Full text of DOI:10.1038/sj.bjp.0704679

British Journal of Pharmacology (2002) 135, 2047 ± 2055
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Involvement of purinergic signalling in central mechanisms of body
temperature regulation in rats
*,1Alexander V. Gourine, 2Ekaterina V. Melenchuk, 2Dmitry M. Poputnikov, 2Valery N. Gourine &
1K. Michael Spyer
1Department of Physiology, Royal Free and University College London Medical School, Rowland Hill Street, London NW3
2PF and Institute of Physiology, National Academy of Sciences of Belarus, Minsk 220725, Belarus
2
1
P2 purinoreceptors are present in hypothalamic and brainstem nuclei that are involved in the
regulation of body temperature (Tb). The role of ATP acting on these P2 receptors in
thermoregulation was investigated by studying the e€ects of the stable ATP analogue a,b-
methyleneATP (a,b-meATP) and P2 receptor antagonists suramin and pyridoxal-5'-phosphate-6-
azophenyl-2',4'-disulphonic acid (PPADS) on Tb when injected intracerebroventricularly (i.c.v.) via a
pre-implanted cannula in conscious rats at various ambient temperatures and during lipopoly-
saccharide (LPS)-induced fever.
2
Depending on ambient temperature, a,b-meATP (0.2 mmol, i.c.v.) induced a fall in Tb (73.38C,
P50.05), no changes in Tb when compared to pre-injection levels, or an increase in Tb (*1.08C,
P50.05) in rats maintained at 108C, 258C and 308C ambient temperature, respectively.
3
Suramin (7 nmol, i.c.v.) induced a lasting (up to 6 h) increase in Tb (on average 1.28C, P50.05)
in rats kept at 258C or 308C, but failed to induce any rise in Tb in rats at 108C ambient temperature.
An increase in Tb was also observed in rats (258C ambient temperature) treated with PPADS
(0.2 mmol, i.c.v.).
4
a,b-meATP (0.2 mmol) injected i.c.v. or directly into the anterior hypothalamus caused a
profound fall in Tb (by 0.98C and 1.08C, respectively; P50.05) during LPS (E.coli; 50 mg kg71)-
induced fever in rats at 258C ambient temperature. Fever was initiated more rapidly in rats treated
with suramin (7 nmol) or PPADS (70 nmol), however its late phase was una€ected. Suramin
(7 nmol) and PPADS (70 nmol) injected at the time when fever was already developed (2.5 h after
LPS injections) did not alter febrile Tb.
5 These data indicate that purinergic signalling may play a signi®cant role in central mechanisms of
Tb regulation at various ambient temperatures and during fever.
British Journal of Pharmacology (2002) 135, 2047 ± 2055
Keywords: ATP; body temperature; fever; hypothalamus; a,b-methyleneATP; P2 receptors; purine; pyridoxal-5'-phosphate-
6-azophenyl-2',4'-disulphonic acid; suramin; thermoregulation
Abbreviations: a,b-meATP, a,b-methylene adenosine 5'-triphosphate; aCSF, arti®cial cerebrospinal ¯uid; ANOVA, analysis of
variance; ATP, adenosine 5'-triphosphate; i.c.v., intracerebroventricular; IL-1b, interleukin-1b; i.p., intraperi-
toneal; LPS, lipopolysaccharide; NTS, nucleus tractus solitarius; PPADS, pyridoxal-5'-phosphate-6-azophenyl-
2',4'-disulphonic acid; s.e., standard error; Tb, body temperature
Introduction
There is growing evidence that extracellular adenosine 5'-
triphosphate (ATP), known as an intracellular source of
energy in metabolism, acting via P2X receptors, is a fast
excitatory neurotransmitter in the central and peripheral
nervous system (Edwards et al., 1992; Burnstock, 1999). P2X
receptors are ATP-gated non-selective cation channels perme-
able to sodium, potassium and calcium ions (Ralevic &
Spyer & Thomas, 2000). There is evidence that ATP, acting
on P2X receptors, within the ventrolateral medulla may play
a role in regulation of vasomotor tone and sympathetic
activity (Horiuchi et al., 1999; Ralevic, 2000), and that P2X
receptors in this area of the brainstem mediate hypercapnia-
induced changes in respiration (Spyer & Thomas, 2000).
There is evidence that purines may also play a role in
cardiovascular and respiratory control through actions within
the nucleus tractus solitarius (NTS) of the medulla oblongata
(Phillis et al., 1997). The role for purinergic signalling in
central cardiovascular and respiratory control is strongly
supported by the results of immunohistochemical and in situ
hybridization studies indicating that P2X receptors of
di€erent types are abundant in the brainstem (Kidd et al.,
1995; Seguela et al., 1996; Vulchanova et al., 1996; Kanjhan
et al., 1999; Yao et al., 2000).
Burnstock, 1998). Seven subtypes of P2X receptors (P2X1 ± 7
)
have been cloned and characterized in terms of agonist/
antagonist selectivity (Buell et al., 1996; Ralevic & Burnstock,
1998).
It has been shown recently that extracellular ATP, acting
at P2X receptors, is involved in the brainstem mechanisms of
cardiovascular and respiratory regulation (Phillis et al., 1997;
Horiuchi et al., 1999; Ralevic et al., 1999; Ralevic, 2000;
It is well known that regulation of body heat exchange to
the environment includes changes in peripheral blood ¯ow,
*Author for correspondence; E-mail: a.gourine@rfc.ucl.ac.uk

2048
A.V. Gourine et al
Purinergic mechanisms and thermoregulation
blood redistribution and changes in respiratory rate.
Although the role of extracellular ATP in brainstem
mechanisms of cardiovascular and respiratory control has
been demonstrated, the possibility that purinergic signalling
may also be involved in central mechanisms of body
temperature (Tb) regulation has not been addressed. This
possibility is supported by the evidence that P2X receptors
are also present in the hypothalamus (Shibuya et al., 1999;
Xiang et al., 1998) ± the primary area of the brain involved
in regulation of body temperature and development of fever.
Recent evidence favours a role for extracellular ATP in the
release of cytokines, such as interleukin (IL)-1b and tumour
necrosis factor (Perregaux & Gabel, 1998; Hide et al., 2000;
Mehta et al., 2001; Solle et al., 2001), essential for the
development of fever (Kluger et al., 1995). Taking into
account evidence that fever is initiated by IL-1b at the level
of the anterior hypothalamus (Klir et al., 1994; Gourine et
al., 1998), we suggest that changes in the level of extracellular
ATP in this area of the brain play a part in the mechanisms
underlying development of the febrile response.
perature-sensitive telemetry transmitter (model E-mitter,
Minimitter, Sunriver, OR, U.S.A.) was implanted into the
abdominal cavity of each rat for continuous monitoring of
Tb. Then a 26-gauge guide injection cannula (Plastic Products
Co., Roanoke, VA, U.S.A.) was stereotaxically implanted
into the third cerebral ventricle or anterior hypothalamus
(stereotaxic co-ordinates: 1.8 mm caudal to bregma, 0.5 mm
lateral to midline and 8.6 mm ventral from the surface of the
skull) according to the atlas of Paxinos & Watson (1986).
Two small screws were placed into the skull, and the cannula
was secured in place by dental acrylic. The guide cannula was
closed with a dummy cannula that extended from the tip of
the guide cannula by 40.2 mm. After the surgery animals
were housed one per cage and were allowed to recover for at
least 7 days before any experiment. At the end of the
experiment the animals were killed humanely by an overdose
(200 mg kg71) of pentobarbitone sodium injected intraper-
itoneally (i.p.), the brain was removed and the location of the
cannula was con®rmed histologically.
In this pharmacological study, experiments have been
designed to provide preliminary evidence on whether ATP
acting on hypothalamic and (or) brainstem P2 receptors is
involved in regulation of Tb at di€erent ambient temperatures
and during fever. The e€ects of the stable ATP analogue a,b-
methyleneATP (a,b-meATP) and P2 receptor antagonists
suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulpho-
nic acid (PPADS) injected intracerebroventricularly (i.c.v.) on
Tb in conscious rats at 10, 25 and 308C ambient temperatures
and during fever induced by bacterial lipopolysaccharide
(LPS) were determined. a,b-meATP, suramin and PPADS
that were injected into the third cerebral ventricle are believed
to reach structures, containing di€erent types of P2X
receptors, in the hypothalamus that are concerned with
thermoregulation as well as in the dorsal brainstem crucial
for cardiovascular control (i.e. NTS). The e€ect of a,b-
meATP microinjected directly into the anterior hypothalamus
on Tb during fever in rats was also determined. Some of the
Tb measurements
Deep Tb (+0.18C) was monitored with implanted telemetry
units (Minimitter). Recordings were made at 1-min intervals
by use of a peripheral processor (VitalView System,
Minimitter) connected to an IBM PC.
Intracerebroventricular, intrahypothalamic and
intraperitoneal injections
Animals were conditioned to handling for 5 min once a day
for 6 days prior to the experiment. Microinjections into the
third cerebral ventricle or into the anterior hypothalamus were
made over a period of 1 ± 2 min using an internal injection
cannula connected to PE-50 tubing attached to a 50 ml or
10 ml syringe, respectively (Hamilton, Reno, NV, U.S.A.). The
injection cannula was removed 2 ± 3 min after the injection.
The volumes of injections were 1 ml for the hypothalamus and
5 ml for the third ventricle. In the control experiments drugs
were also injected i.p. in a volume of 0.1 ml.
results obtained have been reported previously in
preliminary abstract (Gourine et al., 2001).
a
Fever
Methods
Puri®ed LPS (Escherichia coli endotoxin 0111 : B4, Sigma
Chemical, St. Louis, MO, U.S.A.) was dissolved in pyrogen-
free saline and injected i.p. at a dose of 50 mg kg71. Control rats
received an equivalent volume of sterile pyrogen-free saline.
Animals
Two hundred and sixty-two adult male Wistar rats weighing
280 ± 320 g were used in this study. They were housed in a
room maintained at a constant temperature of 25+18C, a
temperature slightly below the thermoneutral zone of rats,
and in a 12 : 12 h light ± dark cycle with light onset at 0600 h.
Drinking water and laboratory rodent chow were provided ad
libitum. All studies on conscious rats were conducted in
facilities of the Institute of Physiology, National Academy of
Sciences of Belarus and were approved by the Institutional
Animal Care and Use Committee.
Drugs
a,b-methylene-adenosine 5'-triphosphate (SIGMA Chemical),
suramin sodium salt (SIGMA Chemical) and PPADS (Tocris
Cookson Ltd, Bristol, U.K.) were dissolved in arti®cial
cerebrospinal ¯uid (aCSF) to the designed amount in 5 ml of
aCSF. The aCSF used for injections consisted of (in mM):
NaCl 145.0, KCl 3.3, CaCl2 1.3 and MgCl2 1.0 dissolved in
sterile pyrogen-free water.
Surgery
Experimental design
Rats were anaesthetized with
hydrochloride (87.0 mg kg71
a
mixture of ketamine
and xylazine hydrochloride
)
Experiment 1. E€ect of i.c.v. injection of the ATP analogue
a,b-meATP on Tb in rats maintained at 258C a,b-meATP
(13.0 mg kg71) injected intramuscularly. A miniature tem-
British Journal of Pharmacology vol 135 (8)

A.V. Gourine et al
Purinergic mechanisms and thermoregulation
2049
(0.02 mmol and 0.2 mmol) was injected into the third cerebral
ventricle of afebrile rats maintained at 258C ambient
temperature between 0900 and 1000 h. Tb was monitored
for 3 h before and 9 h after the injections. Control animals
were injected i.c.v. with aCSF (5 ml). To make sure that the
e€ect of a,b-meATP on Tb was not due to its leakage into the
circulation and action on the periphery, in control studies we
investigated whether a,b-meATP a€ects thermoregulation
when injected i.p. at the same amount (0.2 mmol) that was
shown to in¯uence Tb after i.c.v. injection.
treatment on fever was determined in order to con®rm that
suramin e€ect on the development of the febrile response is
not due to its leakage into circulation and action on the
periphery. Tb was monitored for 3 h before and 9 h after LPS
injections, which were performed between 0900 and 1000 h.
Statistical analysis
Data are reported as mean+standard error (s.e.). Compar-
isons between one experimental and one control group were
made using one-way analysis of variance (ANOVA). For
pairwise comparisons among more than two groups, data
were examined for statistical signi®cance using ANOVA
followed by the method of Fischer least signi®cant di€erence.
A value of P50.05 was considered to be signi®cant.
Experiment 2. E€ect of i.c.v. injection of the P2 receptor
antagonists suramin and PPADS on Tb in rats maintained at
258C Suramin (7 nmol), PPADS (7 nmol, 70 nmol and
0.2 mmol) or aCSF (5 ml) were injected into the third cerebral
ventricle of afebrile rats kept at 258C ambient temperature
between 0900 and 1000 h. Tb was monitored for 3 h before
and 9 h after the injections. To con®rm that the e€ect of
suramin on Tb is not due to its leakage into the circulation
and action on the periphery, we investigated whether this P2
receptor antagonist induces hyperthermia when injected i.p.
at the same amount (7 nmol) that was shown to in¯uence Tb
after i.c.v. injection.
Results
Experiment 1
E€ect of i.c.v. injection of the ATP analogue a,b-meATP on Tb
in rats maintained at 258C There was an initial rise in Tb
when an i.c.v. injection of aCSF was given, representing a
stress-induced rise in Tb, caused by the handling and injection
procedure (Figure 1). This stress-induced rise in Tb was
attenuated by a,b-meATP injected at a dose of 0.02 mmol and
was completely blocked by this ATP analogue at a dose of
0.2 mmol (Figure 1). Deep Tb of rats injected i.c.v. with a,b-
meATP (0.2 mmol) was signi®cantly (P50.05) lower than in
rats injected with aCSF 5 ± 45 min after the injections. It is
important to note that while this stress-induced rise in Tb was
blocked by a,b-meATP, the Tb of rats injected with this ATP
analogue was not di€erent from pre-injection values (Figure
1). Since the mechanisms responsible for the stress-induced
rise in Tb are believed to be similar to those responsible for
fever (Kluger et al., 1987), these data are consistent with the
results obtained in experiments in febrile rats that showed
a,b-meATP to inhibit fever (Experiment 4). Although, there
were no statistically signi®cant di€erences in Tb between rats
treated i.c.v. with aCSF and a,b-meATP 45 min after the
Experiment 3. E€ect of i.c.v. injection of the ATP analogue
a,b-meATP or of the P2 receptor antagonist suramin on Tb in
rats at low and high ambient temperatures Rats in their
home cages (one rat per cage) were placed in a room at a
constant temperature of either 10+18C or 30+18C at 0900 h.
a,b-meATP (0.2 mmol), suramin (7 nmol), a mixture of a,b-
meATP (0.2 mmol) with suramin (7 nmol), or aCSF (5 ml)
were injected into the third cerebral ventricle of these rats
between 0900 and 1000 h on the following day, i.e. after 24 h
of exposure to the ambient temperature of 108C or 308C. Tb
was monitored for 1.5 h before and 3 h after the injections.
Experiment 4. E€ect of i.c.v. or intrahypothalamic injection of
the ATP analogue a,b-meATP on Tb during LPS-induced fever
in rats The results of the experiments 1 and 3 showed that
the duration of a,b-meATP-induced changes in Tb is less than
2 h. Because in rats LPS induces delayed fever, a,b-meATP
was injected at the time when a febrile response had
developed but not prior to LPS treatment. a,b-meATP
(0.2 mmol), a mixture of ab-meATP (0.2 mmol) with suramin
(7 nmol) or aCSF (5 ml) were injected into the third cerebral
ventricle 2.5 h after i.p. injection of LPS. a,b-meATP
(0.2 mmol) or aCSF (1 ml) were also injected into the anterior
hypothalamus 2.5 h after injection of LPS. In the control
experiment, to con®rm that the e€ect of a,b-meATP on
febrile Tb is not due to an action on the periphery, we
investigated whether a,b-meATP a€ect fever when injected
i.p. at the same amount (0.2 mmol) that was shown to
in¯uence febrile Tb after i.c.v. or intrahypothalamic injection.
Tb was monitored for 3 h before and 9 h after LPS injections,
which were performed between 0900 and 1000 h.
Figure 1 E€ect of intracerebroventricular injection of the ATP
analogue a,b-methyleneATP (a,b-meATP; 0.02 mmol and 0.2 mmol)
or arti®cial cerebrospinal ¯uid (aCSF) on body temperature in rats at
258C ambient temperature. Data are presented as mean+s.e.
Numbers in parentheses indicate sample sizes. Arrowhead indicates
time of injections. *Signi®cant di€erence in body temperature
between rats treated into the third cerebral ventricle with a,b-meATP
(0.2 mmol) and rats treated with aCSF, P50.05.
Experiment 5. E€ect of i.c.v. injection of the P2 receptor
antagonists suramin and PPADS on Tb during LPS-induced
fever in rats Suramin (7 nmol), PPADS (70 nmol) or aCSF
(5 ml) were injected into the third cerebral ventricle
immediately before, or 2.5 h after, an i.p. injection of LPS.
Suramin (7 nmol) was also injected i.p. and the e€ect of this
British Journal of Pharmacology vol 135 (8)

2050
A.V. Gourine et al
Purinergic mechanisms and thermoregulation
injection and thereafter, in ®ve out of 13 rats the Tb notably
increased 120 ± 150 min after a,b-meATP administration
(0.2 mmol). As shown in Figure 3, i.p. injection of a,b-
meATP (0.2 mmol) had no e€ect on Tb.
Experiment 3
E€ect of i.c.v. injection of the ATP analogue a,b-meATP or of
the P2 receptor antagonist suramin on Tb in rats at low and
high ambient temperatures These studies indicate that the
e€ects of ATP analogue (a,b-meATP) and P2 receptor
antagonist (suramin) on Tb depend on ambient temperature
(Figure 4). As described above, at 258C ambient temperature
a,b-meATP (0.2 mmol) blocks the stress-induced rise in Tb
(i.e. that elicited by the injection procedure), without inducing
any signi®cant changes in Tb relative to the pre-injection
values (Figures 1 and 4B). At 108C ambient temperature rats
treated i.c.v. with a,b-meATP (0.2 mmol) developed a marked
and lasting (90 min) decrease in Tb (Figure 4A). At this
ambient temperature the Tb of rats treated i.c.v. with a,b-
meATP was 36.33+0.308C (P50.05), 34.69+0.528C
(P50.05) and 35.90+0.698C (P50.05) 30, 60 and 90 min
after the injections, respectively. This e€ect of a,b-meATP on
Tb was completely blocked by suramin (Figure 4A). At 108C
ambient temperature the Tb of rats injected with both a,b-
meATP (0.2 mmol) and suramin (7 nmol) was similar to that
observed in rats treated with aCSF. At 308C ambient
temperature, rats showed an increase in Tb after i.c.v.
administration of a,b-meATP at a dose of 0.2 mmol (Figure
4C). The Tb of rats kept at 308C reached a maximum of
38.86+0.368C 75 min after i.c.v. injection of a,b-meATP,
some 1.18C higher than Tb of rats injected i.c.v. with aCSF
(37.77+0.228C, P50.05; Figure 4C).
Experiment 2
E€ect of i.c.v. injection of the P2 receptor antagonists suramin
and PPADS on Tb in rats maintained at 258C Injection into
the third cerebral ventricle of suramin (7 nmol) induced an
immediate, long-lasting and profound rise in Tb (Figure 2A).
Tb reached its maximal level of 39.17+0.228C 135 min after
injection of suramin ± a level of Tb some 1.68C higher than
Tb of rats injected i.c.v. with aCSF (P50.05, Figure 2A). The
Tb of rats treated with suramin was signi®cantly (P50.05)
higher (on average by 1.28C) than in rats treated with aCSF
60 ± 360 min after the injection (Figure 2A). As shown in
Figure 2B, i.c.v. injection of PPADS at doses of 7 nmol and
70 nmol had no signi®cant e€ect on Tb. PPADS injected into
the third cerebral ventricle at a dose of 0.2 mmol induced a
short-lasting (1 h) but profound increase in Tb. Thirty min
after injection of PPADS (0.2 mmol) the Tb peaked at
38.97+0.298C, which was 1.48C higher than in rats treated
with aCSF (P50.05, Figure 2B). Suramin (7 nmol) injected
i.p. had no signi®cant e€ect on Tb in rats maintained at 258C
(Figure 3).
As shown already, at a 258C ambient temperature suramin
(7 nmol) induced a long-lasting increase in Tb after injection
into the third cerebral ventricle (Figures 2 and 4E). At 308C
ambient temperature rats treated i.c.v. with suramin (7 nmol)
also developed hyperthermia (Figure 4F), which was similar
to that observed in rats injected with this P2 receptor
antagonist at 258C. However, i.c.v. administration of suramin
(7 nmol) did not induce any signi®cant Tb rise in rats
maintained at 108C ambient temperature (Figure 4D). At this
ambient temperature suramin blocked completely the stress-
induced rise in Tb (caused by injection procedure), without
inducing a signi®cant Tb change compared to the pre-
injection values (Figure 4D). At 108C ambient temperature,
the Tb of rats treated i.c.v. with suramin was signi®cantly
Figure 2 E€ect of intracerebroventricular injection of the P2
receptor antagonists suramin (7 nmol (A)) and pyridoxal-phos-
phate-6-azophenyl-2',4'-disulphonic acid (PPADS; 7 nmol, 70 nmol
and 0.2 mmol (B)) or arti®cial cerebrospinal ¯uid (aCSF) on body
temperature in rats at 258C ambient temperature. Data are presented
as mean+s.e. Numbers in parentheses indicate sample sizes. Arrow-
head indicates time of injections. *Signi®cant di€erence in body
temperature between rats treated into the third cerebral ventricle with
suramin (A) or PPADS (B) and rats treated with aCSF, P50.05.
Figure 3 E€ect of intraperitoneal injection of the ATP analogue
a,b-methyleneATP (a,b-meATP; 0.2 mmol), P2 receptor antagonist
suramin (7 nmol) or arti®cial cerebrospinal ¯uid (aCSF) on body
temperature in rats at 258C ambient temperature. Data are presented
as mean+s.e. Numbers in parentheses indicate sample sizes. Arrow-
head indicates time of injections.
British Journal of Pharmacology vol 135 (8)

A.V. Gourine et al
Purinergic mechanisms and thermoregulation
2051
Figure 4 E€ect of intracerebroventricular injection of the ATP analogue a,b-methyleneATP (a,b-meATP; 0.2 mmol (A, B, C)),
P2 receptor antagonist suramin (7 nmol (D, E, F)), mixture of a,b-meATP (0.2 mmol) with suramin (7 nmol (A)) or arti®cial
cerebrospinal ¯uid (aCSF) on body temperature in rats at 108C (A, D), 258C (B, E) and 308C (C, F) ambient temperatures.
Data are presented as mean+s.e. Numbers in parentheses indicate sample sizes. Arrowhead indicates time of injections.
*Signi®cant di€erence in body temperature between rats treated into the third cerebral ventricle with a,b-meATP (B, C) or
suramin (D, E, F) and rats treated with aCSF, P50.05. {Signi®cant di€erence in body temperature between rats treated into the
third cerebral ventricle with a,b-meATP and rats treated with aCSF or rats treated with mixture of a,b-meATP with suramin,
P50.05.
(P50.05) lower (on average by 0.98C) than in rats treated
with aCSF 30 ± 75 min after the injection (Figure 4D).
than the Tb of febrile rats 40 min after intrahypothalamic
administration of aCSF (38.42+0.108C; P50.05). As shown
in Figure 5C, i.p. injection of a,b-meATP (0.2 mmol) at the
peak of the LPS-induced fever had no signi®cant e€ect on
Tb.
Experiment 4
E€ect of i.c.v. or intrahypothalamic injection of the ATP
analogue a,b-meATP on Tb during LPS-induced fever in
rats Following intraperitoneal injection of LPS a fever
reached a maximal Tb (around 398C) 2.5 h after injection
(Figure 5). a,b-meATP (0.2 mmol) injected i.c.v. or into the
anterior hypothalamus at the peak of the LPS-induced fever
caused profound decrease in febrile Tb (Figure 5A, B). Ten
min after i.c.v. injection of a,b-meATP Tb of febrile rats
decreased to 37.84+0.198C, some 0.98C lower that the Tb of
febrile rats 10 min after i.c.v. treatment with aCSF
(38.74+0.198C; P50.05). There were no di€erences in Tb
between febrile rats treated with either a,b-meATP or aCSF
25 min or later after the injections (Figure 5A). The decrease
in Tb of febrile rats induced by intrahypothalamic injection
of a,b-meATP developed slower and lasted longer (Figure
5B) compared to the response evoked by this ATP analogue
injected into the third cerebral ventricle. Forty min after
injection of a,b-meATP into the anterior hypothalamus Tb
of febrile rats decreased to 37.43+0.388C, some 1.08C lower
The e€ect of a,b-meATP injected i.c.v. on Tb of febrile rats
was attenuated signi®cantly by the P2 receptor antagonist
suramin (Figure 6). Ten min after i.c.v. injections, Tb of rats
treated with a,b-meATP was 37.85+0.168C, whereas the Tb
of rats treated with aCSF was 38.67+0.158C (P50.05), and
the Tb of rats injected with mixture of a,b-meATP and
suramin was 38.42+0.178C (P50.05).
Experiment 5
E€ect of i.c.v. injection of the P2 receptor antagonists suramin
and PPADS on Tb during LPS-induced fever in rats Treat-
ment with suramin (7 nmol) or PPADS (70 nmol) resulted in
an enhanced initiation of LPS-induced fever in rats (Figure
7A,B). The Tb of rats treated with LPS and aCSF (i.c.v.)
were 37.69+0.158C, 37.32+0.158C and 37.29+0.118C, 20, 40
and 60 min after injection, respectively. At these time points,
Tb of LPS-treated rats given suramin (i.c.v.) were
38.33+0.138C (P50.05), 38.27+0.148C (P50.05) and
British Journal of Pharmacology vol 135 (8)

2052
A.V. Gourine et al
Purinergic mechanisms and thermoregulation
Figure 6 E€ect of intracerebroventricular injection of the ATP
analogue a,b-methyleneATP (a,b-meATP; 0.2 mmol), mixture of a,b-
meATP (0.2 mmol) with P2 receptor antagonist suramin (7 nmol) or
arti®cial cerebrospinal ¯uid (aCSF) on body temperature at the peak
of fever induced by E.coli lipopolysaccharide (LPS, 50 mg kg71) in
rats (258C ambient temperature). Data are presented as mean+s.e.
Numbers in parentheses indicate sample sizes. a,b-meATP, mixture of
a,b-meATP with suramin or aCSF were injected 2.5 h after
intraperitoneal injection of LPS (the time of LPS injection not
shown). Arrowhead indicates time of injections. *Signi®cant
di€erence in body temperature between LPS-treated rats injected
into the third cerebral ventricle with a,b-meATP and rats injected
with aCSF, P50.05. {Signi®cant di€erence in body temperature
between LPS-treated rats injected into the third cerebral ventricle
with a,b-meATP and rats injected with mixture of a,b-meATP with
suramin, P50.05.
Discussion
In the present study, the role of extracellular ATP, acting on
P2 receptors, in central mechanisms of thermoregulation was
investigated by studying the e€ects of both a stable ATP
analogue and two P2 receptor antagonists injected into the
third cerebral ventricle on Tb in conscious rats at various
ambient temperatures and during fever.
It was found that the e€ect of activating or antagonizing
brain P2 receptors on Tb in rats was dependent on the
ambient temperature, and also on whether or not the animals
were febrile. Since deep Tb is similar at 108, 258 and 308C
ambient temperatures in rats, the e€ects of the ATP analogue
and P2 receptor antagonists, appears to depend on the level
of a€erent information received by the thermoregulatory
centres of the hypothalamus. The magnitude of the changes
of Tb evoked by activation and blockade of P2 receptors
during fever (when Tb is elevated) was signi®cantly di€erent
from that observed in afebrile rats at the same ambient
temperature, suggesting that the e€ects of a,b-meATP and P2
receptor antagonists on Tb are also in¯uenced by pyrogenic
stimulation.
As a brief summary of all the results, it was shown that the
stable ATP analogue a,b-meATP after injection into the third
cerebral ventricle decreases Tb in rats only when the activity
of mechanisms responsible for heat production is enhanced
and the activity of mechanisms responsible for heat loss is
suppressed (i.e. at low ambient temperature and during
fever). On the other hand, P2 receptor antagonists induce an
increase in Tb in conditions at or above thermoneutrality,
when heat production is minimal and the mechanisms
responsible for heat loss are active or facilitated. In addition,
suramin and PPADS injected at the time when fever was
already developed did not alter febrile Tb, suggesting that
Figure 5 E€ect of intracerebroventricular (A), intrahypothalamic
(B) and intraperitoneal (C) injection of the ATP analogue a,b-
methyleneATP (a,b-meATP; 0.2 mmol) or arti®cial cerebrospinal ¯uid
(aCSF) on body temperature at the peak of fever induced by E.coli
lipopolysaccharide (LPS, 50 mg kg71
)
in rats (258C ambient
temperature). Data are presented as mean+s.e. Numbers in
parentheses indicate sample sizes. a,b-meATP or aCSF were injected
2.5 h after intraperitoneal injection of LPS. Arrowheads indicate time
of injections. *Signi®cant di€erence in body temperature between
LPS-treated rats injected into the third cerebral ventricle (A) or into
the anterior hypothalamus (B) with a,b-meATP and rats injected with
aCSF, P50.05.
37.94+0.168C (P50.05) 20, 40 and 60 min after injections,
respectively (Figure 7A). Similarly, 40 min after LPS injection
Tb of rats pretreated i.c.v. with PPADS increased to
38.48+0.208C, some 1.08C higher than the Tb of LPS-
injected rats 40 min after i.c.v. administration of aCSF
(37.47+0.148C, P50.05; Figure 7B). These di€erences in
Tb between rats treated with suramin and aCSF and between
rats treated with PPADS and aCSF disappeared 80 ± 100 min
and 55 ± 65 min following injection of LPS, respectively,
indicating that the late phase of fever was una€ected by these
P2 receptor antagonists (Figure 7A,B). The changes in Tb in
response to LPS followed at 2.5 h by i.c.v. administration of
suramin are shown in Figure 7C. Suramin (Figure 7C) and
also PPADS (data not shown) injected when LPS-induced
fever was already developed (i.e. 2.5 h after LPS injections)
did not induce signi®cant changes in febrile Tb. Intraper-
itoneal injection of suramin (7 nmol) at the peak of the LPS-
induced fever also had no signi®cant e€ect on Tb (data not
shown).
British Journal of Pharmacology vol 135 (8)

A.V. Gourine et al
Purinergic mechanisms and thermoregulation
2053
Hardy, 1974). Hypothalamic warm-sensitive neurones also
provide descending control of thermoregulatory e€ectors and
are believed to be the site of action of pyrogens, which cause
fever by reducing their discharge (for recent reviews, see
Boulant, 1999; 2000). The above hypothesis is supported
strongly by the results showing that during fever a,b-meATP
induces signi®cant decrease in febrile Tb when injected not
only into the third ventricle but also directly into the anterior
hypothalamus. Although, most of the data from this study ®t
well within this hypothesis, it is clear that further studies are
needed to determine whether hypothalamic warm-sensitive
neurones are indeed the site of action of ATP analogue and
P2 receptor antagonists on Tb regulation.
When a,b-meATP and suramin were injected i.p. at the
same amounts that were shown to in¯uence Tb after i.c.v.
injection, they failed to induce any changes in Tb in either
afebrile or febrile rats. These data suggest that the e€ects of
the ATP analogue and P2 receptor antagonist on Tb are
evoked within the central nervous system and not on the
periphery (due to a possible leakage into the circulation).
There is evidence that P2X2 receptors are dense in the
hypothalamus (Xiang et al., 1998; Kanjhan et al., 1999).
Using in situ hybridization and RT ± PCR analysis P2X2,
P2X3, P2X4, P2X6 and P2X7 subunits, but not P2X1
subunits, have been also shown to be expressed in neurones
of the hypothalamic supraoptic and paraventricular nuclei
(Shibuya et al., 1999). These data indicate that the e€ects of
a,b-meATP and P2 receptor antagonists on Tb may be a
result of their direct action on the hypothalamic neurones.
However, at present we can only speculate about which P2
receptors are a€ected by a,b-meATP, suramin and PPADS
and responsible for changes in Tb when drugs are
administered into the third cerebral ventricle. a,b-meATP
was used in this study because, in contrast to the endogenous
ligand ATP, it is relatively stable to hydrolysis by ecto-
ATPases (Welford et al., 1986). This stability of the agonist
was important for our study, since changes in Tb develop
relatively slow. The e€ects we observed with a,b-meATP,
suramin and PPADS may suggest the involvement of P2X1
and/or P2X3 receptors (for recent review see North &
Surprenant, 2000). On the other hand, these receptors are
known to be desensitized rapidly by a,b-meATP (for reviews,
see Ralevic & Burnstock, 1998; North & Surprenant, 2000).
However, in the majority of our experiments the e€ects of
a,b-meATP on Tb were opposite to the e€ects of suramin
(and PPADS) and at low ambient temperature and during
fever were blocked by suramin, suggesting that these e€ects
are due to a,b-meATP-induced activation of certain P2
receptors, rather than desensitisation. On the other hand, in
rats maintained at 308C a,b-meATP induced changes in Tb
which were similar in direction (i.e. an increase) to that
observed after injection of suramin, suggesting that this e€ect
of ATP analogue may be a result of desensitization of certain
P2 receptors, rather then their activation. Also, it was found
that in ®ve out of 13 rats maintained at 258C the Tb notably
increased 120 ± 150 min after a,b-meATP (0.2 mmol) admin-
istration (although the mean di€erence was not statistically
signi®cant). It is possible that this late response is mediated
by P2 receptors with di€erent pharmacology than those
mediating the initial response (perhaps by the receptors from
the P2Y family) or by the P2 receptors located in distant
brain regions (e.g. caudally), or both. Interestingly, it was
Figure 7 E€ect of intracerebroventricular injection of the P2
receptor antagonists suramin (7 nmol (A)) and pyridoxal-phos-
phate-6-azophenyl-2',4'-disulphonic acid (PPADS; 70 nmol (B)) or
arti®cial cerebrospinal ¯uid (aCSF) on body temperature during fever
induced by E.coli lipopolysaccharide (LPS, 50 mg kg71) in rats (258C
ambient temperature). Suramin was injected immediately before (A)
or 2.5 h after (C) intraperitoneal injection of LPS. PPADS was
injected immediately before (B) intraperitoneal injection of LPS.
Data are presented as mean+s.e. Numbers in parentheses indicate
sample sizes. Arrowhead indicates time of injections. *Signi®cant
di€erence in body temperature between LPS-treated rats injected into
the third cerebral ventricle with suramin (A) or PPADS (B) and rats
injected with aCSF, P50.05.
development of the febrile response prevents the e€ects of P2
receptor antagonists on thermoregulation. Potentiation of the
initial phase of fever when P2 receptor antagonists were given
i.c.v. before LPS injection was rather minor and changes in
Tb of rats treated with suramin or PPADS followed by LPS
did not exceed changes observed in rats treated with either of
these antagonists alone.
These data are consistent, if it is assumed that ATP, acting
on P2 receptors, modulates transmission in a hypothalamic
neuronal pathway relaying a€erent information from skin
warm-receptors to the neurones responsible for activation of
heat loss. Analysis of these results allow us to suggest, that
hypothalamic warm-sensitive neurones are the likely targets
for the action of ATP revealed by the use of the ATP
analogue and P2 receptor antagonists in our experiments.
About 30% of the anterior hypothalamic neurones are warm-
sensitive, many receiving input from a€erent pathways
relaying information from skin thermoreceptors (Boulant &
British Journal of Pharmacology vol 135 (8)

2054
A.V. Gourine et al
Purinergic mechanisms and thermoregulation
also found that the time course of the hyperthermia induced
by suramin in afebrile rats was markedly di€erent from that
induced by PPADS (Figure 2). This could be due to several
factors such as the rate of antagonist di€usion to the target
structure, stability of the antagonist, pro®le of P2 receptors
a€ected, or degree of suppression of the nucleotidase activity
induced by these compounds (Hourani & Chown, 1989;
Ziganshin et al., 1995). It is clear that the development of
new highly selective P2X receptor agonists and antagonists is
needed to investigate further the e€ects of purines on Tb.
We suggested initially that extracellular ATP acts to induce
the release of cytokines to play an important role in the
mechanisms for the development of fever. This was based on
the evidence that fever is initiated by IL-1b (and probably
other cytokines) at the level of the anterior hypothalamus
(Klir et al., 1994; Gourine et al., 1998) and that ATP is a
potent inducer of IL-1a, IL-1b and tumour necrosis factor
release (Perregaux & Gabel, 1998; Hide et al., 2000; Mehta et
al., 2001; Solle et al., 2001). The e€ect of ATP on cytokine
release is thought to be mediated via P2X7 receptors (Hide et
al., 2000; Mehta et al., 2001; Solle et al., 2001) which are
present in the hypothalamus (Shibuya et al., 1999). P2X7
receptors are sensitive to the antagonists suramin and
PPADS (North & Surprenant, 2000). If our hypothesis is
correct, and ATP, via cytokine release, plays a role in fever
development, an attenuation of fever by suramin and PPADS
would have been expected. However, fever was virtually
una€ected, or even exaggerated, following administration of
these P2 receptor antagonists into the brain, suggesting that
at the hypothalamic level either ATP is not involved in the
mechanisms of cytokine release, or alternatively that ATP-
induced cytokine production is not essential for the
development of fever following LPS challenge.
purinergic signalling in the febrile response could be obtained
in experiments, investigating the development of fever in
conditions when speci®c P2X7 receptor agonists or antago-
nists are applied peripherally in amounts sucient to a€ect
function of these receptors.
In conclusion, this report describes the involvement of
purinergic signalling in central mechanisms of thermoregula-
tion. Since regulation of Tb is severely disrupted under
anaesthesia, this initial pharmacological study was conducted
in conscious rats investigating the e€ects of a stable ATP
analogue and P2 receptor antagonists injected into the third
cerebral ventricle on Tb at various ambient temperatures and
during fever. Analysis of the di€erences in Tb responses
observed following administration of a,b-meATP, suramin
and PPADS into the third cerebral ventricle at di€erent
ambient temperatures and during fever, allow us to suggest
that extracellular ATP, acting on P2 receptors is involved in
central mechanisms of Tb regulation, perhaps by modulating
transmission in the hypothalamic neural pathway from
peripheral warm-receptors to the neurones responsible for
the stimulation of heat loss. We speculate, that hypothalamic
warm-sensitive neurones are the likely site of action of ATP
analogue and P2 receptor antagonists in relation to
regulation of Tb.
For further understanding of the role of purinergic
signalling in the central mechanisms of thermoregulation,
the e€ects of P2 receptor agonists and antagonists applied
into the cerebral ventricles on the peripheral mechanisms of
heat loss (e.g. peripheral (tail) blood ¯ow) and heat
production (e.g. thermogenesis in the brown fat) could be
investigated in anaesthetized animals. The hypothesis that
population of hypothalamic warm-sensitive neurones is the
site of action of ATP analogue and P2 receptor antagonists
on Tb, can be tested e€ectively only in a more reduced
preparation, such as hypothalamic slice, where P2X receptor
pro®le as well as the e€ects of ATP, selective P2X receptor
agonists and antagonists on activity and thermosensitivity of
the hypothalamic neurones could be determined.
It is possible that P2X7 receptors in the peripheral tissues
may play an important role in mediating LPS-induced
responses, including cytokine production and fever. It has
been found recently, that P2X7 receptor contains a conserved
LPS-binding domain (Denlinger et al., 2001), suggesting that
LPS may have a direct e€ect on P2X7 function and therefore
on ATP-induced responses mediated by this receptor,
including cytokine release. Study of the potential role of
peripheral P2 receptors in fever was not a subject of the
current study. However, exciting new data on the role of
We thank Drs Brian King and Andrew J. Lawrence for critical
reviews of the manuscript.
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(Received October 18, 2001
Revised December 21, 2001
Accepted February 14, 2002)
British Journal of Pharmacology vol 135 (8)