effect of acute and chronic tramadol on [ 3h]-norepinephrine-uptake in rat cortical synaptosomes

ELSEVIER

proS Neuro-Psychophomawl (k EfioL Psychtat. 1999, Vol. 23, pp. 485496

Copyright 8 1999 Elsevier Science Inc.

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PI1 50278-5546(99)00010-X

EFFECT OF ACUTE AND CHRONIC TRAMADOL ON [3HJ-NOREPINEPHRINE-UPTAKE IN RAT CORTICAL SYNAPTOSOMES

DAVIDE FRANCESCHINI, MARIA LIPARTITI and PIETRO GIUSTI

Department of Pharmacology, University of Padua, Padua, Italy

(Final form, March 1999)

Abstract

Franceschini, Davide, Maria Lipartiti and Pietro Giusti: Effect of Acute and Chronic Tramadol on [3H]- Norepinephrine-Uptake in Rat Cortical Synaptosomes. Prog. Neuro-Psycopharmacol. and Biol. Psychiat., 1999, a, pp. 488496.01999 Elsevier Science Inc.

Tramadol hydrochloride is a centrally acting opioid analgesic whose efficacy and potency is only five to ten times lower than that of morphine. Opioid, as well as non-opioid mechanisms, may participate in the analgesic activity of tramadol. [3H]-NE uptake in isolated rat cortical synaptosomes was studied in the presence of tramadol, desipramine, methadone, and morphine. Desipramine and tramadol inhibited synaptosomal [3H]-

NE uptake with apparent Kis of 7.3 f 0.66 and 1.4’ f 0.0045 pM, respectively. Methadone was

active at a lo-fold higher concentration (Ki: 87 i 5.6 PM). In contrast, morphine essentially failed to inhibit [3H]-5-HT uptake (K,: 0.75 f 0.40 M). Methadone, morphine, and tramadol were active in the hot plate test with EDsos of 6.2, 9.3, and 40 mg kg-‘, respectively. [3H]-NE uptake was examined in synaptosomes prepared from rats 30 min after receiving a single dose of morphine, methadone or tramadol. Only tramadol(3 1 mg kg-‘, i.p.) decreased uptake of the transmitter, with an EDso equal to that in the hot plate test. Animals were chronically treated for 15 days with increasing doses of tramadol(20 to 125 rn? kg-‘, i.p.). Twenty-four hours after the last drug injection, a challenge dose of tramadol(40 mg kg- , 1.p.) was administered. Chronic tramadol was still able to reduce [3H]-NE uptake by 35%. These results further support the hypothesis that [3H]-NE uptake inhibition may contribute to the antinociceptive effects of tramadol. The lack of tolerance in [‘HI-NE uptake, together with the absence of behavioural alteration after chronic tramadol treatment proposes that tramadol holds potential over classical opioids in the treatment of pain disorders.

Kevwords: antinociception, [3H]-NE uptake, methadone, morphine, opioids, pain, tramadol.

Abbreviations: 5-hydroxytryptamine (5-HT), norepinephrine (NE).

486 D. Franceschini et al.

Introduction

Morphine is the milestone drug in the treatment of severe pain, In addition to its analgesic effects,

morphine possesses other relevant clinical actions, e.g. relief of anxiety and sleep promotion. Morphine is

considered to be the standard analgesic compound, although its pharmacological use produces a wide

range of unwanted side-effects. Moreover, morphine may not relieve certain types of pain, for instance,

neuropatic pain (McQuay, 1988).

Numerous synthetic and semisynthetic opioidergic compounds have been developed in order to expand

the analgesic properties of morphine while minimising its adverse effects. In particular, tramadol

hydrochloride exhibits an analgesic and antinociceptive potency that is five- to ten-fold lower than

morphine (Lehmann et al., 1990), being comparable to opioid compounds such as codeine, pentazocine,

and dextropropoxyphene (Hermies et al., 1988). Furthermore, tramadol presents an unique profile among

opioid analgesics. Its acute therapeutic use is not associated with sedation, constipation, and respiratory

depression (Mtiller-Limmroth and Krueger, 1978; Vogel et al., 1978; Lehmann et al., 1990). Long-term

clinical trials indicate that tramadol is almost devoid of abuse liability (Preston et al, 1991) or euphoric

effects (Huber, 1978).

Opioid affinity at p receptors parallels their antinociceptive potency in viva Tramadol-induced

antinociception, however, more closely approximates a model which includes not only its interaction with

opioid receptors (Friderichs et al., 1978; Hennies, 1988), but also its capability to inhibit central

monoaminergic uptake (Codd et al., 1995). In fact, naloxone only partially inhibited tramadol analgesic

activity (Kayser et al., 1991), whereas tramadol inhibited in vitro [3H]-1abeled norepinephrine (NE) and 5-

hydroxytryptamine (5HT) uptakes (Driessen and Reimann, 1992; Raffa et al., 1992, Reimann and

Hennies, 1994). Furthermore, it has been proven the acute and chronic administration in rats of

antinociceptive doses of tramadol produced an accumulation of [3H]-5-HT in isolated synaptosomes

(Giusti et al., 1997). On the other hand, the analgesia induced by tricyclic antidepressants may be related

to their inhibition of monoaminergic uptake (Sharov et al., 1987; Magni, 1991).

The present study was undertaken to ascertain the contribution of NE to the antinociceptive effects of

tramadol. [3H]-NE uptake accumulation was investigated after acute and chronic administration of

Tramadol inhibition of 13H)-NE uptake 487

tramadol in rats. Methadone, morphine, and desipramine were included for comparative purposes.

Methods

Animals

Adult male Sprague-Dawley rats (200-250 g) were obtained from Harlan-Nossan (S. Pietro Natisone,

UD, Italy) and housed under controlled temperature (23 = 2°C) and illumination (12 h light - 12 h dark)

(I 8 h 00 min - 00 h 00 min). Experiments were performed between IO h 00 min and 17 h 00 min.

Drugs and Solutions

( + )-Tramadol hydrochloride was a generous gift of Formenti S.p.A. (Milan, Italy). ( + )-Methadone

hydrochloride and ( f )-morphine hydrochloride were from S.A.L.A.R.S., S.p.A. (Como, Italy) and

desipramine was obtained from Sigma (St. Louis, MO, U.S.A.). [‘HI-NE was obtained from NEN (Boston,

MA, U.S.A.). All drug concentrations and doses refer to the free base. Compounds were dissolved in

isotonic saline and administered i.p. in a vol. of I ml kg-‘. All other reagents were of analytical grade.

Systemic Drug Treatment

Five animals were used for each acute treatment group, with a single i.p. injection of methadone,

morphine or tramadol, dissolved in saline. Drug doses chosen corresponded to their respective hot plate

EDSo. Methadone and morphine were also administered at their EDlnr (i.e., 37 and 70 mg kg-‘,

respectively). Control animals received saline only. Thirty minutes later animals were sacrificed and

synaptosomes prepared. Chronic treatment (eight animals per group) consisted of single daily injections

(i.p.) for 15 days. The initial dose of tramadol was 20 mg kg-‘. The tramadol doses were then increased

daily by 7.5 mg kg-’ until reaching 125 mg kg-‘. Control rats (n= 16) received daily injections of saline.

Seventy-two hours after the last tramadol or saline injection, 5 rats from each group were taken for

preparation of synaptosomes. The remaining rats chronically treated with methadone or tramadol (n = 5

per group) received, 24 h after the last injection. a challenge dose of tramadol (40 mg kg-‘). Tramadol (40

mg kg-‘) was likewise administered to chronic saline-treated rats. Animals were sacrificed 30 min later and

synaptosomal [‘HI-NE uptake assessed.

488 D. Franceschini et al.

Svnaptosomal Preparation

Brain synaptosomes were isolated according to Whittaker and Barker (1972). Rats were decapitated, the

brains rapidly excised, and the frontal cortex dissected out on ice. The tissue was homogenized in 10

volumes (w/v) of ice-cold 0.32 M sucrose and then centrifuged at 1800 g for 10 min (4°C). The pellet was

discarded and the supematant centrifuged at 17000 g for 60 min (4°C). The pellet was resuspended in a

small volume of 0.32 M sucrose, layered on a 0.8-1.2 M sucrose gradient and centrifuged at 37000 g for

60 min (4°C).

The synaptosomal fraction at the interface between 0.8 and 1.2 M sucrose was collected and further

centrifuged at 17000 g for 30 min. The pellet was resuspended in buffer (composition in mM: NaCl, 115,

KCl, 4.97, CaC12, 1, MgS04, 1.22, KH2P04, 1.2, NaHC03, 25, and glucose, 11.1, pH 7.4, plus 0.01 mM

pargyline) and immediately used. Protein content was determined according to Lowry et al. (1951) with

bovine serum albumin as standard.

r3H1-NE Uotake in Svnaptosomes

Synaptosomes (200-250 pg protein) were preincubated for 5 min at 37°C in the absence or presence of

different concentrations of drug. Uptake was started by addition of [3H]-NE and incubation continued for 6

min. Control samples were incubated at 0°C to evaluate membrane diffusion. The reaction was stopped by

cooling the tubes in ice. The samples were then filtered through Whatman GF/C glass fibre filters

(Whatman Inc., Clifton, NJ, U.S.A.) and washed twice with 150 mM Tris HCl, pH 7.4. Filter-bound

radioactivity was counted by liquid scintillation spectrometry with Filter Count (Packard). The difference

in [‘HI-NE accumulation at 37°C and 0°C was taken as a measure of active uptake. Ki ( f s.e. mean)

values were calculated by a computer-assisted curve fitting programme (EBDA) (McPherson, 1987).

Hot Plate Test

The hot plate test of Eddy and Leimbach (1953) was utilized. Rats (5 animals per group) were placed

individually on a plate maintained at 55 f 0.5”C by feedback from a surface-mounted thermocouple. The

response latency was evaluated on the basis of either hind paw lick or jump reaction, following contact

Tramadol inhibition of [3H]-NE uptake 489

with the plate. After three readings at 30 min intervals, response changes were assessed 30 min after i.p.

drug administration.

The percentage of antinociceptive maximal possible effect (MPE) was calculated from the formula:

%MPE = 100 (test latency-predrug latency)/(cut-off time of predrug latency) by use of the predrug

latency of each animal and a cut-off time of 45 s.

Data Analysis

EDs0 and ED9a (doses of drug giving 50% and 90% of MPE, respectively) in the hot plate test were

derived from the percentage protection data by Probit analysis according to Finney (1971). The confidence

limits were referred to P=O.O5. Uptake data were expressed as means f se. mean; differences between

means were determined by one way ANOVA followed by REGWF test. The probability level chosen was

PCO.05

J3H1-NE Uptake in Isolated Svnaptosomes

Results

Incubation of isolated rat cortical synaptosomes with tramadol, methadone, and desipramine produced a

concentration-dependent decrease in [3H]-NE uptake. Estimated Kis of 1.4 + 0.045, 7.3 f 0.66, and 87 f

5.6 pM were obtained for tramadol, desipramine, and methadone, respectively (Table 1). Conversely,

morphine did not influence the [3H]-NE uptake (Ki > 300,000 PM).

Hot Plate Test

The antinociceptive efficacy against thermal nociception of acute i.p. administered methadone,

morphine, and tramadol was next examined. The EDsos (mg kg“) were 6.2 (2.7 - 14), 9.3 (5.1 - 17) and 40

(27 - 53) for methadone, morphine, and tramadol, respectively (Table 2). The antinociceptive action of

tramadol was 6.4 (confidence limits: 3.2 - 12) and 4.2 (confidence limits: 1.6 - 11) times lower than for

methadone and morphine, respectively. The highest tramadol dose tested was 100 mg kg-‘.

490 D. Franceschini et cd.

Table 1

Drug-Induced Inhibition of [3H]-NE Uptake in Rat Cortical Synaptosomes

Drug Ki (PM)

Tramadol 1.4 * 0.045 Desipramine 7.3 * 0.66 Methadone 87 f 5.6 Morphine ~300,000

Data are means f s.e.means from three different experiments, each performed in triplicate. For experimental details, see Methods.

Table 2

Antinociceptive Effects of Methadone, Morphine and Tramadol in The Hot Plate Test

Methadone 6.2 (2.7 - 14) Morphine 9.3 (5.1 - 17) Tramadol 40 (27 - 53)

Compounds were administered (i.p.) 30 min prior to the test. Data are means with confidence limits in brackets (five animals per group). For experimental details see Methods.

Effects of Acute Drug Administration on 13H1-NE Untake

The capability of methadone, morphine, and tramadol to influence [3H]-NE uptake was investigated in

rat cortical synaptosomes after acute i.p. administration of the opioids. The doses chosen were derived

from the EDSOs in the hot plate test (Table 2). Methadone and morphine did not affect [3H]-NE uptake at

either lower (6.0 and 9.0 mg kg-‘, respectively) (EDso in the hot plate test), or higher doses (40 and 70 mg

kg-‘, respectively) (ED90 in the hot plate test). In contrast, tramadol at 40 mg/kg i.p. (ED50 in hot plate test)

significantly inhibited [3H]-NE uptake (73 f 5.4 %) (Table 3).

Effects of Chronic Administration of Tramadol on 13H1-NE Wake

Rats injected for 15 days with increasing doses of tramadol were sacrificed 72 hours after the last

administration. Uptake of [3H]-NE in cortical synaptosomes fiorn these rats did not differ from the

chronic-saline group. A challenge dose of tramadol (40 mg kg“, i.p.), given 24 hours after last drug

injection in rats chronically treated with showed a significantly reduced [3H]-NE uptake (Table 4). This

Tramadol inhibition of [3H]-NE uptake 491

reduction was not significantly different from that elicited by acute tramadol administration (Table 3).

Table 3

Effect of Acute Administration of Opioids on [‘HI-NE Uptake by Rat Cortical Synaptosomes

Drug Dose (mg kg-‘) [‘HI-NE uptake inhibition (%)

Methadone 6.0 98 * 5.7 Methadone 37(*) 95 + 5.3 Morphine 9.0 101 + 3.8 Morphine 70(*) 102 * 3.6 Tramadol 40 73 f 5.4”

Compounds were administered i.p. 30 min before the test. The doses used were either the ED50 or the ED,(*) in the hot-plate test, respectively. Values are means * s.e.mean (n = 5). Mean values having different superscript letters indicate significant differences (PcO.05) between groups. For experimental details see Methods.

Table 4

[3H]-NE Uptake in Cortical Synaptosomes from Tramadol Chronically- Treated Rats

Treatment group

% uptake

Drug deprivation Tramadol challenge

Saline 100 f 4.8” 75 l 3.gb

Tramadol 103 f 6.2” 65 f 7.3b

Saline and tramadol refer to groups of rats treated with single daily injections of saline and tramadol for 15 days, respectively. The starting dose of tramadol was 20 mg kg-’ i.p., increasing daily by 7.5 mg kg-’ until the animals received 125 mg kg-‘. Drug deprivation indicates animals killed 72 h following the last drug injection. Tramadol challenge denotes rats receiving a challenge dose of tramadol (40 mg kg-‘, i.p.) 24 h after the last chronic administration and killed 30 min later. Data are means f s.e.mean of 4 animals per group, and are expressed relative to the respective saline- treated groups (0.35 pmol NE mg protein-’ = 100 %). Mean values having different superscript letters indicate significant differences (P<O.Ol) between groups. For experimental details see Methods.

Behaviour

Relevant behavioural changes were observed in rats acutely treated with high doses of methadone (37

mg kg-‘) or morphine (70 mg kg-‘). The former appeared cataleptic and showed body rigidity, absence of

spontaneous movements, and a preserved righting reflex. Morphine produced sedation with a classical

492 D. Franceschini et al.

Straub tail reaction. Tramadol (100 mg kg-‘), given acutely produced only a slight sedation. Rats

chronically treated with tramadol were devoid of gross behavioural abnormalities.

Pharmacological Pronerties of Tramadol

Discussion

The pharmacological profile of tramadol, i.e. analgesia with weak classical opioid side effects, is

probably attributable to a mutual action between both the opioid and other systems involved in the control

of pain transmission, e.g. aminergic system. In the present study, tramadol produced a dose-dependent

antinociception in the hot plate test that was, not unexpectedly, 6.4- and 4.2-fold less potent than

methadone and morphine, respectively. These results are in agreement with the tail flick response in rats

(Hennies et al., 1988) and in clinical studies using patient-controlled analgesia (Lehmann et al., 1990).

Only a slight sedation was observed in acute and chronic tramadol-treated rats, unlike the typical

behavioural signs of opiate agonist administration, i.e. catalepsy and Straub tail phenomena. The latter

were seen in methadone- and morphine-treated rats. The above data concur with the low incidence of

tramadol side effects in humans (Huber, 1978; Barth et al., 1987).

Tramadol reportedly has low affinity for opioid receptors with dissociation constants (Ki) in the

micromolar range (Hennies et al., 1988), and either a lack of selectivity for p, K and 6 binding sites

(Henries et al., 1988) or some selectivity for p-receptors (Raffa et al., 1992). Despite a lOOO-fold lower

affinity for u-opiate receptors, tramadol has been found to induce antinociception with a potency only 5- to

1 O-fold less than that of morphine. While the analgesic efficacy of tramadol could be due to generation of

the active metabolite O-demethyl tramadol (Ml), the only tramadol metabolite with high affinity for p-

opioid receptors (K,: 0.22 nM) (Frink et al., 1996) seemed not to contribute to the tramadol analgesic

effect (Lee et al., 1993).

Analgesia and Aminergic Untake

Analgesia can also be produced by tricyclic antidepressants (amitriptyline or desipramine) (Sharav et

al., 1987; Magni, 1991), which inhibit 5-HT or NE uptakes, or adrenergic agonists (clonidine) (Yaksh,

Tramadol inhibition of [3H]-NE uptake 493

1985). Some synthetic non-phenantrene opioids (such as methadone and tramadol) were as potent as

desipramine in inhibiting [3H]-5-HT uptake when added to isolated rat cortical synaptosomes (Giusti et al.,

1997). However, only tramadol displayed 5-HT uptake inhibition and antinociceptive activity in a

correlative fashion at pharmacologically relevant doses.

Here, we report that tramadol was as potent as desipramine in inhibiting [‘HI-NE uptake when added to

rat isolated cortical synaptosomes. The other synthetic non-phenantrene opioid studied, i.e. methadone,

inhibited transmitter uptake only at 10 ~ 15-fold higher concentrations. Furthermore, in synaptosomes

prepared from rats acutely treated with equieffective doses (i.e., the EDsa in the hot plate test) of

methadone or tramadol, only the latter significantly reduced [3H]-NE uptake.

To investigate the influence of chronic tramadol administration on pharmacological responses, a

challenge dose of this drug was administered 30 min before synaptosomal preparation from chronically

drug-treated rats. In this experimental condition the challenge dose of tramadol, following chronic

administration of the drug, inhibited [3H]-NE uptake as effectively as when given acutely (Table 4).

Tramadol was further differentiated from classical opioids by its action in chronically-treated, drug-

deprived rats. NE uptake was not altered 72 hours after termination of chronic treatment with increasing

doses of tramadol. Clearly, under the present experimental conditions neither withdrawal nor a

compensatory increase in NE uptake occur after chronic tramadol.

Role of NE in Analgesia

The role of NE in analgesia is controversial. NE may increase the analgesia produced by systemic

opioids by activation of the bulbospinal noradrenergic pathways (Xu et al., 1997). Increased NE release in

the spinal cord might contribute to the inhibitory effects of morphine through a synergistic interaction

between opioid and adrenergic receptors. For instance, the potency of morphine can be attenuated by

spinal administration of adrenergic antagonists, presumably by blocking the action of endogenously

released NE (Yaksh, 1979). However, NE might also produce hyperalgesia via an az-adrenergic receptor

mechanism. In this regard the as-adrenergic agonist clonidine and the more selective az-agonist UK 14304

were reportedly analgesic (Khasar et al., 1995), whereas administration of the a2antagonist yohimbine

494 D. Franceschini et al.

produced hyperalgesia and decreased the antinociceptive effects of opiates (Raffa et al., 1991). These data

propose that the role of NE in analgesia may be due, at least in part, to participation of different a2 receptor

subtypes. For instance, a2a receptors mediates NE hyperalgesia whereas the a2c receptor might be

responsible of the antinociceptive effect of a2 agonists (Khasar et al., 1995). Differential coupling to

second messenger systems and cell-type specific localization can contribute to the observed complexity of

responses. In light of these data it is hard to understand whether tramadol-induced increase in NE can

favour or attenuate tramadol-induced analgesia produced through a different mechanism. Further studies

are in progress to address this issue.

Conclusions

The antinociceptive action of tramadol is concomitant with effects on the serotoninergic and

noradrenergic systems. Together with its opioid activity, these effects may indeed participate in tramadol-

induced analgesia, thus differentiating its molecular mechanism of action from that of pure opioids - as

suggested by the absence of noticeable opioid behavioural signs. The lack of tolerance in 5-HT and NE

uptakes, and the absence of behavioural alterations after chronic tramadol treatment suggest that this drug

may provide superior therapeutic management of long-term pain in comparison to other opioid analgesics

like methadone and morphine.

Acknowledgement

We thank Dr. Stephen D. Skaper for critically reading this manuscript.

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Inquires and reprint requests should be addressed to:

Prof. Pietro Giusti Department of Pharmacology University of Padua L.go Meneghetti, 2 35 13 1 Padova, Italy Tel +39-49-827-5 103 Fax +39-49-827-5093 Email piusti@uxl .unind.it