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Release studies with rat brain cortical synaptosomes indicate that tramadol is a 5-hydroxytryptamine uptake blocker and not a 5-hydroxytryptamine releaser

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Abstract

Tramadol is a centrally acting opioid analgesic whose mechanism of action could also involve an increase in central serotoninergic transmission. Thus, tramadol inhibits synaptosomal serotonin (5-hydroxytryptamine, 5-HT) reuptake and induces tritium release from [3H]5-HT-preloaded slices. We investigated the effect of (±)-tramadol in release studies with superfused rat brain cortex synaptosomes preloaded with [3H]5-HT. Tramadol had no releasing effect up to 30 μM, whereas at 10 μM tramadol significantly inhibited by 45% d-fenfluramine-induced [3H]5-HT release. At 100 μM, tramadol showed a slight releasing effect in the absence or in the presence of pargyline, which was not augmented in synaptosomes pre-exposed to Ro 04-1284 (2-ethyl-1,3,4,6,7,11b-hexahydro-3-isobutyl-9,10-dimethoxy-2H-benzo [a]quinolizin-2-ol hydrochloride), a reserpine-like compound that enhances cytoplasmic 5-HT levels. In summary, (±)-tramadol behaved as a classical 5-HT uptake blocker (like citalopram) and not as a substrate of the 5-HT carrier with indirect 5-HT mimetic properties (like d-fenfluramine).

Introduction

Tramadol is a centrally acting opioid analgesic with affinity for μ-opioid receptors in the low micromolar range (Hennies et al., 1988; Raffa et al., 1992). It was, however, suggested that an increase in monoaminergic transmission might also contribute to its antinociceptive effects (Driessen and Reimann, 1992; Raffa et al., 1992; Giusti et al., 1997). Thus, it was shown that tramadol inhibited the uptake of serotonin (5-hydroxytryptamine, 5-HT) and noradrenaline (but not dopamine) into rat brain synaptosomes in the low micromolar range (Driessen and Reimann, 1992; Raffa et al., 1992; Driessen et al., 1993; Giusti et al., 1997). In release studies with [3H]noradrenaline-preloaded slices, tramadol (mainly the (−) enantiomer) mimicked the effects of cocaine, suggesting that it is a classical noradrenaline uptake blocker (Driessen et al., 1993). However, similar studies with [3H]5-HT-preloaded slices suggested that tramadol (mainly the (+) enantiomer) is a substrate of the 5-HT carrier with 5-HT-releasing activity, thus resembling an indirect 5-HT mimetic agent (like d-fenfluramine) more than a classical 5-HT uptake blocker (Driessen and Reimann, 1992; Reimann and Schneider, 1998). In particular, these authors found that tramadol was active in inducing tritium release at concentrations as low as 1–10 μM and that this effect was antagonized by 6-nitro-quipazine, a well-known 5-HT uptake blocker.

In the present study, we reconsidered this proposed mechanism by studying the effects of (±)-tramadol in release studies with superfused [3H]5-HT-preloaded synaptosomes. With this experimental approach, and differently from [3H]5-HT-preloaded slices, the neurotransmitter released by the drug is immediately removed and cannot be retaken up (Raiteri et al., 1974; Gobbi et al., 1992). Thus, the releasing effect is completely distinct from uptake inhibition. This is particularly important when studying the effects of those compounds that are believed to act both as releasers and reuptake inhibitors (Crespi et al., 1997). Moreover, the previous studies were carried out in the presence of pargyline (Driessen and Reimann, 1992; Reimann and Schneider, 1998), which is likely to increase the cytoplasmic 5-HT concentration (Fitzgerald and Reid, 1993; Reimann and Schneider, 1998). In order to have data more related to the physiological situation, we carried out these experiments in the absence of the monoamine–oxidase inhibitor, so that 5-HT was probably mainly stored in synaptic vesicles.

Section snippets

Experimental procedure

Male CRL:CD(SD)BR rats (Charles River, Italy) weighing about 150 g were used. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L. n. 116, G.U., suppl. 40, Feb. 18, 1992) and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, Dec.12, 1987; Guide for the Care and Use of Laboratory Animals, US National Research Council, 1996).

The rats were killed by decapitation and their

Results

In the absence of pargyline, (±)-tramadol showed no releasing activity up to 30 μM while at 100 μM it induced a very low release of tritium (2.2±1.4%, mean±S.E., n=3 experiments), much lower than the release induced by 5 μM d-fenfluramine (15.2±1.2%) (Fig. 1A). The classical 5-HT uptake blocker citalopram, tested at a concentration (1 μM) which should completely inhibit 5-HT transport, had no releasing activity (Fig. 1A), thus confirming that, with the superfusion apparatus used the released

Discussion

It was previously described that (±)-tramadol inhibits synaptosomal [3H]5-HT uptake in the low micromolar range (1-3 μM) (Driessen and Reimann, 1992; Raffa et al., 1992; Driessen et al., 1993; Giusti et al., 1997). This effect could be due to a citalopram-like effect (i.e., an action as a pure 5-HT uptake blocker) or to a d-fenfluramine-like effect (i.e., an action as a 5-HT carrier substrate). The present data, obtained using [3H]5-HT preloaded synaptosomes, support the former possibility.

Acknowledgements

The technical assistance of Mr. Fabio Dalla Valle and Ms. Manuela Moia was greatly appreciated. We thank Dr. E. Borroni (Hoffmann-La Roche, Basel, Switzerland) for the generous gift of Ro 04-1284 and Dr. Monica Casiraghi for the revision of the English style.

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