q Birkhäuser Verlag, Basel, 1999
Inflamm. res. 48 (1999) 120–127
1023-3830/99/030120-08 $ 1.50+0.20/0
Original Research Papers
The potentiation of analgesic activity of paracetamol plus morphine
involves the serotonergic system in rat brain
M. Sandrini1, G. Vitale2, A. Ottani1 and L.-A. Pini2
1
Dept of Biomedical Sciences, Section of Pharmacology, University of Modena, Italy
2
Clinical Pharmacology Unit, Dept. of Internal Medicine, University of Modena, Via del Pozzo, 71, I-41100 Modena, Italy, Fax þ39 59 424069,
e-mail: pinila@unimo.it
Received 7 July 1998; returned for revision 31 August 1998; accepted by K. Brune 2 November 1998
Abstract. Objective and Design: We investigated the
antinociceptive effect of subactive doses of paracetamol
and morphine, given in combination.
Material and Treatment: Male Wistar rats were injected with
paracetamol (50 or 100 mg/kg i.p.) and morphine (2, 3 or 5
mg/kg s.c.) 10 min later and subjected to algesimetric tests
20 min thereafter.
Methods: Pain threshold was evaluated in the hot-plate and
formalin tests. 5-HT2 receptor binding capacity and 5-HT
and 5-HIAA levels were measured in cortical and pontine
areas of the brain by means of radioligand binding technique
and by HPLC, respectively. Statistical analysis was done
using Student-Neuman-Keul’s test and 2 × 2 factorial
analysis.
Results: Only when given in combination, paracetamol (100
mg/kg) and morphine (2 and 3 mg/kg) were able to evoke an
antinociceptive effect in both tests associated with an increase
in 5-HT levels and a decrease in 5-HT2 receptors in the cortex.
These effects were prevented by i.p. pretreatment with
naloxone (1 mg/kg i.p.).
Conclusions: Subactive doses of paracetamol and morphine
exert an analgesic effect when given in combination in the rat
and indicate an involvement of both serotonergic and
opiatergic systems.
Key words: Paracetamol – Morphine – Antinociception –
5-Hydroxytryptamine (5-HT) – 5-HT2 receptors – Brain
Introduction
The mechanism of action of non-steroidal anti-inflammatory
drugs (NSAIDs) is believed to result from suppression of
Correspondence to: L.-A. Pini
m
Inflammation Research
prostaglandin synthesis owing to cyclooxygenase inhibition.
However, whereas the anti-inflammatory potency of
NSAIDs can be correlated with their inhibition of cyclo-
oxygenase, the relationship between the antinociceptive
activity of these drugs and their capacity for cyclooxygenase
inhibition is controversial [1–3]. It has been observed that
phenazone and paracetamol significantly inhibit prostaglan-
din synthesis in the central nervous system (CNS), and some
findings suggest that paracetamol has a weak inhibitory
activity on the synthesis of peripheral prostaglandin, but is a
potent inhibitor of prostaglandin biosynthesis within the
CNS [4, 5]. Recently the description of two structurally
different forms of cyclooxygenase enzyme (COX-1 and
COX-2) indicated a possible explanation for the different
analgesic and anti-inflammatory activities of many NSAIDs
[6], and inhibition of COX-2 represents for some authors the
most likely mechanism of action for NSAID-mediated
analgesia [7, 8], mainly at spinal level as for indomethacin
[9] or peripherically as for meloxicam [10].
Other authors have demonstrated that paracetamol has
only a weak antinociceptive effect when injected intracer-
ebroventricularly (i.c.v.) in rats [11]. This lack of activity
may result from its inability to inhibit prostaglandin
synthesis in rat CNS tissue [12].
The central monoaminergic and serotonergic pathways
are involved in pain modulation [13] and a possible
connection between the analgesia induced by some
NSAIDs and the increase in the turnover rate of dopamine,
noradrenaline and serotonin in the rat CNS has been
proposed [14, 15]. It has been proposed that many types of
analgesic drugs (opiates, NSAIDs, etc) act through an
increase in brain serotonin levels. The serotonergic system
can regulate nociception in different ways, depending on the
receptor subtypes involved, and serotonin has been claimed
to exert its central antinociceptive effect in defined brain
areas through its receptor subtypes, notably 5-HT1A [16],
5-HT2 [17] and 5-HT3 at the spinal level [18].
Vol. 48, 1999 Potentiation of analgesic activity of paracetamol plus morphine
The bulk of the data suggests that stimulation of 5-HT1
receptors reduces nociceptive sensitivity, whereas activation
of 5-HT2 receptors increases nociceptive responsiveness
after long term treatment [19].
The serotonergic system can play a role in the
antinociceptive mechanism of some NSAIDs; it has been
also proposed that other neurotransmitter systems, including
opiatergic pathways, may be involved in the central
analgesic effect of this class of drugs [13, 20–22].
Previous observations by our group suggested that the
antinociceptive effect of phenazone, acetylsalicylic acid
(ASA) or paracetamol (PARA) in the hot-plate test in rats
was associated with a decrease in the number of serotonin
receptors in some brain areas [23–25].
Morphine stimulates 5-HT release via a supraspinal
action [26] and the latter depletion in the CNS decreases the
analgesic effect of morphine [27]; thus, we can draw the
conclusion that morphine exerts its analgesic effect, at least
in part, through the serotonergic system [28, 29]. Recently,
our group [30] demonstrated that PARA binds to [3H]nalox-
one sites, although with less potency than morphine; the
maximum binding of PARA was 70% of that of morphine.
Since morphine and PARA may share common pathways in
their mechanism of action, it could be expected that the
combination of the two drugs, at subactive doses, should
result in a potentiation of analgesic efficacy, as has been well
documented in clinical practice with combinations of
analgesics and mild opiates.
The aim of this work was to assess:
1) the analgesic effect of combinations of PARA and
morphine in rats used in subactive doses, in the hot-
plate and formalin tests;
2) the changes in serotonin and 5-HIAA levels and 5-HT2
receptors in rat brain membranes;
3) the influence of pretreatment with naloxone on the
behavioural and biochemical changes induced by drug
combinations.
Materials and methods
Animals
Adult male rats (Harlan-Nossan, SPF, Correzzano, Italy) weighing
180–200 g at the beginning of the experiments, were housed in
Plexiglas cages, four per cage, with free access to food and water, and
maintained on a 12 h dark/light cycle (light on at 7:00 a.m.) under
controlled environmental conditions (temperature, 22 6 1 8C; humidity,
60%). The ethical guidelines for investigation of experimental pain in
conscious animals were followed in all tests, and the procedures were
carried out according to the EEC ethical regulation for animal research
(EEC Council 86/609; D.L.27/01/1982, No. 116).
Drug treatment
Rats, divided into groups of 8 animals, were injected intraperitoneally
(i.p.) with paracetamol at the dose of 50 or 100 mg/kg (dissolved in a
vehicle, which consisted of 12.5% of 1,2-propanediol in sterile saline)
or vehicle. Morphine 2, 3 and 5 mg/kg s.c. dissolved in saline, or saline
was injected, 10 min after PARA administration. Rats were subjected to
the algesimetric tests 20 min thereafter. Other groups of rats were
pretreated with naloxone (1 mg/kg i.p.) in sterile saline or saline 10 min
before PARA (100 mg/kg) or morphine (2 or 3 mg/kg) combination and
m
121
followed the same experimental procedure. At the end of the
experiments the rats were anaesthetized, decapitated and their blood
and brains were removed and stored until required for analysis. Two
additional groups of rats were tested for motor activity with the use of
either PARA 100 mg/kg plus morphine 3 mg/kg or vehicle plus saline.
Test procedures
Hot-plate test. The hot-plate test (HP) consisted of an electrically-
heated surface (Socrel DS-35, Ugo Basile, Comerio, VA, Italy) kept at a
constant temperature of 54 6 0:8 8C. The latencies for paw licking or
jumping were recorded for each animal. The baseline latency in the hot-
plate test ranged from 5:9 6 0:8 to 6:3 6 0:9 sec (ANOVA, p > 0.5).
The analgesic efficacy of the drugs was evaluated as the percentage of
the maximum possible effect (% MPE) according to the formula: (TL-
BL)/(45-BL) × 100, where TL¼test latency; BL¼baseline latency;
45¼cutoff time in seconds.
Immediately after the last pain threshold measurement, the animals
were anaesthetized with ethyl ether and decapitated; the blood was
collected and serum was stored at ¹20 8C. The brains were removed,
weighed and stored at ¹80 8C until required for assays.
Formalin test. Two hours before testing the animals were placed
individually in standard cages and, after the adaptation period, 50 ml of
5% formalin solution was injected subcutaneously into the dorsal
surface of the left hindpaw by use of microsyringe with a 26-gauge
needle. Pain behaviour was monitored for a period of 40 min, 5-min
intervals starting at the time 0. Two phases of spontaneous flinching
behaviour were observed: phase 1 began immediately after formalin
injection to 10 min thereafter, phase 2 began at time 11 and terminated
40 min after formalin injection.
To avoid possible interference of room temperature on skin
temperature, all experiment were performed at room temperature of
22 6 1 8C [31].
Motor activity. The experiments were performed between 9:00 and
12:00 a.m. in a sound proof room by experienced observers unaware of
the treatments. Motor activity was measured in an activity cage by
means of an ultrasound apparatus (Cibertic, S. A., Barcelona, Spain)
placed on the lid of the cage. The number of movements were recorded
continuously 1 h after an adaptation period of 30 min.
Paracetamol assay
Paracetamol were determined in the sera by fluorescence polarization
immunoassay (FPIA). A TDx analyzer was used for drug evaluation
(Abbot Laboratories, Chicago, IL, USA).
Serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA)
determination
The brain areas were assayed for 5-HT and 5-HIAA determinations by
reverse-phase high-performance liquid chromatography (HPLC),
according to Grossi and coworkers [32] with modifications.
The thawed areas were homogenized with an ultrasonic dismem-
branator, in 0.1 M HClO4 containing 4 mM NaHSO4 (10 ml per mg of
wet weight) and centrifuged at 2000 × g for 15 min at 4 8C. After
centrifugation, the acid supernatant was filtered on 0.22 mm filters
before analysis.
5-HT and 5-HIAA assays were performed with a Beckman System
Gold high-performance liquid chromatograph (Beckman Instruments
Inc., San Ramon, CA, USA) equipped with an ESA Coulochem II
Multi-Electrode high sensitivity electrochemical detector (ESA Inc.,
Bedford, MA, USA) with conditioning cell set at ¹0.75 V, detector set
at þ0.05 V and detector 2 set at þ0.25 V, response 2, gain 10 × 5. A
reverse phase C-18 10 cm × 4,6 cm Hypersil column (Labservice
Analytical, Bologna, Italy) packed with 3 mm ODS was used.
122 M. Sandrini et al. Inflamm. res.

The mobile phase composed of methanol 15%, acetonitrile 8% and

Nal þ Para þ Morp5

50 mM NaH2PO4, pH 2.8, with 0.2 mM ethylendiaminetetracetic acid
disodium salt and 200 mg/l sodium octyl sulfate, was pumped at a rate
of 1 ml/min
3,4-Dihydroxybenzylamine (DHBA), the internal standard, and

8:6 6 2:3
5-HT and 5-HIAA were used as standards. Standards and samples are
evaluated according to analite/DHBA ratio in a calibration curve.

Binding assay

Nal þ Para þ Morp3

The characteristics of 5-HT1A binding sites were evaluated according to
Gulati and Bhargava [33] using 6 concentrations of [3H]8-OH-DPAT

9:2 6 4:7
(0.18-6nM) (specific activity 142.9 Ci/mmol).
The characteristics of 5-HT2 binding sites were evaluated
according to Leysen and coworkers [34] with minor modifications.
Brain regions were homogenized in 5 ml of ice-cold 0.25 M sucrose (12
strokes of a Teflon pestle at 120 rpm) and centrifuged at 1300 × g for 10
min at 4 8C. This procedure was repeated, the combination of sucrose

Sal þ Para þ Morp5

Table 1. Influence of naloxone (Nal) treatment on the antinociceptive effect of paracetamol (Para) and morphine (Morp) in the hot-plate test.
supernatants was diluted with 10 ml of 50 mM Tris HCl, pH 7.7, and the
suspension was centrifuged at 3500 × g for 10 min. The pellet was

39:7 6 5:7¬;a
resuspended in 20 ml of Tris-HCl buffer and centrifuged once at
50,000 × g for 10 min; the pellet was then homogenized and diluted in

Nal (1 mg/kg i.p.) or saline (Sal, 2 ml/kg) were injected 10 min before Para (100 mg/kg i.p.) plus Morp (3 or 5 mg/kg s.c.) treatment.
Tris-HCl (about 300 mg protein/ml). Aliquots of membranes (800 ml)
were placed in plastic test tubes containing [3H]ketanserin (six
concentrations in 10% ethanol), methysergide (10 mM, dissolved in
Tris-HCl buffer to define nonspecific binding) or Tris buffer at 37 8C for
15 min. The mixture was filtered under reduced pressure through

Sal þ Para þ Morp3

Whatman GF/B glass fiber filters, previously soaked for 5 min in 0.5%
poliethylenimine with use of a Millipore vacuum pomp and rapidly

31:9 6 10:4¬;a
rinsed twice with 5 ml ice-cold Tris buffer. The filters were transferred
to plastic vials containing 6 ml of Packard Optifluor and shaken. The
vials were stored for 20 h at 4 8C in the dark.

Hot-plate started 20 min after the last treatment. % MPE ¼ percentage of the maximum possible effect.
The following concentrations were used: 0.05 to 4 nM [3H]ketan-
serin (specific activity, 87.5 Ci/mmol). The specific binding was 60 to
70% of the total binding for [3H]ketanserin and 80 to 90% for [3H]8-
Nal þ Veh þ Sal

OH-DPAT.
2:1 6 2:2

Statistics

The results obtained from motor activity were analyzed with Student’s
t-test. The results of binding experiments were analyzed according to
Sal þ Veh þ Morp5

the method of Rosenthal [35]. The equilbrium dissociation constant

ANOVA, *p < 0.05 vs. Sal þ Veh þ Sal. ap < 0.05 vs. Sal þ Veh þ Morp.

(kD) and maximum number of binding sites (Bmax) were evaluated

individually for each sample with six concentrations of labeled drug. A
20:9 6 2:5¬

two-way analysis of variance followed by 2 × 2 factorial analysis by

means of orthogonal comparisons was used to analyze the effect of
PARA þ morphine treatment, naloxone treatment and their com-
bination [36].
The data were examined by ANOVA followed by Student-
Newman-Keuls’ test when the effects of naloxone and PARA þ
Sal þ Veh þ Morp3

morphine were being evaluated separately.

7:2 6 0:9

Drugs

Paracetamol, was kindly provided by ACRAF SpA. Ancona, Italy, while

morphine hydrochloride, 5-HT, 5-HIAA, DHBA and naloxone were
purchased from Sigma Chemicals Co., St. Louis, MO, USA.
Sal þ Veh þ Sal

[3H]8-hydroxy-2-(D-n-proply-amino)tetralin ([3H]8-OH-DPAT)
and [3H]ketanserin were from Du Pont NEN, Co. Ltd, Milan, Italy.
Treatment

0:8 6 1:7

Formalin was obtained through Bracco Chemical Co., Milan, Italy.

Results
% MPE

Neither PARA alone (50 or 100 mg/kg) nor the single dose of
2 or 3 mg/kg or morphine was able to modify the reaction

m
Vol. 48, 1999 Potentiation of analgesic activity of paracetamol plus morphine
Fig. 1. Antinociceptive effect of paracetamol (PARA) and morphine
(MORP) combination in the hot-plate test. % MPE represents the
percentage of the maximum possible effect. MORP (0–5 mg/kg s.c.)
was administered 10 min after PARA (50–100 mg/kg i.p.) and the rats
were tested 20 min after morphine. Values are means 6 SEM of 8 rats
for each group. *p < 0.05 vs. control group (VEH þ MORP 0 mg/kg);
P
p < 0.05 vs VEH þ MORP (ANOVA followed by Student-Newman-
Keuls’ test).
time in the HP test. Nor was PARA at the dose of 50 mg plus
2 and 3 mg/kg of morphine able to modify the % MPE in this
test. The combinations of 100 mg/kg of PARA with 2 or 3
mg/kg of morphine, on the other hand, significantly increased
% MPE and exerted an antinociceptive effect (Fig. 1).
The last combination of the two drugs showed a
potentiation effect, demonstrated by the factorial analysis
[F(1-32)¼10.4; p < 0.01], [F(1-32)¼7.6; p < 0.01], for 2 and 3
mg/kg of morphine, respectively. Morphine, at the dose of
Fig. 2. Antinociceptive effect of a combination of paracetamol (PARA)
and morphine (MORP) in the formalin test. PARA (100 mg/kg i.p.) and
MORP (2 or 3 mg/kg s.c.) were administered 10 min after each other
and the rats were tested 20 min after morphine. Each histogram
represents the total number of flinches (mean 6 SEM) in phase 1 and in
phase 2. *p < 0.05 vs. control values (ANOVA followed by Student-
Newman-Keuls’ test).
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123
Fig. 3. Influence of naloxone (NAL) pretreatment on the antinocicep-
tive effect of a combination of paracetamol (PARA) and morphine
(MORP) in the formalin test. NAL (1 mg/kg i.p.) or saline (SAL, 2 ml/
kg) were injected 10 min before PARA (100 mg/kg i.p.) plus MORP (3
mg/kg s.c.) treatment. Each histogram represents the total number of
flinches (mean 6 SEM) in phase 1 and in phase 2. *p < 0.05 vs. control
values (ANOVA followed by Student-Newman-Keuls’ test).
5 mg/kg, provoked a significant increase in % MPE that was
potentiated by the combination with PARA (100 mg/kg)
[F(1-32)¼6.2; p < 0.05].
Naloxone (1 mg/kg) significantly prevented the anti-
nociceptive effect of PARA plus morphine (3 or 5 mg/kg) in
the % MPE (Table 1); the factorial analysis showed a
negative interaction [F(1-28)¼5.4; p < 0.05]; [F(1-28)¼4.4;
p < 0.05].
PARA 100 mg/kg and morphine 2 or 3 mg/kg were
chosen to test the antinociceptive activity in the formalin
test, as these doses did not exert a significant antinociceptive
effect when given alone; a combination of the two drugs
seemed to be the most suitable way of assessing eventual
synergy. The combinations of PARA 100 mg/kg and MORP
2 or 3 mg/kg had a significant effect in both phases of the
formalin test (Fig. 2). On the other hand, the interaction test
showed significant potentiation in the first phase
[F(1-20¼11.5; p < 0.01]; [F(1-20)¼4.8; p < 0.05] for the two
doses of morphine, whereas, in the second phase, only the
combination of PARA plus the dose of 3 mg/kg morphine
caused the potentiation effect [F(1-20)¼6.34; p < 0.05];
[F(1-20)¼1.1; p > 0.05] for the combination of 2 mg/kg.
Naloxone prevented the effect of the combination of
PARA 100 mg/kg and morphine 3 mg/kg in both phases of
the test [F(1-20)¼5.07; p < 0.05], first phase, and [F(1-20)¼8.5;
p <0.01], second phase. Naloxone alone slightly increased
the number of flinches (Fig. 3). None of these combinations
affected motor activity, the number of movements being
1274 6 60 for PARA 100 mg/kg plus morphine 3 mg/kg and
1243 6 48 for the control group (p > 0.05).
In order to exclude any pharmacokinetic interaction
between naloxone, PARA and morphine we evaluated the
levels of paracetamol in the sera; the concentrations of
PARA were 12:8 6 2:3, 15:2 6 1:6 and 13:1 6 2:1 mg/ml for
saline þ PARA (100 mg/kg)-treated, saline þ PARA þ
124
Fig. 4. The effect of the combination of paracetamol (PARA) and
morphine (MORP) on serotonin content in cortical and pontine
membranes. PARA (100 mg/kg i.p.) and MORP (3 mg/kg s.c.) were
administered 10 min after each other. The rats were killed at the end of
the hot-plate test and brain areas were weighed and frozen at ¹80 8C
until assayed. Values are expressed as mean 6 SEM for 6 rats for each
group. *p < 0.05 vs. control values (ANOVA followed by Student-
Newman-Keuls’ test).
morphine (3 mg/kg)-treated and naloxone (1 mg/kg) þ
PARA þ morphine-treated rats, respectively (p > 0.05).
The levels of serotonin in the cortex and in the pons were
significantly increased by the antinociceptive combination of
PARA 100 mg/kg and morphine 3 mg/kg, while they did not
change the values of controls when given alone (Fig. 4); the
interaction test showed a potentiation in the two areas
[F(1,20)¼15.2; p < 0.01], [F(1,20)¼28.2; p < 0.01] for pons and
cortex respectively. Pretreatment with naloxone prevented
the increase of 5-HT in both areas (Fig. 5); [F(1,20)¼10.7;
p < 0.01], [F(1,20)¼12.2; p < 0.01] for pons and cortex
respectively.
The levels of 5-HIAA in the cerebral cortex and in the
pons increased but not significantly; this lack of significance
could be due to the wide variability of the data obtained with
respect to size of the sample (Fig. 5).
The combination of the same doses of PARA and
morphine provoked a decrease in the number of 5-HT2
receptors in the cortex (Table 2), [F(1-20)¼7.0; p < 0.01], but
not in the pons, while the two drugs did not change the
maximum number of binding sites in the two areas when
given alone. Naloxone partially prevented the decrease in the
Bmax but the interaction test showed no significance
[F(1-20)¼1.76; p >0.05]. The affinity constant remained
unchanged. The characteristics of 5-HT1A receptors never
changed, either in the cortex or in the pons (Table 3).
Discussion
The evaluation of antinociceptive drug activity in animal
models depends on several factors, such as the type of
m
M. Sandrini et al. Inflamm. res.
Fig. 5. Influence of naloxone (NAL) pretreatment on the effect of
paracetamol (PARA) and morphine (MORP) on serotonin (5-HT) and
5-hydroxyindolacetic acid (5-HIAA) levels in the rat brain. NAL (1 mg/
kg i.p.) or saline (SAL, 2 ml/kg) was administered 10 min before PARA
(100 mg/kg) and 20 min before MORP (3 mg/kg); the rats were killed at
the end of the hot-plate test and brain areas were weighed and frozen at
¹80 8C until assayed. Each value represents the mean 6 SEM of 6
separate experiments. *p < 0.05 vs. control values (ANOVA followed
by Student-Newman-Keuls’ test).
stimulus, species and strain of animals, the type of recording
device involved in the experimental model.
Nevertheless, in previous studies the antinociceptive
effect of phenazone [23] and paracetamol [30] has been
tested by the hot-plate and formalin tests, which are
generally considered a reliable method of measuring
NSAID antinociceptive activity. Indeed, the hot-plate test
has been widely employed to study nociception, and, in a
review concerning this test, Hunskaar and coworkers [37]
demonstrated similar potencies for ASA and morphine in the
rats.
The behavioural response to the injection of formalin is
biphasic, with an acute phase followed by a quiescent period
and then a prolonged response with a maximum between 10
and 40 min [3]. It has been suggested that the early phase is
caused by a direct effect of formalin on nociceptors, whereas
the second phase is a tonic response in which inflammatory
processes are involved and neurons of the dorsal horn of the
spinal cord are activated [38]; no obvious inflammatory state
has been found 10 min after formalin injection. Thus, the
Vol. 48, 1999 Potentiation of analgesic activity of paracetamol plus morphine
Table 2. Influence of naloxone (Nal) treat-
ment on the effect of a combination of
paracetamol (Para) and morphine (Morp) on
the characteristics of 5-HT2 receptors in the Treatment
rat brain.
Sal þ Veh þ Sal
Nal þ Veh þ Sal
Sal þ Para þ Sal
Sal þ Morp þ Sal
Sal þ Para þ Morp3
Nal þ Para þ Morp3
Rat were injected with saline (Sal, 2 ml/kg), vehicle (Veh, 2 ml/kg), Nal (1 mg/kg i.p.), Para (100
mg/kg i.p.) and Morp (3 mg/kg s.c.). They were killed at the end of the experiment and the brain
were stored at ¹80 8C until assayed. Each value represents the mean 6 SEM of 6 separate
experiments and were derived from Rosenthal plot. Bmax, maximum binding capacity; kD,
equilibrium dissociation constant. ANOVA, p < 0.05 vs. Sal þ Veh þ Sal.
antinociceptive non-antiinflammatory properties of drugs
can be evaluated immediately after formalin injection and
for 10 minutes thereafter [39]. In this paper we demonstrate
that PARA and morphine, when given in combination, even
in subactive doses, exert a significant analgesic effect in both
the hot-plate and the formalin test.
The combination of doses of morphine and PARA that
are ineffective, when given alone, may be of interest from the
therapeutic point of view, for the combination showed actual
additional antinociceptive effects. This combination is largely
used in the treatment of mild to moderate pain in humans.
Our data support the hypothesis that the analgesic
activity of PARA and morphine in association might occur
as the result of opiatergic activation, it having been
demonstrated that naloxone blocks the potentiation effect.
We have previously shown that naloxone significantly
prevents the action of PARA in the hot-plate test and in
the first phase of the formalin test but does not affect PARA
activity in the second phase, while the effect of morphine is
prevented in both phases of the test [30], which suggests that
the mechanism of action may be different in the second
phase of the formalin test where inflammatory processes are
involved. In the present paper it has been shown that
naloxone prevents this effect of PARA and morphine
combinations in all the phases of the formalin test.
Table 3. Effect of a treatment with paracetamol (Para) and morphine
(Morp) on the characteristics of 5-HT1A receptors in the cortex of the rat
brain.
Treatment Bmax kD
(fmol/mg prot) (nM)
Veh þ Sal 264:3 6 21:3 1:8 6 0:3
Para þ Sal 253:1 6 10:7 1:7 6 0:3
Veh þ Morp 212:7 6 18:4 1:8 6 0:3
Para þ Morp 266:8 6 22:8 2:1 6 0:3
Rat were injected with saline (Sal, 2 mg/kg), vehicle (Veh, 2 ml/kg i.p.),
paracetamol (Para, 100 mg/kg i.p.) and morphine (Morp, 3 mg/kg s.c.).
They were killed at the end of the experiment and the brains were stored
at ¹80 8C until assayed. Each value represents the mean 6 SEM of 6
separate experiments and were derived from Rosenthal plot. Bmax,
maximum binding capacity (fmol/mg protein); kD, equilibrium
dissociation constant (nM).
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125
Cortex Pons
Bmax kD Bmax kD
(fmol/mg prot) (nM) (fmol/kg prot) (nM)
221:8 6 6:3 1:9 6 0:2 33:4 6 3:9 1:3 6 0:10
259:4 6 10:3 1:6 6 0:10 35:4 6 3:9 1:3 6 0:1
222:3 6 13:7 1:6 6 0:30 n.d. n.d.
238:5 6 7:3 1:5 6 0:3 38:4 6 5:4 1:51 6 0:10
155:0 6 7:1¬ 20 6 0:2 31:4 6 3:0 1:2 6 0:10
202:7 6 9:4 1:9 6 0:1 n.d. n.d.
It has been previously shown that m opiate receptor
agonists suppress the activity of formalin in all the phases of
the test [40], so we may hypothesize that the block of
morphine activity in the second phase of the formalin test
induced by naloxone can explain also naloxone antagonism
on the effect of PARA and morphine in combination. On the
other hand, all the doses used were subactive, and when the
activity of morphine was blocked by naloxone the anti-
nociceptive activity of PARA did not make itself felt because
of the low dose.
In a recent paper it was suggested that the mechanism of
action by which some NSAIDs exert their analgesic effect
involves a number of neurotransmitter systems, particularly
adrenergic, serotonergic and opiatergic. An increase in 5-HT
levels in brain areas has been observed by many authors after
NSAIDs or PARA injection. Morphine has also been shown
to act on serotonergic pathways in a way similar to that of
PARA [41], increasing the concentration of 5-HT in the
cerebral cortex and decreasing the number of 5-HT2
receptors in the same area. In our experimental conditions
it is unlikely that 5-HT1A receptors may be involved, at least
in the cerebral cortex, because of the different distribution of
this type of receptors in CNS. Indeed some authors showed
that 5-HT1A and 5-HT2 receptors are involved in antinoci-
ception at spinal level [42, 43].
PARA and morphine in combination may activate opiate
receptors, which in turn increase 5-HT levels, at least in the
cerebral cortex and pons. Naloxone could therefore block the
opiatergic pathways that in turn activate the serotonergic
system [30].
Opioid inhibition of GABAergic transmission is poten-
tiated by inhibitors of the enzymes cyclooxygenase and
5-lipoxygenase [44] and the sinergistic analgesic action of
opioids and NSAIDs in the CNS may be mediated by COX1
rather than COX2 [45].
Although the mechanism by which PARA interact with
opiatergic and serotonergic systems has still not been
completely elucidated, we suggest that PARA and morphine
might act through common pathways in the CNS (i.e.
serotonergic and opiatergic) that are involved in pain
modulation, with selective effects in discrete brain areas
[46]. In a recent paper we observed that PARA was also able
to bind to [3H]-naloxone binding sites, although with minor
potency than morphine, where it might exert a morphine-like
126
action, the final effect being a potentiation of the analgesic
activity when the two drugs are given in combination [30].
Many findings indicate the complexity of the central pain
modulating system, where a network of neurotransmitter
pathways (i.e. monaminergic and peptidergic) may interact
to modulate and perform many behavioural changes
including nociception [22].
The results obtained using this experimental procedure
could thus probably be transferred to human beings; in fact,
in human therapeutics as well there are products indicated
for mild pain containing PARA and opiate derivatives (like
codeine) at ineffective doses when used alone [47].
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