A specific taurine recognition site in the rabbit brain is responsible for taurine effects on thermoregulation

Article abstract of DOI:10.1038/sj.bjp.0705274

1. Taurine and GABA are recognized as endogenous cryogens. In a previous study, some structural analogues of taurine, namely 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (TAG), 2-aminoethylarsonic (AEA), 2-hydroxyethanesulfonic (ISE) and (

Full text of DOI:10.1038/sj.bjp.0705274

British Journal of Pharmacology (2003) 139, 487–494
&
2003 Nature Publishing Group All rights reserved 0007–1188/03
$25.00
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A specific taurine recognition site in the rabbit brain is responsible
for taurine effects on thermoregulation
,
1
3
Maria Frosini, Casilde Sesti, Simona Saponara, Lorenzo Ricci, Massimo Valoti, Mitri Palmi,
1
1
1
1
*
2
1
Fabrizio Machetti & Giampietro Sgaragli
1
2
Istituto di Scienze Farmacologiche, Universita
`
Istituto di Chimica dei Composti Organo Metallici-CNR c/o Dipartimento di Chimica Organica ‘U. Schiff’, Universita
di Siena, Nuovo Polo Scientifico, Viale A. Moro 2, lotto C, Siena 53100, Italy and
di Firenze,
`
Firenze, Italy
1
Taurine and GABA are recognized as endogenous cryogens. In a previous study, some structural
analogues of taurine, namely 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (TAG),
-aminoethylarsonic (AEA), 2-hydroxyethanesulfonic (ISE) and (7)cis-2-aminocyclohexane sulfonic
2
3
acids (CAHS) have been shown to displace [ H]taurine binding from rabbit brain synaptic membrane
preparations, without interacting either with GABA-ergic systems, nor with taurine uptake
mechanism, thus behaving like direct taurinergic agents.
2
To answer the question whether the role of taurine as an endogenous cryogen depends on the
activation of GABA receptors or that of specific taurine receptor(s), taurine or the above structural
analogues were injected intracerebroventricularly in conscious, restrained rabbits singularly or in
combination and their effects on rectal (RT)- and ear – skin temperature and gross motor behavior
(
GMB) were monitored.
Taurine (1.2 ꢀ 10ꢁ – 4.8 ꢀ 10 mol) induced a dose-related hypothermia, vasodilation at ear
6
ꢁ5
3
ꢁ5
vascular bed and inhibition of GMB. CAHS, at the highest dose tested (4.8 ꢀ 10 mol) induced a
taurine-like effect either on RT or GMB. On the contrary ISE, injected at the same doses of taurine,
induced a dose-related hyperthermia, vasoconstriction and excitation of GMB. AEA and TAG caused
ta r ed a ot sme -e rne tl .a ted hyperthermia, but at doses higher than 1.2 ꢀ 10ꢁ mol caused death within 24 h after
7
ꢁ
5
ꢁ6
4
CAHS (4.8 ꢀ 10 mol) antagonized the hyperthermic effect induced by TAG (1.2 ꢀ 10 mol),
ꢁ ꢁ5
8
AEA (1.2 ꢀ 10 mol) or ISE (4.8 ꢀ 10 mol).
5 In conclusion, these findings may indicate the existence of a recognition site specific for taurine,
responsible for its effects on thermoregulation.
British Journal of Pharmacology (2003) 139, 487 – 494. doi:10.1038/sj.bjp.0705274
Keywords: Taurine; taurine derivatives; thermoregulation; taurine binding site; taurine agonist; taurine antagonist
Abbreviations: AEA, 2-aminoethylarsonic acid; b-ALA, b-alanine; CAHS, (7)cis-2-aminocyclohexane sulfonic acid;
ISE, 2-hydroxyethanesulfonic acid; TAG, 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide;
TAHS, (7)trans-2-aminocyclohexane sulfonic acid
Introduction
Amino acids have recently received increased attention with
regard to their role in thermoregulation as neurotransmitters
within the central nervous system (CNS) (Zhang et al., 1995;
Monda et al., 1998). This is particularly true for GABA and
taurine as endogenous cryogens (Frosini et al., 2000). The
pathways that down regulate body temperature are of over-
whelming interest in view of the effectiveness of hypothermia
in protecting from ischemia-reperfusion damage to the brain of
patients surviving cardiac arrest (Safar & Kochanek, 2002).
Nevertheless, the physical and pharmacological treatments
so far adopted to attain a mild hypothermia have required
such a long time (8 – 12h) (The Hypothermia after Cardiac
Arrest Study Group, 2002) that they may compromise its
beneficial effects. It would therefore be valuable for
emergency-care medicine to develop new drugs that could
rapidly and safely induce hypothermia. To this end, it is
important to improve the knowledge about the role of
endogenous cryogens.
A recent study performed in the conscious rabbit has
demonstrated that heat stress modifies brain metabolism of
taurine and GABA so that increases in cerebrospinal fluid
(CSF) concentrations of these amino acids were induced. This
rise, which could not be ascribed to blood contamination
resulting from a transient opening of the blood – CSF barrier,
was probably aimed at counteracting the hyperthermia
promoted by exposure to heat. Moreover, under these
conditions, taurine appeared to play a prominent role in
comparison to GABA. When the time course for heat-stress-
induced changes in rectal temperature (RT) and CSF taurine
contents were compared, in fact, it was found that RT
*
Author for correspondence; E-mail: frosinim@unisi.it
3
Current address: Department of Pharmacology, Cornell University,
New York, NY, U.S.A.

488
M. Frosini et al
Taurine and thermoregulation in the rabbit brain
decreased as soon as taurine CSF contents rose, while CSF
GABA levels were significantly higher just before RT had
regained basal values (Frosini et al., 2000). Furthermore,
central or systemic injection of either GABA or agonists of
(St Louis, MA, U.S.A.). TAG, synthesized as described by
Girard et al. (1982), was a generous gift of Dr G.G. Yarbrough
from Merk Frosst Laboratories (Quebec, Canada). The purity
1
of all compounds were evaluated above 95% by H NMR or
high-perfomance liquid chromatography.
either GABA
A
or GABA
B
receptors usually diminishes core
temperature, whereas such injection of antagonists of either
class induces hyperthermia (Serrano et al., 1985). Yakimova
et al. (1996), however, have demonstrated that the GABA
Animals
effect on thermoregulation is essentially mediated by GABA
receptors. This is confirmed by the fact that mice lacking
the GABAB(1) subunit of the GABA receptors did not
become hypothermic when GABA agonists were applied
Schuler et al., 2001). Taurine, as well as GABA, when
B
Adult male New Zealand albino rabbits (Charles River, Calco,
Como, Italy) weighing 2.0 – 2.5 kg were kept in large
individual cages under a 12: 12h day – night cycle at 201C
B
B
a
ambient temperature (T ). Drinking water and conventional
(
laboratory rabbit food were available ad libitum. Before the
experimental session, the animals were habituated to restraint
and to the rectal probe in order to minimize the stress
response.
injected intracerebroventricularly (i.c.v.) induces dose-related
hypothermia accompanied by depression of gross motor
behavior and peripheral vasodilation (Sgaragli et al., 1981),
whereas the taurine antagonist 6-aminomethyl-3-methyl-4H-
1,2,4-benzothiadiazine-1,1-dioxide (TAG), increases core
temperature (Sgaragli et al., 1994). It is not clear, however,
Surgery
which receptor(s) mediates the hypothermic effect of taurine.
Since taurine binds both to GABA
All the experiments were performed in strict compliance with
the recommendations of the EEC (86/609/CEE) for the care
and use of laboratory animals and were approved by the
Animal Care and Ethics Committee of the University of Siena,
Italy.
A B
and GABA receptors
(
Krogsgaard-Larsen et al., 1980; Kontro & Oja, 1990;
Frosini et al., 2003), it may affect body temperature by
interacting with GABA-ergic systems. However, there is a
large body of evidence that taurine elicits a number of
neurophysiological effects distinguishable from those afforded
by GABA (Hayes et al., 1975; Huxtable, 1989; Kamisaki et al.,
At least 3 weeks before the experiment, animals were
implanted with a cannula for i.c.v. injection at the level of the
lateral ventricle. The stereotaxic technique has been described
elsewhere (Palmi et al., 1994). Anesthesia was induced by
1
993; Hussy et al., 2001; Tuz et al., 2001). Moreover,
ꢁ1
taurine desynchronizes while GABA, muscimol and baclofen
synchronize motor and limbic cortex activity patterns,
suggesting that their effects could depend on the interaction
with different neuronal pathways afferent to the cortical
areas monitored (Sgaragli et al., 1978; Sesti et al., 1999).
Thus, it is conceivable that taurine exerts its biologic
activity by interacting with a specific receptor. In a previous
study, some direct taurinergic compounds were identified
intramuscular (i.m.) injection of xylazine chloride (10 mg kg ,
s
Rompun Vet., Bayer AG, Germany) and ketamine hydro-
ꢁ1
s
chloride (35 mg kg , Ketavet , Parke Davis/Warner-Lam-
bert, U.S.A.). Under sterile precautions, the dorsal structure of
the skull was exposed by a midline skin incision and, after
drilling a hole near the bregma according to stereotaxic
coordinates (Sawyer et al., 1954), a thin stainless-steel cannula
was inserted. Spontaneously, CSF outflow indicated the
correct position of the cannula. The cannula was fixed to the
skull with stainless-steel screws and dental acrylic cement,
occluded with an obturator, and the skin incision was sutured
around the cannula.
(
(
Frosini et al., 2003). 2-Aminoethylarsonic acid (AEA),
7)cis-2-aminocyclohexane sulfonic acid (CAHS), 2-hydro-
xyethanesulfonic acid (ISE) and TAG proved to displace
3
[
H]taurine binding without interacting either with both
GABA receptors or taurine- and GABA-uptake systems
and not affecting GABA-transaminase activity. The effects
of these compounds on body temperature have been studied,
and the findings so far collected may indicate that the effects of
After surgery, rabbits were injected for at least 5 days with
s
the following drugs: prednisolone acetate (Novosterol ,
ꢁ1
Vetem S.p.A., Italy), 10 mg day i.m.; enrofloxacin, (Bay-
s
ꢁ1
tril , Bayer AG, Germany) 25 mg day i.m. The animals were
allowed to recover for at least 15 days.
taurine on thermoregulation is mediated by
taurinergic pathway.
a specific
Experimental protocol
Methods
Conscious animals were individually housed in a thermo-
statted chamber set at neutral temperature (201C). Concur-
rently, ear – skin temperature (EST) and RT were measured to
an accuracy of 0.11C, using thermocouples attached to the ear
and inserted 10 cm deep into the rectum, respectively, every
5 min. The thermocouple thermometer was connected to a
personal computer with an Isothermex program (Columbus
Instr., Columbus, OH, U.S.A.). Temperature was monitored
for at least 1 h before the experimental session and up to
325 min after the treatment. RT was again monitored 24 h after
i.c.v. injection.
Materials
Taurine and ISE were purchased by Sigma. AEA was prepared
by periodate oxidation of 2-[(2-hydroxyethyl)amino]ethylarso-
nic acid, itself made by treating 2-chloroethylarsonic acid with
ethanolamine (Geoghegan & Dixon, 1989). CAHS was
prepared from 2-aminobenzenesulfonic acid by catalytic
hydrogenation as reported by Egli & Eugster (1975). TAHS
was prepared from cyclohexene by sulfur monochloride
addition, followed by oxidation to 2-chlorosulfonic acid and
substitution of chlorine as reported by Machetti et al. (2000).
b-alanine (b-ALA) was purchased from Sigma Chemical Co
To isolate any experimental artefacts arising from animal
restraint, manipulation or i.c.v. injection, all the rabbits used
in the present study were injected with 10 ml pyrogen-free water
British Journal of Pharmacology vol 139 (3)

M. Frosini et al
Taurine and thermoregulation in the rabbit brain
489
(
vehicle) and RT was monitored as previously described. Only
lasting motor incoordination and loss of righting reflex.
Excitation of GMB: +, alertness; ++, alertness and increased
panting rate; +++, alertness and elevated panting rate,
++++, alertness, sustained panting rate and episodic
convulsive attacks.
those animals that did not exhibit significant changes in RT
were used in the subsequent experiments. Among all the
rabbits used in this study, six were randomly selected to form
the control group in which the vehicle was injected i.c.v. and
RT monitored.
Compounds dissolved in pyrogen-free water were adminis-
tered i.c.v. (range of doses: 1.2 ꢀ 10ꢁ – 4.8 ꢀ 10 mol) in a
final volume of 10 or 20 ml with an Agla micrometer-operated
syringe (Burroughs Wellcome and Co., London, U.K.) to
randomly selected rabbits (four to six animals/group/dose).
After i.c.v. injections, gross motor behavior (GMB) was
observed during the entire experimental session. The following
arbitrary scale to describe GMB changes was chosen: 0 ¼ no
effect. Depression of GMB: ꢁ, sedation and light motor
incoordination; ꢁꢁ, sedation, drowsiness and motor incoor-
dination; ꢁꢁꢁ, sedation, drowsiness and long-lasting motor
incoordination; ꢁꢁꢁꢁ sedation, drowsiness, severe and long-
8
ꢁ5
Statistical analysis
Values are expressed as mean7s.e.m. and reported as changes
in RT or EST (DRT and DEST). The area under the
experimental curve (AUC) relative to RT was calculated by
a combined linear logarithmic trapezoidal method using
Graphpad-Prism II program (GraphPad Software Inc.,
San Diego, CA, U.S.A.). The comparison between AUC(0 –
2 4 h) of control (vehicle-injected) vs treated (taurine- or
its derivatives-injected) animals or between the AUC(0 – 24 h)
relative to different doses of the compounds, was performed
Figure 1 Mean (7 s.e.m.) changes in rabbit rectal- (RT) and ear – skin temperature (EST) following i.c.v. administration of
ꢁ
6
ꢁ5
ꢁ5
taurine (panel a) and TAG (panel b). The doses injected for taurine were 1.2 ꢀ 10 (K, n ¼ 4), 1.2 ꢀ 10 (~, n ¼ 4), 2.4 ꢀ 10 (&,
ꢁ5
ꢁ7
ꢁ6
ꢁ5
n ¼ 6) and 4.8 ꢀ 10 mol (^, n ¼ 6); for TAG 1.2 ꢀ 10 (!, n ¼ 6), 1.2 ꢀ 10 (K, n ¼ 4), 1.2 ꢀ 10 (~, n ¼ 3) mol. Vehicle alone
pyrogen-free water, dotted line) was injected at the same volume used for the compounds to a group of six rabbits. EST was
measured in animals treated with the highest dose ( , dotted line). To improve clarity, s.e.m. of vehicle RT values and EST values are
(
*
not depicted. Arrows indicate time of i.c.v. injection. In the right panels, the area under the experimental curve (AUC(0 – 24 h)) of RT
changes relative to each dose/compound is shown. The comparison between AUC(0 – 24 h) of vehicle- vs taurine- or TAG-injected
rabbits or between AUC(0 – 24 h) of the different doses of each compound was performed by using Student’s t-test. *Po0.05,
#
##
*
*Po0.01, ***Po0.001 vs vehicle. Po0.05, Po0.01 vs the previous dose.
British Journal of Pharmacology vol 139 (3)

490
M. Frosini et al
Taurine and thermoregulation in the rabbit brain
5
using Student’s t-test. A P-value o0.05 was considered
of 4.8 ꢀ 10ꢁ mol, it was marked and long lasting, peaked with
a ꢁDRT value of 2.570.31C at 100 min after injection and was
accompanied by increase in EST. This peaked at 50 min with a
DEST value of 6.372.81C. Subsequently, EST decreased
owing to a steady vasocostriction beyond basal values with a
significant.
Results
ꢁ
DEST value of 3.672.11C at 125 min, then remained almost
Effects of i.c.v. injection of pyrogen-free water on RT and
EST
stable during the entire experimental session. EST shift marked
the start of the slow recovery of RT to baseline values that
were attained at 300 min. After 24 h, all taurine-treated
animals were normothermic. Taurine was shown to induce
depression of GMB. This depression depended on the
dose of taurine and was characterized by sedation and
light motor incoordination (1.2 ꢀ 10ꢁ mol dose; arbitrary
scale, ꢁꢁ), sedation, drowsiness and motor incoordination
To isolate any experimental artefacts arising from animal
restraint, manipulation or i.c.v. injection, in a group of six
rabbits treated by i.c.v. injection of 10 ml pyrogen-free water,
RT and EST were monitored. RT and EST basal values were,
respectively, 39.070.1 and 20.870.21C. Pre- and postinjection
values of temperatures showed no statistically significant
differences (data not shown). GMB was not affected by the
treatment.
5
ꢁ
5
(2.4 ꢀ 10 mol dose; arbitrary scale, ꢁꢁꢁ) and, finally,
sedation, drowsiness, long-lasting motor incoordination
ꢁ5
and loss of righting reflex (4.8 ꢀ 10 mol dose; arbitrary
scale, ꢁꢁꢁꢁ). After 24 h, the behavior was comparable to that
of controls.
Effects of i.c.v. injection of taurine, TAG, AEA, ISE,
CAHS, TAHS and b-ALA on RT and EST
ꢁ7
As shown in panel b, TAG, at the dose of 1.2 ꢀ 10 mol, did
modify RT by inducing hyperthermia. RT increased
regularly peaking with an increment of 1.370.31C at 100 min
(AUC(0 – 24 h) TAG vs control, Po0.01, n ¼ 6). After 24 h,
As reported in Figure 1, (panel a) taurine induced a dose-
related hypothermia, accompanied by muscle tone reduction
and vasodilation in the vascular bed of the ear. Hypothermia
was significant at 1.2 ꢀ 10ꢁ mol dose and, at the highest dose
ꢁ6
RT had regained basal values. The 1.2 ꢀ 10 mol dose (n ¼ 4)
5
produced a sustained and long-lasting hyperthermia that
Figure 2 Mean (7 s.e.m.) changes in rabbit rectal- (RT) and ear –
skin temperature (EST) following i.c.v. administration of AEA. The
Figure 3 Mean (7 s.e.m.) changes in rabbit rectal- (RT) and ear –
skin temperature (EST) following i.c.v. administration of ISE. The
ꢁ8
ꢁ7
ꢁ7
ꢁ6
doses injected were 1.2 ꢀ 10 (*, n ¼ 6), 1.2 ꢀ 10 (!, n ¼ 5),
doses injected for ISE were 1.2 ꢀ 10 (n ¼ 4), 1.2 ꢀ 10 (K, n ¼ 4),
ꢁ
6
ꢁ5
ꢁ5 ꢁ5
1
(
.2 ꢀ 10
pyrogen-free water, dotted line) was injected at the same volume
used for the compounds to a group of six rabbits. EST was measured
in animals treated with the highest dose ( , dotted line). To improve
(K, n ¼ 4), 1.2 ꢀ 10
(~, n ¼ 3) mol. Vehicle alone
1.2 ꢀ 10 (~, n ¼ 4) and 4.8 ꢀ 10 mol (^, n ¼ 4). Vehicle alone
(pyrogen-free water, dotted line) was injected at the same volume
used for the compounds to a group of six rabbits. EST was measured
*
*
in animals treated with the highest dose ( , dotted line ). To improve
clarity, s.e.m. of vehicle RT values and EST values are not depicted.
Arrows indicate time of i.c.v. injection. In the inset the area under
the experimental curve (AUC(0 – 24 h)) of RT changes relative to each
dose is shown. The comparison between AUC(0 – 24 h) of vehicle- vs
AEA-injected rabbits or between AUC(0 – 24 h) of the different doses
was performed by using Student’s t-test. *Po0.05, **Po0.01,
clarity, s.e.m. of vehicle RT values and EST values are not depicted.
Arrows indicate time of i.c.v. injection. In the inset, the area under
the experimental curve (AUC(0 – 24 h)) of RT changes relative to each
dose is shown. The comparison between AUC(0 – 24 h) of vehicle- vs
ISE-injected rabbits or between AUC(0 – 24 h) of the different doses
was performed by using Student’s t-test. *Po0.05, **Po0.01,
#
##
#
##
*
**Po0.0001 vs vehicle. Po0.05, Po0.01 vs the previous dose.
***Po0.001 vs vehicle. Po0.05, Po0.01 vs the previous dose.
British Journal of Pharmacology vol 139 (3)

M. Frosini et al
Taurine and thermoregulation in the rabbit brain
491
peaked with a DRT value of 2.270.21C at 325 min, and
was still maintained after 24 h (DRT value of 1.670.41C); at
48 h after treatment, however, RT had returned to normal
values (data not shown). At the highest dose tested of
ꢁ
5
1
.2 ꢀ 10 mol, TAG induced a sharp rise in RT, increasing
by 2.770.11C at 175 min. At this time, however, all rabbits
of this group (n ¼ 3) died. TAG did not modify GMB at
ꢁ
7
ꢁ6
the dose of 1.2 ꢀ 10 mol, while at 1.2 ꢀ 10 mol dose, during
the hyperthermic phase, alertness and sustained panting
rate were observed (arbitrary scale, +++). At 1.2 ꢀ 10ꢁ mol
dose, alertness, sustained panting rate and episodic seizures
together with intense kicking were observed (arbitrary
scale, ++++).
5
ꢁ8
ꢁ7
As reported in Figure 2, AEA at 1.2 ꢀ 10 , 1.2 ꢀ 10
,
ꢁ6
ꢁ5
1
.2 ꢀ 10
and 1.2 ꢀ 10 mol dose did remarkably affect
thermoregulation, inducing hyperthermia and vasoconstriction
at ear vascular bed. After the lowest dose injected, RT
increased regularly peaking with an increment of 1.670.31C at
3
25 min (AUC(0 – 24 h) AEA vs control, Po0.01, n ¼ 6). After
ꢁ7
2
4 h, RT had regained basal values. The 1.2 ꢀ 10 mol dose
produced a bimodal effect. Immediately after the injection RT
decreased peaking with a DRT value of 0.470.21C at 50 min
and rose gradually thereafter. At 325 min rabbits were
hyperthermic with a DRT value of 1.270.21C. After 24 h,
hyperthermia was even greater with
1
a DRT value of
.670.41C; after 48 h, however, RT had returned to basal
values (data not shown). None of the five animals injected with
this dose died. The statistical analysis demonstrated a
significant difference of the AUC(0 – 24 h) of AEA vs control
and vs the lowest dose tested (Po0.01 and o0.05, respec-
Figure 4 Mean (7 s.e.m.) changes in rabbit rectal- (RT) and ear –
skin temperature (EST) following i.c.v. administration of CAHS.
ꢁ
7
ꢁ6
The doses injected were 1.2 ꢀ 10 mol (!, n ¼ 4), 1.2 ꢀ 10 (K,
ꢁ5
ꢁ5
n ¼ 4), 1.2 ꢀ 10 (~, n ¼ 4) and 4.8 ꢀ 10 mol (^, n ¼ 6) mol.
Vehicle alone (pyrogen-free water, dotted line) was injected at the
same volume used for the compounds to a group of six rabbits. EST
ꢁ6
tively). The dose of 1.2 ꢀ 10 mol, injected in four rabbits,
induced a marked rise in RT. The increase observed at the end
of the experimental session was 1.870.11C. With this dose two
rabbits died during the subsequent night. The autoptic
examination of their brain did not reveal macroscopic
modifications. The other surviving animals were still hy-
perthermic 24 h after treatment showing a DRT value of
*
was measured in animals treated with the highest dose ( , dotted
line). To improve clarity, s.e.m. of vehicle RT values and EST values
are not depicted. Arrows indicate time of i.c.v. injection. In the inset,
the area under the experimental curve (AUC(0 – 24 h)) of RT changes
relative to each dose is shown. The comparison between AUC(0 – 24 h)
of vehicle- vs CAHS-injected rabbits or between AUC(0 – 24 h) of the
different doses was performed by using Student’s t-test. *Po0.05,
#
##
*
*Po0.01, ***Po0.001 vs vehicle. Po0.05, Po0.01 vs the
2
.270.21C, but returned normothermic within 48 h (data not
previous dose.
ꢁ5
shown). At the highest dose tested (1.2 ꢀ 10 mol), AEA
induces a biphasic effect. After i.c.v injection, RT decreased by
.470.11C after 50 min and sharply rose thereafter showing a
0
1.2 ꢀ 10ꢁ mol (Figure 4). At the 4.8 ꢀ 10 mol dose,
however, it induced hypothermia that peaked with a ꢁDRT
value of 0.870.11C after 100 min (AUC(0 – 24 h) CAHS vs
5
ꢁ5
DRT value of 2.770.11C at 175 min. All animals (n ¼ 3)
injected with this dose died after reaching a DRT value
of 2.770.21C. AEA did not modify GMB at the doses
ꢁ
8
ꢁ7
ꢁ6
ꢁ5
of 1.2 ꢀ 10
,
1.2 ꢀ 10
,
1.2 ꢀ 10 mol, while after
control and vs 1.2 ꢀ 10 mol, Po0.01, n ¼ 6). Hypothermia
ꢁ5
1
.2 ꢀ 10 mol dose, during the hyperthermic phase, it caused
was accompanied by vasodilation at ear vascular bed,
which caused increase in EST, subsequently followed by a
moderate decrease which peaked with a ꢁD value of
3.371.41C at 225 min. RT regained basal values at 150 min.
CAHS slightly affected GMB with sedation and light motor
incoordination during the decrease of RT. These effects were
comparable to those induced by 1.2 ꢀ 10ꢁ mol dose of taurine
(arbitrary scale, ꢁ).
alertness, sustained panting rate and episodic seizures (arbi-
trary scale, ++++).
ꢁ7
ꢁ6
ISE was tested at the doses range of 1.2 ꢀ 10 , 1.2 ꢀ 10
,
ꢁ5
ꢁ5
1
1
.2 ꢀ 10 and 4.8 ꢀ 10 mol (Figure 3, panel b). Both doses of
ꢁ7 ꢁ6
.2 ꢀ 10 and 1.2 ꢀ 10 mol did not modify RT (AUC(0 – 24 h)
5
ISE vs control, NS, n ¼ 4/dose). On the contrary, at either
ꢁ5
ꢁ5
1
.2 ꢀ 10 and 4.8 ꢀ 10 mol doses, it induced a significant
rise in body temperature that peaked with a DRT value of
.870.11C at 325 min and 1.370.41C at 275 min, respectively
b-ALA and TAHS did not induce significant changes of both
ꢁ
8
ꢁ7
0
RT or GMB at all the doses injected (1.2ꢀ 10 , 1.2ꢀ 10
,
1.2 ꢀ 10ꢁ , 1.2 ꢀ 10 , 4.8ꢀ 10 mol) (data not shown).
6
ꢁ5
ꢁ5
(
AUC(0 – 24 h) ISE vs control, Po0.01, n ¼ 4/dose). At
ꢁ5
4
.8 ꢀ 10 mol dose, EST increased, peaking with a D value
of 1.370.41C, which was maintained constant until the end of
the experimental session. After 24 h, RT had gained basal
values. It was only at the highest dose that ISE slightly
stimulated GMB (arbitrary scale, +). CAHS did not affect
significantly RT at the doses of 1.2 ꢀ 10ꢁ , 1.2 ꢀ 10ꢁ6 and
Effects of coadministration of CAHS with TAG, ISE or
AEA
CAHS, injected at the dose of 4.8 ꢀ 10ꢁ mol partially
5
7
ꢁ6
antagonized the hyperthermic effect induced by 1.2 ꢀ 10 mol
British Journal of Pharmacology vol 139 (3)

492
M. Frosini et al
Taurine and thermoregulation in the rabbit brain
Figure 5 Effects of CAHS on TAG-, ISE- and AEA-induced hyperthermia. Data are reported as mean 7s.e.m. of changes in
ꢁ
5
ꢁ6
rabbit RT. Black triangles represent the effects of CAHS (4.8 ꢀ 10 mol) injected 10 min before TAG (1.2 ꢀ 10 mol, n ¼ 4, panel
ꢁ5
ꢁ8
a), ISE (4.8 ꢀ 10 mol, n ¼ 4, panel b) or AEA (1.2 ꢀ 10 mol, n ¼ 4, panel c), while open circles represent the hyperthermia elicited
by TAG, ISE or AEA injected alone. Arrows indicate time of TAG, ISE or AEA i.c.v. injection. In the right panels, the area
under the experimental curve (AUC(0 – 24 h)) of RT changes relative to the different treatments is shown. The comparison between
AUC(0 – 24 h) relatives to the different treatments was performed by using Student’s t-test. **Po0.01 vs vehicle. Po0.001 vs TAG,
#
##
ISE or AEA alone.
TAG. As reported in Figure 5, panel a, in fact, after the
coadministration of the two compounds, RT decreased slightly
in the first 75 min after the treatment, and rose slowly
thereafter, peaking to 1.270.21C at the end of the experi-
mental session. After 24 h, RT regained basal values. During
the experimental session, GBM was comparable to that of
control animals. The AUC(0 – 24 h) relative to CAHS+TAG
treatment was significantly lower (Po0.001) than that of TAG
injected alone at the same dose as that used in combination
with CAHS. However, as reported in the inset of Figure 5, the
AUC(0 – 24 h) relative to the coadministration of the two above-
mentioned drugs was significantly higher than that relative to
the injection of vehicle (Po0.01).
CAHS was able to fully antagonize the hyperthermic
effect of both AEA and ISE. As reported in Figure 5,
ꢁ
5
panels b and c, respectively, the injection of 4.8 ꢀ 10 mol of
ꢁ5
ꢁ8
CAHS together with 4.8 ꢀ 10 mol ISE or 1.2 ꢀ 10 mol
AEA did not significantly modify RT (AUC(0 – 24 h)
CAHS+ISE and CAHS+AEA vs AUC(0 – 24 h) controls, NS)
that remained around basal values during the entire
experimental session. Also GMB was not affected by these
treatments.
British Journal of Pharmacology vol 139 (3)

M. Frosini et al
Taurine and thermoregulation in the rabbit brain
493
Discussion
Taurine is a highly flexible molecule and both amine and
sulfonic moiety have free rotation around the central
methylene linkage; therefore, it can attain many low-energy
conformations that allow its binding either to GABA receptors
or taurine binding sites (Liebowitz et al., 1987; Lombardini
et al., 1989). Since the configuration of taurine at its binding
sites is unknown, more restricted analogues of taurine, like its
cyclic derivatives, have been considered. In CAHS, the amine
and sulfonic groups must have equatorial and axial position,
respectively (Liebowitz et al., 1987). Since CAHS and taurine
exert similar effects in vivo, we conclude that the amine and
sulfonic groups need to be in the gauche position for the
compound to induce hypothermia. It is worth noting that
THAS, in which these two groups are both equatorial, is
inactive. The difference in pharmacological activity of TAHS
and CAHS suggests that CAHS effect is mediated by a specific
recognition site. The lower potency of CAHS in comparison to
taurine in inducing hypothermia, could be explained by
considering that CAHS was administered as a racemate. Since
the difference in pharmacological activity between enantiomers
could be great, in some cases, the resolution of CAHS
racemates might lead to an optical isomer provided with
considerable potency. Braghiroli et al. (1996), have shown, in
fact, that the hypotensive effect elicited by i.c.v. injection of the
2-methyltaurine racemate was mainly because of (S)-enantio-
mer, the (R)-form being inactive.
The results of the present study when combined with data
relative to the interaction of these compounds with GABA-
ergic or taurinergic systems of the rabbit brain (Frosini et al.,
2
003), can shed light on the question whether the role of
taurine as an endogenous cryogen depends on the activation of
GABA or specific taurine receptor(s).
We have demonstrated that AEA, ISE and CAHS, and the
taurine antagonist TAG were able to modify RT of conscious
rabbit. In particular, while CAHS induced hypothermia, TAG,
AEA and ISE caused hyperthermia, TAG and AEA being
effective even at a dose of 1.2ꢀ 10ꢁ mol, but were also very
neurotoxic causing death of animals few hours after adminis-
tration. All the above-mentioned compounds, as described by
Frosini et al. (2003) did not interact with GABA-ergic systems,
8
3
but were able to inhibit [ H]taurine binding to washed synaptic
membranes of rabbit brain. Furthermore, CAHS was able to
antagonize the hyperthermia induced by AEA, ISE and TAG,
suggesting that the effects of the latter compounds are mediated
by taurine binding sites. In the light of these results, the
hypothesis of the existence of a specific taurinergic pathway
involved in the central mechanisms of thermoregulation in the
rabbit which parallels a GABA-ergic system, can be advanced.
AEA and ISE that induced hyperthermia similar to TAG,
can be considered as novel taurine antagonists. Interestingly, it
has been demonstrated that AEA antagonizes taurine-stimu-
lated calcium uptake by rat liver mitochondria in a competitive
fashion, suggesting the presence of a common recognition site
for both compounds (Palmi et al., 1999). CAHS, which
induced a taurine-like effect on body temperature, can be
considered a taurine agonist. Liebowitz et al. (1987) have
demonstrated that this compound, like taurine, is able to
stimulate ATP-dependent calcium uptake in rat retina.
To explain the different effects of taurine derivatives on
thermoregulation, we can postulate the existence of a recogni-
tion site specific for taurine in rabbit brain. Taking into account
the conformation of the compounds used in the present study,
we can hypothesize a receptor possessing two regions, one
negatively and the other positively charged, which interact with
the amine and the sulfonic group, respectively, of taurine. The
distance between these two sites corresponds to that of two
bonded carbon atoms. The interaction of taurine with the
receptor at both groups is essential, but the receptor binding of
the sulfonate group is specific for its tetrahedral shape, since b-
ALA, with its planar carboxylate group, had no effect.
Taurine was able to bind sites in rabbit whole brain synaptic
membrane preparations with a higher affinity than that shown
for GABA
taurine-induced hypothermia depends on its interaction with
taurine binding sites and, to a certain extent, also with GABA
receptors. The hypothesis that part of the effect of taurine is
mediated by GABA receptors, however, is hampered by the
B
receptors (Frosini et al., 2003). This suggests that
B
The hypothesis of a taurine receptor might help to explain
why some of the taurine analogues (i.e. ISE, AEA and TAG)
elicited hyperthermia. If we admit that they are able to block
the receptor without activating it, then they will prevent
endogenous taurine from exerting a tonic influence on
thermoregulatory centers that are directed towards dissipation
of body heat. Without this influence the system will be
unbalanced and hyperthermia will develop.
B
finding that b-ALA did not affect thermoregulation in the
rabbit, although it exhibited [ H]GABA displacing activity at
3
B
micromolar concentrations from GABA receptors in rabbit
brain synaptic membrane preparations (Frosini et al., 2003).
At physiological pH, taurine is present as a zwitterion, while
alkylsulfonic acids such as ISE are present almost in the anion
form. Aminoalkylarsonic acids might be expected to have pK
values of about 4 and about 9. Hence, at neutral pH, they will
be almost in the charged form, being monoanion predominating
over the dianion form. These considerations, together with the
effects elicited by these compounds on body temperature,
suggest that both sulfonic and amine groups are essential for
taurine to induce the hypothermic effect. In fact, the substitu-
tion of sulfonic group by one with similar tetrahedral shape and
size such as the arsonate group gives rise to AEA that induced a
hyperthermia. Also the substitution of the entire amine with an
hydroxy group shifted the biological activity of the resulting ISE
toward hyperthermia. The substitution of sulfonic with a planar
carboxylic group, moreover, gives rise to b-ALA which, in
contrast with its effectiveness in the rat (Sgaragli & Pavan,
In conclusion, the existence of a specific taurinergic system
that is involved in the central mechanisms of thermoregulation
in the rabbit can be advanced. The future development of new
taurine derivatives will be crucial for the characterization of
the brain receptor responsible for the physio-pharmacological
activity of taurine. This is the premise for the development of
new drugs, able to induce rapidly and safely hypothermia, to
be used in the emergency care medicine.
This work was supported by contributions of Ministero degli Affari
Esteri (Rome, Italy) under law 212/92 and by MURST, Cofin. ‘98, EU
COST Action D13 (WG number 13-0011-00) and Fondazione Monte
dei Paschi di Siena. The technical assistance of R. Borghi is gratefully
acknowledged. A preliminary account of this study was presented at
the International Taurine Symposium 1999 held in Siena, Italy, on
August 4 – 8, 1999 and published as short report in Della Corte et al.
(2000).
1
972), was inactive in the rabbit.
British Journal of Pharmacology vol 139 (3)

494
M. Frosini et al
Taurine and thermoregulation in the rabbit brain
References
BRAGHIROLI, D., TRUZZI, C., BRANDOLI, C., BARALDI, M.,
GAMBERONI, G. & DI BELLA, M. (1996). Effect of 2-methyltaurine
and its enantiomers on arterial blood pressure. Pharmacol. Res., 34,
PALMI, M., FROSINI, M., BECHERUCCI, C., SGARAGLI, G.P. &
PARENTE, L. (1994). Increase of extracellular brain calcium
involved in interleukin-1 beta-induced pyresis in the rabbit:
antagonism by dexamethasone. Br. J. Pharmacol., 112, 449 – 452.
PALMI, M., YOUMBI, T.G., FUSI, F., FROSINI, M., SGARAGLI, G.P.,
3
3 – 36.
DELLA CORTE, L., HUXTABLE, R.J., SGARAGLI, G.P. & TIPTON, K.F.
eds) (2000). Taurine 4: Taurine and Excitable Tissues. New York:
Plenum Press.
EGLI VON, R. & EUGSTER, C.D. (1975). U
Reduktion von substituierten Anilinen zu substituierten Cyclo-
hexylaminen und von Benzol-bwz. Phenyl-alkan-sulfonsauren zu
Cyclohexan-bzw. Cyclohexylalkan-sulfonsauren. Helv. Chim. Acta
8, 2321–2346.
2
+
(
DIXON, H.B.F. & TIPTON, K.F. (1999). Taurine potentiates Ca
sequestration by rat liver mitochondria. Biochem. Pharmacol., 58,
1123 – 1131.
¨
ber die selektive katalytische
SAFAR, P.J. & KOCHANEK, P.M. (2002). Therapeutic hypothermia
after cardiac arrest. N. Engl. J. Med., 346, 612– 613.
SAWYER, C.H., EVERETT, J.W. & GREEN, J.D. (1954). The rabbit
diencephalons in stereotaxic coordinates. J. Comp. Neurol., 101,
801 – 824.
SCHULER, V., LUSCHER, C., BLANCHET, C., KLIX, N., SANSIG, G.,
KLEBS, K., SCHMUTZ, M., HEID, J., GENTRY, C., URBAN, L.,
FOX, A., SPOOREN, W., JATON, A.L., VIGOURET, J., POZZA, M.,
KELLY, P.H., MOSBACHER, J., FROESTL, W., KASLIN, E.,
KORN, R., BISCHOFF, S., KAUPMANN, K., VAN DER PUTTEN,
¨
¨
5
FROSINI, M., SESTI, C., DRAGONI, S., VALOTI, M., PALMI, M.,
DIXON, H.B.F., MACHETTI, F. & SGARGLI, G.P. (2003). Interac-
tion of taurine and structurally related analogues with the
GABAergic system and taurine binding sites of rabbit brain. Br.
J. Pharmacol., 138, 1163–1171.
FROSINI, M., SESTI, C., PALMI, M., VALOTI, M., FUSI, F.,
MANTOVANI, P., BIANCHI, L. & DELLA CORTE, L. & SGAR-
AGLI, G.P. (2000). Heat-stress-induced hyperthermia alters CSF
osmolality and composition in conscious rabbits. Am. J. Physiol. –
Regul. Integ. Comp. Physiol., 279, 2095 – 2103.
H.
& BETTLER, B. (2001). Epilepsy, hyperalgesia, impaired
memory, and loss of pre- and postsynaptic GABA(B) responses in
mice lacking GABA(B(1)). Neuron, 31, 47 – 58.
SERRANO, J.S., MINANO, F.J., SANCIBRIAN, M. & DURAN, J.A.
(1985). Involvement of bicuculline-insensitive receptors in the
hypothermic effect of GABA and its agonists. Gen. Pharmacol.,
16, 505 – 508.
GEOGHEGAN, K.F. & DIXON, H.B. (1989). Synthesis of 2-amino-
ethylarsonic acid. A new synthesis of primary amines. Biochem. J.
1
5, 295 – 296.
GIRARD, Y., ATKINSON, J.G., HAUBRICH, D.R., WILLIAMS, M. &
YARBROUGH, G.G. (1982). Aminomethyl-1,2,4-benzothiadiazines
as potential analogues of gamma-aminobutyric acid. Unexpected
discovery of a taurine antagonist. J. Med. Chem., 25, 113 – 116.
SESTI, C., FROSINI, M. & SGARAGLI, G.P. (1999). Rabbit rectal
temperature and ECoG spectrum changes induced by intracereb-
roventricular injection of GABA, taurine and analogues. Pharma-
col. Res., 39, 61.
`
HAYES, K.C., CAREY, R.E.
degeneration associated with taurine deficiency in the cat. Science,
88, 949 – 951.
HUSSY, N., BRES, V., ROCHETTE, M., DUVOID, A., ALONSO, G.,
DAYANITHI, G. MOOS, F.C. (2001). Osmoregulation of
&
SCHMIDT, S.Y. (1975). Retinal
SGARAGLI, G.P., CARLA, V., MAGNANI, M. & GALLI, A. (1981).
Hypothermia induced in rabbits by intracerebroventricular taurine:
specificity and relationship with central serotonin (5-HT) systems.
J. Pharmacol. Exp. Ther., 219, 778 – 785.
1
`
&
SGARAGLI, G.P., CARLA, V., MAGNANI, M. & GIOTTI, A. (1978).
vasopressin secretion via activation of neurohypophysial nerve
terminals glycine receptors by glial taurine. J. Neurosci., 21, 7110 –
Homotaurine and muscimol mimic taurine and GABA effects on
muscle tone and temperature regulation. Naunyn-Schmiedebergs
Arch. Pharmacol., 305, 155 – 158.
7
116.
HUXTABLE, R.J. (1989). Taurine in the central nervous system and the
mammalian actions of taurine. Progr. Neurobiol., 32, 471 – 533.
KAMISAKI, Y., MAEDA, K., ISHIMURA, M., OMURA, H. & ITOH T.
SGARAGLI, G.P., FROSINI, M., PALMI, M., BIANCHI, L. & DELLA
CORTE, L. (1994). Calcium and taurine interaction in mammalian
brain metabolism. Adv. Exp. Med. Biol., 359, 299 – 308.
(
1993). Effects of taurine on depolarization-evoked release of amino
acids from rat cortical synaptosomes. Brain Res., 627, 181 – 185.
KONTRO, P. OJA, S.S. (1990). Interactions of taurine with
GABA binding sites in mouse brain. Neuropharmacology, 29,
43 – 247.
SGARAGLI, G.P. & PAVAN, F. (1972). Effects of amino acid
compounds injected into cerebrospinal fluid spaces, on colonic
temperature, arterial blood pressure and behaviour of the rat.
Neuropharmacology, 11, 45 – 56.
&
B
2
THE HYPOTHERMIA AFTER CARDIAC ARREST STUDY
GROUP (2002). Mild therapeutic hypothermia to improve the
neurologic outcome after cardiac arrest. N. Engl. J. Med., 346,
549 – 556.
KROGSGAARD-LARSEN, P., FALCH, E., SCHOUSBOE, A., CURTIS,
D.R. & LODGE, D. (1980). Piperidine-4-sulphonic acid, a new
specific GABA agonist. J. Neurochem., 34, 756 – 759.
LIEBOWITZ, S.M., LOMBARDINI, J.B. & SALVA, P.S. (1987). Cyclic
TUZ, K., ORDAZ, B., VAA, L., QUESADA, O. & PASANTES-
2
+
taurine analogs. Synthesis and effects on ATP-dependent Ca
uptake in rat retina. Biochem. Pharmacol., 36, 2109 – 2114.
MORALES, H. (2001). Isovolumetric regulation mechanisms in
cultured cerebellar granule neurons. J. Neurochem., 79, 143 – 151.
YAKIMOVA, K., SANN, H., SCHMID, H.A. & PIERAU, F.K. (1996).
Effects of GABA agonists and antagonists on temperature-sensitive
neurones in the rat hypothalamus. J. Physiol. (London), 494, 217 –
230.
LOMBARDINI, J.B., LIEBOWITZ, S.M.
& CHOU, T.C. (1989).
Analogues of taurine as stimulators and inhibitors of ATP-
dependent calcium ion uptake in rat retina: combination kinetics.
Mol. Pharmacol., 36, 256 – 264.
MACHETTI, F., CACCIARINI, M., CATRAMBONE, F., CORDERO,
F.M., ROMOLI, S. & DE SARLO, F. (2000). Synthesis of taurine
analogues. Part 1: 2-aminosulfonic acids from alkene – sulfur
monochloride adducts. J. Chem. Res., 120 – 121.
ZHANG, Y.H., YANASE-FUJIWARA, M., HOSONO, T. & KANOSUE,
K. (1995). Warm and cold signals from the preoptic area: which
contribute more to the control of shivering in rats. J. Physiol.
(London), 485, 195 – 202.
MONDA, M., VIAGGIANO, A., SULLO, A. & DE LUCA, V. (1998).
Aspartic and glutamic acids increase in the frontal cortex during
prostaglandin E1 hyperthermia. Neuroscience, 83, 1239 – 1243.
(
Received February 13, 2003
Accepted March 7, 2003)
British Journal of Pharmacology vol 139 (3)