Short communication
Clenbuterol enhances the production of kynurenic
acid in brain cortical slices and glial cultures
El¿bieta Luchowska1, Renata Kloc1, Sebastian Wnuk1, Bartosz Olajossy1,
Marian Wielosz1, Ewa M. Urbañska1,2

Department of Pharmacology and Clinical Pharmacology, Skubiszewski Medical School, Jaczewskiego 8,
PL 20-090 Lublin, Poland
Department of Toxicology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland
Correspondence: Ewa M. Urbañska, e-mail: emurbanska@poczta.onet.pl
Abstract:
The effect of a b -adrenergic agonist, clenbuterol on the production of a glutamate receptor antagonist, kynurenic acid was studied
in vitro. Clenbuterol enhanced the production of kynurenic acid in brain cortical slices (0.1–1.0 mM) and in glial cultures (1–50 mM).
Timolol, a non-selective b-adrenergic antagonist prevented this effect. The presented data indicate a novel mechanism of action of
b -adrenoceptor agonists and suggest that an increased formation of the endogenous glutamate receptor antagonist, kynurenic acid
could partially contribute to their neuroprotective activity.
Key words:
b-adrenergic agonist, kynurenic acid, glial culture, in vitro
Abbreviations: GFAP – glial fibrillary acidic protein, HPLC –
high performance liquid chromatography, KAT – kynurenine
aminotransferase, KRB – Krebs-Ringer buffer, NGF – nerve
growth factor, NMDA – N-methyl-D-aspartate
Introduction
Kynurenic acid is the only known endogenous compound
able to block the glycine site of N-methyl-Daspartate
(NMDA) receptors and to antagonize a7
subtype of cholinergic nicotinic receptors [15]. It is
formed in the brain and in the periphery [10, 15, 18]
from a direct bioprecursor, L-kynurenine, via enzymatic
reaction mediated by kynurenine aminotransferases
I and II (KAT I and II). Kynurenic acid exerts anticonvulsant
and neuroprotective activity in experimental
models, and its disturbed metabolism was suggested
to be one of the causative factors in human neuropathology
[15, 17]. Kynurenic acid may also inhibit glutamate
release, and this effect is not shared by other
NMDA or a7 cholinergic antagonists [2]. It blocks presynaptic
NMDA autoreceptors already at low, submicromolar
concentrations, thereby regulating glutamate
release [12]. Kynurenic acid formation is controlled
by several distinct intra- and intercellular mechanisms,
such as e.g. endogenous excitatory amino acid agonists
or D,L-homocysteine [3, 8, 13, 17]. Here, we demonstrate
that b2-adrenergic agonist clenbuterol enhances
the production of kynurenic acid ex vivo, in rat brain
cortical slices and in mixed glial cultures.
574 Pharmacological Reports, 2008, 60, 574–577
Pharmacological Reports
2008, 60, 574–577
ISSN 1734-1140
Copyright © 2008
by Institute of Pharmacology
Polish Academy of Sciences
Materials and Methods
Animals
Cortical slices were obtained from male Wistar rats,
weighing 220–250 g. Newborn Wistar rats (postnatal
day 1–2) were used for the preparation of glial cultures.
Animals were housed under standard laboratory
conditions, at ambient temperature of 20°C, with food
and water available ad libitum. All experimental procedures
were approved by the Local Ethics Committee
in Lublin and are in agreement with European
Communities Council Directive on the use of animals
in experimental studies.
Substances
L-kynurenine (sulfate salt), kynurenic acid, L-pyruvate,
pyridoxal-5’-phosphate, 2-mercaptoethanol, clenbuterol
and timolol and cellulose membrane dialysis
tubing were obtained from Sigma-Aldrich (St. Louis,
USA), whereas all high pressure liquid chromatography
(HPLC) reagents were obtained from J.T. Baker
Laboratory Chemicals. All other chemicals were purchased
from Sigma-Aldrich (St. Louis, USA).
In vitro experiments – cortical slices
Production of kynurenic acid in freshly prepared cortical
slices was performed as previously described [8].
Each well contained 6 slices, and each concentration
of the drug was studied in 6 wells (N = 6). Briefly,
slices were incubated (2 h, 37°C) in Krebs-Ringer
buffer (KRB), pH 7.4 (118.5 mM NaCl, 4.75 mM
KCl, 1.18 mM MgSO4, 1.77 mM CaCl2, 12.9 mM
NaH2PO4, 3 mM Na2HPO4 and 5 mM D-glucose)
saturated with 95% O2/5% CO2, in the presence of
L-kynurenine (10 mM) and various concentrations of
the studied substances. Kynurenic acid was separated
from the incubation medium and quantified fluorimetrically
using a high performance liquid chromatography
(HPLC) system (Varian, USA) and ESA catecholamine
HR-80, 3 mm, C18 reverse-phase column. The
mean control production of kynurenic acid in the
presence of 10 mM L-kynurenine was 2.98 ± 0.61
pmol/h/well.
In vitro experiments – glial cultures
Mixed glial cultures were obtained from cerebral
hemispheres of rat pups (postnatal day 1–2) as described
by others [5, 19], with our modification.
Briefly, brain tissue of newborn rats was seeded at
a density of one brain/75 cm2 culture flasks (Falcon,
Switzerland) containing 10 ml of Basal Medium Eagle
with Earle’s salts (BME, Biochrom AG, Berlin), supplemented
with 2 mM L-glutamine, penicillin-streptomycin
(500 IU/ml – 500 UG/ml; Gibco-BRL) and
containing 10% heat-inactivated fetal bovine serum
(Gibco, InVitrogen). The medium was replaced twice
a week. Cultures were maintained at 37°C in a 5%
CO2/95% air atmosphere. After 14–15 days, the cells
were replated on poly-L-lysine-coated 24-microwell
plates (Nunc), and the medium was replaced every
3 days. After 1 week, a confluent astrocytic monolayer
developed with scattered oligodendrocytes and
microglia on top. Approx. 75–80% of the cells were
glial fibrillary acidic protein (GFAP) – positive (as revealed
by immunostaining). The production of kynurenic
acid was assessed in tissue cultures after 20–22
days in vitro. On the day of experiment, the culture
medium was changed and the studied substances were
added to cultures. After 22 h, the medium was
changed into the freshly prepared KRB of the above
composition. Cells were further incubated in the presence
of L-kynurenine (final concentration 10 mM; 2 h)
and the studied substances (total incubation time
24 h). Each concentration of the drug was studied in
6 wells (N = 6). The mean control production of kynurenic
acid by glial cells in the presence of 10 mM
L-kynurenine was 11.8 ± 1.25 pmol/h/well.
Activities of kynurenine aminotransferases
(KATs)
The activities of KAT I and KAT II were assayed in
the dialyzed homogenate of cortical brain tissue, as
described before [8]. Briefly, freshly obtained cortical
brain tissue was homogenized (1:9; w/v) in 5 mM
Tris-acetate buffer, pH 8.0, containing 50 mM pyridoxal-5’-phosphate
and 10 mM 2-mercaptoethanol. The
resulting homogenate was centrifuged (12,000 rpm
for 10 min), and the supernatant was dialyzed overnight
at 8°C, using cellulose membrane dialysis tubing,
against 4 l of the buffer composed as above. The
enzyme preparation was incubated in the reaction
mixture containing 2 mM L-kynurenine, 1 mM pyruvate,
70 mM pyridoxal-5’-phosphate, 150 mM Trisacetate
buffer, at pH of 7.0 or 9.5, for KAT II or KAT I,
respectively (all given concentrations are the final
ones) and solutions of the tested drugs at varying conPharmacological
Reports, 2008, 60, 574–577 575
Clenbuterol stimulates kynurenic acid production
El¿bieta Luchowska et al.
centrations. When KAT II activity was assayed, glutamine
(final concentration 2 mM), the KAT I inhibitor,
was added to the samples. Further procedures were
performed as described above. The mean control activity
of KAT I and KAT II in the presence of 2 mM
kynurenine was 1.72 ± 0.09 and 0.78 ± 0.07 pmol of
kynurenic acid/mg of tissue/h, respectively.
Each experiment was repeated at least twice. Statistical
comparisons were preformed using one-way
analysis of variance (ANOVA) with Bonferroni posthoc
test.
Results
Clenbuterol enhanced the kynurenic acid synthesis in
cortical slices (0.25–1.0 mM) and in glial cultures
(1–50 mM) (Fig. 1). The non-selective b-adrenergic
antagonist timolol, used at the 50 mM concentration in
cortical slices and at 5 mM concentration in glial cultures,
has reversed its action in both experiments
(Fig. 1). KAT I and KAT II activities were not altered
by clenbuterol at up to 0.5 mM concentration (Tab. 1).
Discussion
Central noradrenergic system may modulate neuronal
survival, neuroplasticity and neurogenesis [14]. Neurons
and glial cells express several subtypes of functional
a- and b-adrenergic receptors, moreover, b2adrenergic
receptors are located predominantly on the
glial cells [14]. Agonists of b2-adrenoceptors display
neuroprotective effects in vitro and in vivo [7, 14].
Clenbuterol protected mouse cerebral cortex and rat
hippocampus from ischemic damage and attenuated
hippocampal neuronal loss induced by L-glutamate in
culture [16]. These effects seemed to result from the
induction of nerve growth factor (NGF) synthesis [4,
16]. Moreover, the compound was shown to increase
brain levels of tryptophan [11]. Clinically, drugs activating
b2-adrenergic receptors are commonly used to
treat and to prevent the attacks of bronchial asthma
due to their potent peripheral effects, including bronchodilation
and enhancement of mucociliary clearance
[1].
The data presented herein suggest a novel central mechanism
of action of b2-adrenergic agonists involving
an increased production of glutamate receptor antagonist,
kynurenic acid. Timolol, a non-selective b-adrenergic
antagonist reversed the action of clenbuterol in
vitro; KATs activities were not changed by clenbuterol.
Thus, the observed increase in the kynurenic acid
production seems to result from a direct stimulation of
the b2-adrenergic receptors and not from the activation
of kynurenic acid biosynthetic enzymes. The
regulation of kynurenic acid synthesis is governed by
various extracellular and intracellular mechanisms.
Notably, only few drugs, e.g. some antiepileptics, are
able to stimulate kynurenic acid production, but when
used at higher, close to millimolar concentrations [9].
The concentrations of clenbuterol effective in glial
cultures (1–50 mM) are comparable with the neuroprotective
concentrations of clenbuterol (1 mM) re-
576 Pharmacological Reports, 2008, 60, 574–577
Fig. 1. The effect of clenbuterol on the kynurenic acid production
in rat brain cortical slices and in mixed glial cultures. Cortical slices
were incubated for 2 h in KRB, pH 7.4, in the presence of 10 µM
L-kynurenine and 50–1000 µM clenbuterol. Timolol (50 µM) was
added to the incubation media 10 min prior to clenbuterol. Glial cells
were incubated in the presence 0.1–50 µM clenbuterol for 24 h.
L-kynurenine (final concentration 10 µM) was present in the incubationmedia
during thelast2hofincubation. Timolol (5µM)wasadded
to the incubation media 10 min prior to clenbuterol.* p < 0.05 LI.control
(ANOVA); = p < 0.05 LI. clenbuterol (ANOVA)
Tab. 1. The effect of clenbuterol on the activity of kynurenine aminotransferases
(KATs) I and II
Clenbuterol (µM) KAT I (% of control) KAT II (% of control)
10 101.8 ± 4.0 98.5 ± 3.7
50 97.8 ± 5.3 102.7 ± 4.8
100 98.1 ± 5.5 95.2 ± 8.5
500 104.7 ± 4.5 95.7 ± 6.2
Data are presented as the percentage of control values ± SD
ported by others [7]. The underlying cellular mechanism
most probably is associated with an increased
formation of intracellular cAMP, the second messenger
mediating the effects of b2-adrenergic stimulation
[6, 7]. Further experiments will clarify this issue.
An enhanced synthesis of kynurenic acid may be
one of the mechanisms contributing to the neuroprotective
effects of b-adrenoceptor agonists. Development
of drugs modifying noradrenergic receptor activity
and/or downstream signaling, especially in
astrocytes, was suggested for treatment of several
neurological and psychiatric disorders and as a novel
therapy in neurodegeneration [7]. Our current research
focuses on the assessment of the effects exerted
by a broad range of b-adrenergic agonists on the
brain synthesis of kynurenic acid in vitro and identification
of cellular mechanisms underlying this action.
Acknowledgments:
This study was supported by the grant form the State Committee
for Scientific Research, KBN 2P05F 04429 and grant from Medical
University School in Lublin, DS. 450/08.
References:
1. Anderson GP: Interactions between corticosteroids and
b-adrenergic agonists in asthma disease induction, progression,
and exacerbation. Am J Respir Crit Care Med,
2000, 161, S188–196.
2. Carpenedo R, Pittaluga A, Cozzi A, Attucci S, Galli A,
Raiteri M, Moroni F: Presynaptic kynurenate-sensitive
receptors inhibit glutamate release. Eur J Neurosci, 2001,
13, 2141–2147.
3. Chmiel-Perzyñska I, Perzyñski A, Wielosz M, Urbañska
EM: Hyperglycemia enhances the inhibitory effect of
mitochondrial toxins and D,L-homocysteine on the brain
production of kynurenic acid. Pharmacol Rep, 2007, 59,
268–273.
4. Culmsee C, Stumm RK, Schafer MK, Weihe E, Krieglstein
J: Clenbuterol induces growth factor mRNA, activates
astrocytes, and protects rat brain tissue against
ischemic damage. Eur J Pharmacol, 1999, 379, 33–45.
5. Giulian D, Baker TJ: Characterization of ameboid microglia
isolated from developing mammalian brain. J Neurosci,
1986, 6, 2163–2178.
6. Hertz L, Chen Y, Gibbs ME, Zang P, Peng L: Astrocytic
adrenoceptors: a major drug target in neurological and
psychiatric disorders? Curr Drug Targets CNS Neurol
Disord, 2004, 3, 239–267.
7. Junker V, Becker A, Hühne R, Zembatov M, Ravati A,
Culmsee C, Krieglstein J: Stimulation of b-adrenoceptors
activates astrocytes and provides neuroprotection.
Eur J Pharmacol, 2002, 446, 25–36.
8. Kocki T, Luchowski P, Luchowska E, Wielosz M,
Turski WA, Urbañska EM: L-cysteine sulphinate,
endogenous sulphur-containing amino acid, inhibits rat
brain kynurenic acid production via selective interference
with kynurenine aminotransferase II. Neurosci Lett,
2003, 346, 97–100.
9. Kocki T, Wielosz M, Turski WA, Urbañska EM:
Enhancement of brain kynurenic acid production by
anticonvulsants-novel mechanism of antiepileptic
activity? Eur J Pharmacol, 2006, 41, 147–151.
10. Kuc D, Rahnama M, Tomaszewski T, Rzeski W,
Wejksza K, Urbanik-Sypniewska T, Parada-Turska J
et al.: Kynurenic acid in human saliva – does it influence
oral microflora? Pharmacol Rep, 2006, 58, 393–398.
11. Lenard NR, Gettys TW, Dunn AJ: Activation of b - and
b!-adrenergic receptors increases brain tryptophan. J
Pharmacol Exp Ther, 2003, 305, 653–659.
12. Luccini E, Musante V, Neri E, Raiteri M, Pittaluga A:
N-methyl-D-aspartate autoreceptors respond to low and
high agonist concentrations by facilitating, respectively,
exocytosis and carrier-mediated release of glutamate in
rat hippocampus. J Neurosci Res, 2007, 85, 3657–3665.
13. Luchowska E, Luchowski P, Paczek R, Ziembowicz A,
Kocki T, Turski WA, Wielosz M et al.: Dual effect of
DL-homocysteine and S-adenosylhomocysteine on brain
synthesis of the glutamate receptor antagonist, kynurenic
acid. J Neurosci Res, 2005, 79, 375–382.
14. Marien MR, Colpaert FC, Rosenquist AC: Noradrenergic
mechanisms in neurodegenerative diseases: a theory.
Brain Res Brain Res Rev, 2004, 45, 38–78.
15. Németh H, Toldi J, Vecsei L: Role of kynurenines in
the central and peripheral nervous systems. Curr Neurovasc
Res, 2005, 2, 249–260.
16. Semkova I, Schilling M, Henrich-Noack P, Rami A,
Krieglstein J: Clenbuterol protects mouse cerebral cortex
and rat hippocampus from ischemic damage and attenuates
glutamate neurotoxicity in cultured hippocampal
neurons by induction of NGF. Brain Res, 1996, 717,
44–54.
17. Stazka J, Luchowski P, Wielosz M, Kleinrok Z, Urbañska
EM: Endothelium-dependent production and liberation
of kynurenic acid by rat aortic rings exposed to
L-kynurenine. Eur J Pharmacol, 2002, 448, 133–137.
18. Urbañska EM, Luchowski P, Luchowska E, Pniewski J,
WoŸniak R, Chodakowska-¯ebrowska M, £azarewicz J:
Serum kynurenic acid positively correlates with cardiovascular
disease risk factor, homocysteine a study in
stroke patients. Pharmacol Rep, 2006, 58, 501–511.
19. Vincent VAM, Van Dam A-M, Persoons JHA, Schotanus
K, Steinbusch HWN, Schoffelmeer ANM, Berkenbosch
F: Gradual inhibition of inducible nitric oxide synthase
but not interleukin-1b production in rat microglial cells
of endotoxin-treated mixed glial cell cultures. Glia,
1996, 17, 94–102.
Received:
February 27, 2008; in revised form: May 13, 2008.
Pharmacological Reports, 2008, 60, 574–577 577
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