endogenous norepinephrine (NE) (3). Several lines of evidence have shown that tramadol inhibits the reuptake of monoamines, as do antidepressant drugs such as desipramine. Tramadol inhibits the reuptake of NE and serotonin in rat brain cortical slices (4) and rat cortical synaptosomes (5). Thus, tramadol seems to exert its analgesic effect, at least in part, by modulating NE transmission in the brain via inhibition of NE transporters (NETs), although the inhibitory mechanism by tramadol of NE reuptake in the brain tissue remains to be determined.

NETs located in the presynaptic membranes of noradrenergic nerve terminals regulate neurotransmission by taking up NE released into the synaptic cleft (6). Pacholczyk et al. (7) cloned the human NET and reported that its messenger RNA is expressed at high levels in the brainstem and the adrenal medulla. The complementary DNA of bovine adrenal medullary NET cloned by Lingen et al. (8) encodes an amino acid sequence that is approximately 94% identical to that of human NET, and the pharmacological properties of bovine adrenal medullary NET are quite similar to those of central noradrenergic neurons (9). Therefore, bovine adrenal medullary cells are useful models for studying the effects of IV anesthetics on NETs in noradrenergic neurons (10).

In this study, we investigated the mechanism by which tramadol inhibits NET function by assessing the effects of tramadol on [3H]-NE uptake. Moreover, to clear the binding site of tramadol on NET, we studied the effects of tramadol on [3H]-desipramine binding and specific binding sites for [14C]-tramadol on the bovine adrenal medullary NET.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The cells were isolated by collagenase digestion of bovine adrenal medulla slices as described previously (11). The cells were plated at a density of 4 x 106 cells per dish (Falcon 35mm, Becton Dickinson Labware, Franklin Lakes, NJ) in Eagle’s minimum essential medium containing 10% calf serum, 60 µg/mL aminobenzylpenicillin, 100 µg/mL streptomycin, 0.3 µg/mL amphotericin B, and 3.0 µM cytosine arabinoside. The cells were cultured in 5% CO2 and 95% air in an incubator at 37°C and used for experiments after 2 to 4 days of culture.

The cultured cells were incubated at 37°C for 15 min in Krebs-Ringer-HEPES (KRH) buffer containing 100 µM pargyline, 100 µM ascorbic acid, and 500 nM [3H]-NE in the presence or absence of various concentrations of tramadol (0.1–100 µM). KRH buffer consisted of 154 mM NaCl, 5.6 mM KCl, 1.1 mM MgSO4, 2.2 mM CaCl2, 10 mM HEPES, and 10 mM glucose, adjusted to pH 7.4. After incubation, the cells were rapidly washed four times with 1 mL of ice-cold KRH buffer and solubilized in 1 mL of 10% Triton X-100 (Nacalai Tesque, Kyoto, Japan). The radioactivity in the solubilized cells was measured with a liquid scintillation counter (LSC-3500E; Aloka, Tokyo, Japan). To determine saturation kinetics of [3H]-NE uptake, various concentrations (1–30 µM) of [3H]-NE were added in the presence or absence of 10 µM tramadol or 10 µM desipramine, a selective inhibitor of the NET. After incubation, the radioactivity in the solubilized cells was measured by liquid scintillation counting. Nonspecific uptake was determined in the presence of 10 µM desipramine, and the specific [3H]-NE uptake was obtained by subtracting the nonspecific uptake (determined in the presence of desipramine) from the total uptake.

Plasma membranes isolated from bovine adrenal medulla were prepared as described previously (10). The membranes (20 µg protein) were resuspended in 10 mM Tris-HCl (pH 7.4), 135 mM NaCl, 5 mM KCl, and 1 mM MgSO4 (buffer B). The membrane suspension was incubated in a final reaction volume of 250 µL for 30 min at 25°C with [3H]-desipramine (1–24 nM) in the presence or absence of 10 µM tramadol or 10 µM nisoxetine, a selective inhibitor of the NET. After incubation, binding was terminated by the addition of 2 mL of ice-cold buffer B, followed by rapid vacuum filtration of the membrane suspension through Whatman GF/C glass fiber filters (Whatman, Maidstone, UK). The filters were rapidly washed twice with 2 mL of ice-cold buffer B, and the radioactivity retained on the filters was determined by liquid scintillation counting. Specific binding of [3H]-desipramine was defined as the binding inhibited by nisoxetine.

The cells that had been cultured for 3 days were dispersed by 0.05% trypsin, and isolated cells were resuspended in KRH buffer at a cell density of 106 cells per 200 µL. The cells were then incubated for 30 min at 4°C, either with varying concentrations of [14C]-tramadol (5–60 µM) or with desipramine (10 and 30 µM), atropine (10 µM), hexamethonium (10 µM), or naloxone (10 µM), in the presence of [14C]-tramadol (5 µM). After incubation, the cells were centrifuged for 2 min at 10,000 rpm through a 1:1 mixture of dinonyl phthalate and silicon oil. The bottom of the tube containing the cell pellet was cut off, and the cells were solubilized in 400 µL of 10% Triton X-100. The radioactivity in the solubilized cells was measured by liquid scintillation counting. Nonspecific binding was determined in the presence of 1 mM unlabeled tramadol. Specific binding was obtained by subtracting the nonspecific binding from the total binding.

Eagle’s minimum essential medium was obtained from Nissui Pharmaceuticals (Tokyo, Japan). Calf serum, l-norepinephrine, pargyline hydrochloride, l-ascorbic acid, atropine sulfate, and HEPES were obtained from Nacalai Tesque (Kyoto, Japan). Collagenase was obtained from Nitta Zerachin (Osaka, Japan). Desipramine hydrochloride, hexamethonium bromide, and naloxone hydrochloride hydrate were obtained from Sigma (St. Louis, MO). Nisoxetine hydrochloride was obtained from Research Biochemicals International (Natick, MA). L-[7,83H]-norepinephrine (34.0 Ci/mmol) was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). [Benzene ring 10,11-3H]-desmethylimipramine (desipramine) hydrochloride (73.0 Ci/mmol) was obtained from New England Nuclear (Boston, MA). Trypsin was obtained from Difco Laboratories (Detroit, MI). Tramadol hydrochloride and [14C]-tramadol (57.9 mCi/mmol) were donated by Nippon Shinyaku (Kyoto, Japan).

The kinetic variables for [3H]-NE uptake (the apparent Michaelis constant [Km] and maximal velocity [Vmax]) were estimated by Eadie-Hofstee analysis. The kinetic variables for [3H]-desipramine and [14C]-tramadol binding (the apparent dissociation constant [Kd] and maximal binding [Bmax]) were estimated by Scatchard analysis. All values are expressed as mean ± SD. Statistical evaluations were accomplished by one-way analysis of variance. When a significant P value was found by analysis of variance, the Scheffé test for multiple comparisons was performed to identify differences among the groups. Differences were considered to be statistically significant when P < 0.05. Curve fitting and estimation of the 50% inhibitory concentration (IC50) value for concentration-response curves were performed with Prism version 3.0.2 (GraphPad Software, San Diego, CA).


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Tramadol (1–100 µM) inhibited [3H]-NE uptake by the cells in a concentration-dependent manner (Fig. 1). The uptake of [3H]-NE was significantly reduced to 90.6% ± 2.4%, 74.5% ± 3.2%, and 17.1% ± 2.1% of the control value at 1, 10, and 100 µM tramadol, respectively. The IC50 was 21.5 ± 6.0 µM (Fig. 1). Incubation of the cells with increasing concentrations of [3H]-NE (1–30 µM) showed that [3H]-NE uptake was saturable (Fig. 2A). Eadie-Hofstee analysis yielded a Vmax of 329 ± 74 pmol/4 x 106 cells per 15 min and a Km of 5.5 ± 1.6 µM in control cells (Fig. 2B). Tramadol (10 µM) produced an increase in Km (9.5 ± 2.3 µM; P < 0.05) with no change in Vmax (342 ± 84 pmol/4 x 106 cells per 15 min), indicating competitive inhibition (Fig. 2B). The inhibition constant (Ki) of 13.7 µM was calculated from the shift in the Km value. This value is similar to the Ki of 19.7 µM calculated from mean tramadol IC50 values by using the equation of Cheng and Prusoff (12).



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  		Figure 1. Effects of various concentrations of tramadol on desipramine-sensitive [3H]-norepinephrine ([3H]-NE) uptake by the cells. The cells were incubated with 500 nM [3H]-NE and various concentrations of tramadol (0.1–100 µM) for 15 min at 37°C. [3H]-NE uptake is expressed as a percentage of control values. Data shown are means ± SD of five independent experiments performed in duplicate. *P < 0.05, **P < 0.01 compared with control.

 


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  		Figure 2. Titration of desipramine-sensitive [3H]-norepinephrine ([3H]-NE) uptake by the cells. A, The cells were incubated with various concentrations (1–30 µM) of [3H]-NE in the presence or absence of 10 µM tramadol for 15 min at 37°C. Data shown are means ± SD of five independent experiments performed in duplicate. B, Eadie-Hofstee analysis of the data shown in A.

 
To determine the NET site of tramadol action, we examined the effects of tramadol on [3H]-desipramine binding to the membranes. Specific binding of [3H]-desipramine was saturable (Fig. 3A). A single population of binding sites was indicated by Scatchard analysis. The Kd was 8.3 ± 1.3 nM, and the Bmax was 2.6 ± 0.2 pmol/mg protein in control cells (Fig. 3B). Tramadol (10 µM) significantly increased the Kd for [3H]-desipramine binding (15.8 ± 4.0 nM; P < 0.05) without any change in Bmax (2.6 ± 0.3 pmol/mg protein), indicating competitive inhibition (Ki = 11.2 µM).



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  		Figure 3. Specific binding of [3H]-desipramine to the membranes. A, The membranes (20 µg of protein) were incubated with increasing concentrations of [3H]-desipramine (1–24 nM) for 30 min at 25°C in the presence or absence of nisoxetine to determine nonspecific binding and, where indicated, in the presence of 10 µM tramadol. Data shown are means ± SD of specific binding of five independent experiments performed in duplicate. B, Scatchard analysis of the data shown in A.

 
We next characterized tramadol binding to the cells by incubation with increasing concentrations (5–60 µM) of [14C]-tramadol. Specific binding of [14C]-tramadol was saturable (Fig. 4A). Scatchard analysis revealed that the binding site had an apparent Kd of 18.1 ± 2.4 µM and a Bmax of 1.5 ± 0.1 nmol/106 cells (Fig. 4B). Specific binding of [14C]-tramadol was reduced to 79% ± 5% (P < 0.05) and 60% ± 5% (P < 0.01) of the control value at 10 and 30 µM desipramine, respectively. The incomplete inhibition by a large concentration of desipramine indicates that [14C]-tramadol may also bind to sites other than those on the NET. According to previous reports, tramadol binds to µ-opioid receptors (2), and it has been reported that µ-type opioid receptors are found in adrenal medullary cells (13). We next studied the effect of naloxone on [14C]-tramadol binding to the cells. The specific binding of [14C]-tramadol was partially inhibited by naloxone to 82% ± 10% (P < 0.05 versus control binding of [14C]-tramadol). Moreover, muscarinic and nicotinic receptors are found in adrenal medullary cells (11,14). Atropine (10 µM) caused partial inhibition to 86% ± 7% of control binding (P < 0.05). Moreover, specific binding of [14C]-tramadol was also partially inhibited to 84% ± 8% by hexamethonium (10 µM), a nicotinic acetylcholine receptor antagonist (P < 0.01 versus control binding). This is in agreement with previous findings that adrenal medullary cells express muscarinic and nicotinic acetylcholine receptors (11,14).



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  		Figure 4. Specific binding of [14C]-tramadol to the cells. A, The cells were incubated with various concentrations of [14C]-tramadol (5–60 µM) for 30 min at 4°C. Nonspecific binding was determined in the presence of 1 mM unlabeled tramadol. Data shown are means ± SD of specific binding of four independent experiments performed in duplicate. B, Scatchard analysis of the data shown in A.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we demonstrated that tramadol inhibited NET function in the cells. These findings are consistent with previous reports using rat brain cortex (4,5). According to the report by Lintz et al. (15), the tramadol concentration in human serum reaches approximately 600 ng/mL (approximately 2 µM) after IV injection of a 100-mg clinical dose. In this study, tramadol at 1 and 10 µM significantly inhibited NE uptake, reducing it to 90.6% ± 2.4% and 74.5% ± 3.2% of the control value, respectively. As much as 80%–90% of NE released from presynaptic terminals is believed to be physiologically taken up again by the neuron, thereby terminating neurotransmission (16). Therefore, even a slight inhibition of NET activity by clinical concentrations of tramadol may enhance noradrenergic neurotransmission. According to these findings, clinically relevant concentrations of tramadol probably partly suppress the NET function.

To examine the NET site of tramadol action, we studied the effects of tramadol on kinetic variables for [3H]-NE uptake and [3H]-desipramine binding. Tramadol increased the Km for [3H]-NE uptake without altering the Vmax, indicating competitive inhibition. We also examined the effects of tramadol on [3H]-desipramine binding to the membranes isolated from bovine adrenal medulla. Tramadol significantly increased the Kd for [3H]-desipramine binding without changing Bmax, suggesting that tramadol competitively inhibits [3H]-desipramine binding. The 11.2 µM Ki for tramadol calculated from this shift in the Kd is almost identical to the Ki for tramadol inhibition of NE uptake.

The NET substrate recognition site has been controversial over the past 10 to 15 years; the debate centers on whether the NE substrate recognition site is identical to the tricyclic antidepressant-binding site (17). From molecular studies of monoamine transporters, evidence has emerged that there are distinct but overlapping regions within NET molecules that determine substrate recognition, translocation, and antagonist affinity (18,19). In our study, the competitive inhibition of [3H]-NE uptake and [3H]-desipramine binding by tramadol suggests that tramadol may bind to a region that overlaps the sites responsible for NE recognition and binding of antidepressants. More recently, Roubert et al. (18) reported that two residues located in transmembrane domains (TMDs) 6 and 7 of the human NET may play an important role in tricyclic antidepressant interaction and that a critical region in TMD 8 is likely to be involved in the tertiary structure allowing high-affinity binding of tricyclic antidepressants. It will be interesting to determine whether mutation of some residues in TMD 6, TMD 7, or TMD 8 of NET abolishes tramadol action. Such studies are necessary to identify the tramadol-binding site.

In this study, we pharmacologically characterized the binding of tramadol to the cells by use of [14C]-tramadol. We demonstrated the presence of specific and saturable binding of [14C]-tramadol with a Kd of 18.1 ± 2.4 µM. However, only approximately 40% of [14C]-tramadol binding was inhibited by a large concentration (30 µM) of desipramine. Here, the question arises as to why such a large concentration of desipramine did not completely inhibit [14C]-tramadol binding. One possibility may answer this question: i.e., there would be another binding site of [14C]-tramadol that has an affinity similar to that to NET. The cells express several receptors for acetylcholine and opioids such as muscarinic, nicotinic (11,14), and µ-opioid receptors (13). In this study, we examined the effects of various antagonists for these receptors on [14C]-tramadol binding. Atropine, hexamethonium, and naloxone, used as antagonists for muscarinic, nicotinic, and µ-opioid receptors, respectively, each caused approximately 15% inhibition of [14C]-tramadol-specific binding. This suggests that [14C]-tramadol may also, in part, bind to muscarinic, nicotinic, and µ-opioid receptors. Recently, the role of brain muscarinic and nicotinic acetylcholine receptors in antinociception and analgesic actions has been investigated (20,21). In our laboratory, we have studied the effects of tramadol on acetylcholine receptors. Tramadol suppressed the function of nicotinic receptors in the cells (22) and M1 muscarinic receptors expressed in Xenopus oocytes (23). Although more detailed studies are necessary, it would be of interest to study whether muscarinic and nicotinic receptors are one of the sites of antinociception exerted by tramadol.

Several lines of evidence have shown that the descending inhibition system consists of noradrenergic neurons (24). Tricyclic antidepressants such as desipramine are used in clinical practice to treat chronic pain (25). Their antinociceptive effects are partly explained by an enhancement of noradrenergic neurotransmission by NET suppression of the descending inhibitory system of the brain and spinal cord (26). More recently, Bohn et al. (27) reported that the tricyclic antidepressant desipramine enhanced morphine-induced analgesia in wild-type mice, but not in NET knockout mice, suggesting that the antinociceptive effect of desipramine is primarily caused by NET blockade. Our findings provide further evidence in support of NET inhibition as the basis for the antinociceptive effect of tramadol. Indeed, the spinal antinociceptive effects of tramadol are antagonized by yohimbine, an {alpha}2-adrenoreceptor blocker (28). These studies are in accordance with our findings that tramadol has pharmacological properties that are very similar to those of desipramine. To clarify the hypothesis, the animal study with intrathecal tramadol administration is currently under way.

In conclusion, our results suggest that clinically relevant concentrations of tramadol inhibit NET function by blocking desipramine-binding sites.


   Acknowledgments
 
This research was supported by grants-in-aid (11671532, 10770778, 12770851, 11770878, 12671515, 12671516, and 12770849) from the Ministry of Education, Science, and Culture of Japan, a UOEH Research Grant for Promotion of Occupational Health, the Japan Research Foundation for Clinical Pharmacology, the Uehara Memorial Foundation, and the Kanehara-Ichiro Memorial Medical Foundation.

We thank Dr. Heinz Bönisch (Department of Pharmacology and Toxicology, University of Bonn, Germany) for kind discussion and technical suggestions.


   Footnotes
 
Presented in part at the annual meeting of the American Society of Anesthesiologists, San Francisco, CA, October 16, 2000.


   References
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 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication December 11, 2001.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2002 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press