JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2006, 57, Supp 6, 43–54
www.jpp.krakow.pl
W. KORCZYNSKI , M. CEREGRZYN , I. KATO , J. WOLINSKI , R. ZABIELSKI
1
1
2
1
1,3
THE EFFECT OF OREXINS ON INTESTINAL MOTILITY IN VITRO
IN FED AND FASTED RATS
1
Department of Gastrointestinal Physiology, The Kielanowski Institute of Animal Physiology
and Nutrition, Polish Academy of Science, Jab³onna, Poland;
Awakura, Fujinomiya-shi, Japan;
3
2
Yanaihara Institute Inc., 2480-1
Department of Physiological Sciences, Faculty of Veterinary
Medicine, Warsaw Agricultural University, Warsaw, Poland.
Orexin-A and -B (OXA, OXB) are peptides involved in many gastrointestinal (GI)
functions, including motility. Orexins, their precursors and receptors are present in
the
GI
tract.
The
expression
of
orexins
increases
in
the
hypothalamus
and
gastrointestinal tract in response to fasting. We have examined the effect of OXA and
OXB on GI motility in vitro in fed and fasted rats. The intestinal segments were
mounted
in
chambers
filled
with
Krebs
measured in response to acetylcholine (10
-5
orexins (10
-10
-9
-7
SB- 334867 (10
solution.
Isotonic
contractions
were
M), electric field stimulation (EFS), and
M) alone or in the presence of orexin-1 type receptor antagonist,
-5
M), tetrodotoxin (TTX) 10
-6
M, or atropine (10
-5
M). Orexins
caused a dose-dependent increase of intestinal segment contractions with a more
pronounced effect of OXB over OXA. Fasting did not influence orexin-induced
responses. Incubation with SB-334867 led to a marked decrease in orexin-induced
contractions
Atropine
decreased
in
OXA-treated
diminished
response
segments,
contractions
to
orexins
in
only
both
while
in
those
fasted
groups.
of
OXB
animals,
The
were
while
results
not
TTX
show
that
affected.
led
to
OXB
a
is
predominant in inducing gut motility response, that the effect of orexins is not fully
dependent on cholinergic and Na
+
transmitters is possible.
Key
w o r d s : orexin-A,
orexin
tetrodotoxin
B,
orexin-1
transmissions, and that involvement of other
receptor
antagonist,
intestinal
contractions,
44
INTRODUCTION
Orexin-A (OXA) and orexin-B (OXB) (1, 2), also described as hypocretin-I
and -II, are a family of hypothalamic neuropeptides selectively expressed in the
brain, gastrointestinal tract, kidney and testis (3-6). Orexin receptors (OX1R and
OX2R) have been detected in all of the organs mentioned above and in the
adrenal
gland,
pancreas
and
skin
(6-11).
Orexins
are
derived
by
proteolytic
cleavage of the same precursor, prepro-orexin (a 130-amino-acid peptide) and act
through activation of G protein-coupled OX1R and OX2R receptors (1, 12).
Orexins have been shown to be involved in sleep-wakefulness, feeding behavior,
energy expenditure, nociceptive sensations, cardiovascular functions and stress
response
(13-17).
Orexins,
their
receptors,
and
prepro-orexin,
are
present
in
various parts of the gastrointestinal system: the enteric nervous system (ENS)
including the myenteric plexus, submucosal plexus, as well as in mucosa and
smooth
muscles
immunoreactivity
(5,
18-20).
display
leptin
Some
enteric
neurons
immunoreactivity
and
that
share
have
orexin
co-localization
either with vasoactive intestinal peptide (VIP), substance P (SP) or nitric oxide
synthase (NOS) (18, 21). The plasma OXA concentration has been shown to
increase during fasting in rats and the level of its specific mRNA increases in the
hypothalamus (19, 22). That of OXB in the hypothalamus also increases during
fasting (22). The effects of orexins on gastrointestinal tract motility have been
investigated recently in various experimental setups (19, 23, 24). A dual effect of
centrally applied OXA on gastric motility has been recorded in the rat, where
relaxation of the proximal and contraction of distal stomach have been observed
(23). Furthermore, it was shown that this dual excitatory/inhibitory effect of OXA
and OXB depends on the site of central administration (24). Studies on intestinal
motility performed in vivo have shown that orexins slow down intestinal motility
by extending the duration of the of migrating myoelectric complex (MMC) (19,
25). However, orexins have stimulated intestinal and colonic motility in studies
performed in vitro (18, 26, 27). The majority of studies used only OXA in the
investigation of gastrointestinal effects of orexins. No direct comparison of OXA
and OXB on in vitro motor activity of the gut has been made. Moreover, the
possible involvement of orexins in regulation of gut motility in the fasting and fed
states has not been extensively studied yet. Therefore, the aim of the present study
was to determine the effect of OXA and OXB on the in vitro motility of small
intestine segments collected from fasted and non-fasted rats.
MATERIALS AND METHODS
Animals
Male Wistar rats (410 ± 40 g BW) were used for the experiments. The animals were housed
under artificial lighting 12 hours a day in a temperature-controlled room (20-22 °C). They had free
45
access
to
standard
laboratory
chow
and
to
water.
Animals
were
randomly
allocated
to
two
experimental groups: non-fasted and 24 hr-fasted animals. All experiments started at 10 a.m. Rats
were sacrificed by CO2 inhalation in a chamber. The experimental procedures were carried out
according to a protocol approved by the Local Ethics Committee.
Experimental protocol
Whole tissue middle jejunum segments were taken promptly from animals and immediately placed
in cold Krebs solution (in mM: NaCl 118, KCl 4.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.9, NaHCO3 25,
glucose 10.1). Whole jejunum segments (15 mm long) were prepared within approximately 20 minutes.
The same stretch was applied during each preparation. Next, the segments were placed in 25 ml organ
bath chambers (Letica Scientific Instruments, Spain) that were filled with Krebs solution (37°C, pH 7.4)
and continuously saturated with carbogen (95% O2, 5% CO2). The intestinal segments were attached to
isotonic
transducers
(Letica
Scientific
Instruments,
Spain)
under
a
constant
load
of
0.5
g.
The
transducers were coupled with a PowerLab recording system (ADInstruments, Sydney, Australia). The
tissues were allowed to equilibrate for 30 minutes (the solution in chambers was changed once after 15
minutes) to regain spontaneous activity. Then the segments were subjected to a procedure that started
by addition of acetylcholine (ACh) 10
-5
M. ACh was left in the solution for 1 min, next the tissues were
washed and allowed to equilibrate. Electrical field stimulation (EXP-ST-01, Experimetria, Budapest,
Hungary) was then performed (voltage 90 V, duration 10 seconds). Three frequencies were used: 0.5,
5 and 50 Hz with 1 min intervals between each train of pulses. After 10 min equilibration, the tissues
were exposed to OXA and OXB in a cumulative manner. Orexins were added to reach concentrations
from 10
-9
to 10
-7
M. Each concentration of orexin was allowed to act for 5 minutes in the solution then
the next dose of orexin was added. After the last orexin exposure, ACh 10
-5
M was added to the solution
and after 1 minute the chambers were washed. Next, the tissues were allowed to equilibrate for at least
20 minutes. The second step of the experiment was performed after the equilibration. The segments
Fig.
1.
Time
acetylcholine
course
10
-5
M
of
the
(ACh),
experimental
electrical
schedule.
field
Events
stimulation
are
(EFS):
marked
90V,
above
10
s
the
time
duration
of
axis:
three
consecutive trains of pulses with frequencies of 0.5, 5, 50 Hz. Antagonists: tetrodotoxin 10
atropine 10
10
, 10
-9
-5
M or SB 334867 10
, and 10
-8
-7
-5
-6
M,
M. Orexin-A and orexin-B were given at three concentrations:
M in a cumulative manner. Isoproterenol was given at a concentration of 10-
All substances were added to chambers in a volume of 0.1 ml.
5
M.
46
were incubated with atropine (ATR, a non-selective muscarinic receptor antagonist, 10
-5
M), TTX, a
neurotoxin that blocks electrically active sodium channels preventing depolarization and propagation
of action potentials in neurons, 10
-6
10
-5
M) or SB-334867 (pharmacological orexin-1 receptor antagonist,
M) for 5 min, then EFS and orexin challenges were performed in the manner described above. Each
experiment was finished by administration of isoproterenol 10
-5
M in order to control relaxation of
tissues (Fig. 1).
Drugs and solutions
Tetrodotoxin was purchased from Alomone Labs (Jerusalem, Israel), 1-(2-methylbenzoxazol-6yl)-3-[1,5]naphthyridin-4-yl
Tocris
Bioscience
(UK).
urea
hydrochloride
Acetylcholine
(SB-334867)
chloride,
selective
isoproterenol,
OX1R
atropine,
antagonist,
from
dimethylsulfoxide
(DMSO) were purchased from Sigma-Aldrich (Germany). SB-334867 was dissolved in DMSO.
The final concentration of DMSO in the bathing solution was 0.4% and did not itself affect the
spontaneous activity of the investigated tissues. Orexins were synthesized by dr I. Kato (Yanaihara
Institute, Japan). All solutions were applied in a volume of 0.1 ml.
Data analysis
Experimental
data
were
collected
and
analyzed
by
Chart
4.1
software
(ADInstruments,
Australia). The effectiveness of the OX1R antagonist, ATR, and TTX was verified on separate
A
B
Fed
Fasted 24h
WASH
WASH
1 mm
100 s
ACH 10-5 M
ACH 10-5 M
1 mm
100 s
4
Contraction [mm]
C
3
Fig. 2. Representative traces of
2
response to acetylcholine of
isolated rat jejunal segments in fed
(A) and 24 h fasted (B) rats.
1
Comparison of response to
acetylcholine 10-5 M in both
groups (C). Data expressed as
0
fed
fasted 24 h
mean ± SE (n= 20 and 48 for fed
and fasted groups, respectively).
47
preparations displaying normal spontaneous activity using ACh and EFS. In the comparison of
the response to ACh in the fed and fasted groups, the amplitude of contraction was expressed in
absolute values (millimeters). Further data was normalized and presented as a percent of response
amplitude
to
ACh
10
-5
M.
Data
were
statistically
evaluated
using
STATISTICA
software
(StatSoft, Tulsa, USA) with one-way or repeated measurements ANOVA followed by the Scheffe
post-hoc test or one-way Friedman ANOVA followed by the Mann-Whitney test. For two groups
analysis, the Student-t test or Man-Whitney test was used. The coefficient of significance was set
at
P<0.05.
All
results
are
expressed
as
means
±
standard
errors
(SE),
with
the
number
of
experiments given in parentheses.
RESULTS
Intestinal segments showed spontaneous contractile activity within 30 min
from the start of recording in both the fed and fasted groups. Tissues from fasted
and fed animals responded similarly to ACh 10
-5
M (Fig. 2). EFS stimulation
induced a typical biphasic contraction (Fig. 3). Pretreatment with TTX 10
-6
ATR
10
-5
M
significantly
reduced
tissue
contraction
responses
to
M or
EFS
stimulation, however, incubation with SB 334867 had no influence on EFS (5 Hz)
induced phase I and II contractions (Fig. 3). The responses to EFS in the presence
of the OX1R antagonist were not influenced by either OXA, OXA or feeding
status (Fig. 5 A,B). All preparations had the capability of relaxing in response to
isoproterenol at the end of the experiments.
Effect of orexins on intestinal segments contractions
Representative responses of intestinal tissue to OXA and OXB in fed and
fasted
rats
are
shown
in
Fig.
4.
OXA
and
OXB
induced
dose-dependent
contractions (P<0.001) of intestinal segments in fasted and fed animals. The
amplitude of OXB induced contractions tended to be higher than that of OXA in
the fed group at a dose of 10
-8
M (P=0.06), but only at a dose of 10
-7
M did the
Fig. 3. Representative traces of responses to electrical field stimulation (EFS, 10 s, 5 Hz) of isolated
jejunal segments in the presence of atropine 10
-5
Stimulation marked as a horizontal bar.
M, tetrodotoxin (TTX)10
-6
M and SB334867 10
-5
M.
48
Fig. 4. Representative traces of
response to orexin-A (A) and
orexin-B (B) of isolated rat jejunal
segments in fed rats. Comparison
of the response (C) of the tissue to
OXA (white symbols) and OXB
(black symbols) in fasted (circles)
and fed (squares) animals. Data
expressed as a percentage of
contraction induced in each tissue
by acetylcholine 10
-5
M. Data is a
mean value ± SE (n=15-26). Main
effect ANOVA (10
-10
-9
M, OXA,
-7
OXB) - P<0.001. *,**
significantly different at P<0.05.
difference reach statistical significance (P<0.05). Fasting had no significant effect
on response to OXA or OXB.
Effect of SB-334867 on orexin stimulation
Incubation with SB-334867 10
-6
10
-9
M led to a marked decrease in OXA 10
-7
and
M induced intestinal segment contractions in fed (reduction by 22-42%) and
fasted animals (reduction by 37-62%), however, only at 10
-8
M was this reduction
significant in both groups as compared with the respective controls (P<0.05) (Fig.
5C). Pretreatment with SB-334867 failed to significantly diminish the stimulatory
effect of OXB 10
-10
-9
-7
M (Fig. 5D).
Effect of atropine on orexin stimulation
Intestinal segments from fasted animals preincubated with atropine 10
M had
-5
reduced contractions in response to OXA 10
-9
P<0.01), 10
-7
M (13.30 ± 3.36 vs. 3.18 ± 0.45,
M (64.37 ± 6.06 vs. 42.77 ± 3.19, P<0.05), and OXB 10
-9
M (13.76
± 2.58 vs. 5.76 ± 1.58, P< 0.05). Tissue responses of fed animals were also
reduced, but the difference was not significant.
49
Fig. 5. The effect of
SB334867 on phase 1 (white
columns) and phase 2 (black
columns) of response to
electrical field stimulation
(5 Hz, 10 s) in fed (A) and
fasted (B) rats and the effect
of SB334867 on orexin A
(C) and orexin B (D) effects
in fed (circles) and fasted
(squares) animals. Data
expressed as a percentage of
contraction induced in each
tissue by acetylcholine 10
-5
M. Data is a mean value ±
SE (n=6-26). *,** different
from respective group P<0.05.
Effect of TTX on orexin stimulation
TTX
reduced
sensitivity
concentrations of 10
-9
to
OXA-the
contractile
effects
M (13.30 ± 3.36 vs. 2.80 ± 0.55) and 10
-8
of
OXA
at
M (34.54 ± 6.08
vs. 11.67 ± 4.20) were significantly inhibited in fasted (P<0.05) and, at the same
concentrations, in fed animals (11.81 ± 2.32 vs. 2.88 ± 0.32, P<0.01), and (32.11
± 1.81 vs. 21.21 ± 3.19, P<0.05), respectively. The effect of OXB was blocked
only at 10
-9
M in both the fed (13.76 ± 2.58 vs. 3.94 ± 1.05, P<0.05) and fasted
group (17.89 ± 3.90 vs. 4.18 ± 1.57, P<0.01), respectively.
DISCUSSION
In the present study there were no significant differences in responses to
orexins between fasted and fed animals. It has been shown that 24 hours fasting
50
is an adequate time to achieve a central orexin response and influence feeding
pattern (1, 28). In response to fasting the OX1R receptor-specific mRNA in the
rat hypothalamus increased, as did the OXA and OXB contents (1, 22). In another
study, the hypothalamic OXA level was not changed in hypoglycemic, fasted rats,
whereas the OXB level was 10-fold higher (29). Therefore, possible involvement
of orexins in the regulation of gastrointestinal motility during the fasting and fed
states occurs on the central nervous system level.
Pharmacodynamic studies have revealed that OXA readily enters the brain by
passive diffusion, while OXB is metabolized in the blood and lacks blood-brain
barrier crossing properties (30). Additionally, OXB is less metabolically stable
than OXA (31).
The present experiments showed that both OXA and OXB have a stimulatory
effect on rat small intestine motility in vitro. These findings confirm in vitro data
obtained from guinea pig (18, 26) and mice (27). At the concentrations tested
here, OXA- and OXB-induced contractions were not fully inhibited by TTX and
ATR. Previously it was shown that TTX in vitro totally abolished the OXA
stimulatory effect in guinea pig ileal strips and segment preparations (18, 26).
However, in another study only partial inhibition was shown in orexin-induced
contraction of rat ileal strips in vitro (32). TTX is a neurotoxin that impairs transmembrane sodium transport and thus nerve conductance, therefore TTX does not
block
Ca
2+
cellular
transients
or
Ca
2+
sensitive
K
+
channels
following
OXR
activation (33, 34). Other studies confirmed this mechanism showing that the
excitatory
effect
preganglionic
of
OXA
neuron
persisted
preparations
in
the
and
presence
brain
of
slices
TTX
in
(35-37).
insensitive neuronal firings are blocked by a non-selective Ca
2+
sympathetic
These
TTX-
blocker, while the
blocker alone does not change the amplitude of orexin-induced depolarization.
This may indicate that postsynaptic orexin activation is required to achieve these
effects (38). Therefore, the mechanism of OXA- and OXB-induced contraction in
rat
small
intestine
transmission
and
involves
most
both
likely
activation
Ca
of
-dependent
2+
neuronal
(sodium-dependent)
conductance.
Additionally,
the
direct effect of orexins on smooth muscle can not be excluded.
It has been postulated that the OXA effect on GI tract smooth muscle motility
is mediated by ACh release from the intestinal tissue, both by presynaptic and
postsynaptic
demonstrated
action
that
in
in
ENS
the
(18,
26).
presence
of
Surprisingly,
atropine
and
Satoh
et
al.
guanethidine
(27)
have
(adrenergic
receptor antagonist), OXA induced dose-dependent relaxation of mouse intestinal
segments that was abolished by TTX. In the present study the effect of exogenous
orexins was not completely inhibited by atropine, which indicates other possible
neuroendocrine pathways. Our results suggest that orexins stimulate motility via
neural and myogenic mechanisms.
It
is
reasonable
to
assume
that
other
neurotransmitters
such
as
SP,
noradrenaline, 5-hydroxytryptamine (5-HT) are involved in the orexin-induced
response (19, 27, 32). It is also possible that orexins act directly on their receptors
51
present on smooth muscle. Therefore, the physiological pathway most likely
involves orexins released from enterochromaffin cells and/or orexins released
from
ENS
neurons
that
influence
GI
tract
motility.
Moreover,
the
balance
between the neuronal and muscular effect of orexins varies depending on species.
Orexins act through activation of OX1R and OX2R receptors; OXB has a 10
times higher affinity for OX2R than for OX1R, while OXA shares the same
affinity to OX1R and OX2R (1). Interestingly, Yazdani et al. (32) have observed
that food deprivation for 24 h causes a decrease in OX1R expression in the rat
intestine. Feeding resulted in partial re-establishment of the OX1R pool with little
effect on OX2R. This indicates that feeding status differently influences orexin
receptors. On the other hand, three days starvation led to a higher density of OXA
containing cells in the intestinal submucosal ganglia of guinea pig (18). Thus, it
is difficult to speculate if the inhibitory effect of orexins on fasted rats intestinal
motility in vivo is strictly of peripheral origin (19, 25).
Blocking the OX1R receptor with SB 334867 reduces food intake in mice and
rats (39, 40), 2-Deoxy-D-glucose induced gastric secretion (41), and GI tract
motility
(27).
In
the
present
study
only
partial
inhibition
of
OXA-induced
contraction was observed in the presence of OX1R antagonist, SB 334867, as
reported in mice (27). OXB-induced contractions were not influenced at all. It
should be pointed out that despite the at least 50-fold higher affinity of SB334867 to OX1R over OX2R receptors, OXA and OXB have an equal affinity to
OX1R (42). Thus, it seems that in the present study the effect of OX1R blockage
was overridden by OX2R stimulation. Therefore, it must be determined in detail
which receptor subtype (OX1R or OX2R) is dominant in terms of function and
density in the enteric nervous system and other structures in the gastrointestinal
tract before a final conclusion can be drawn.
Flemström et al. (43) have shown that OXA given after overnight fasting
causes downregulation of muscarinic receptors, resulting in decreased duodenal
secretion in rats. We did not observe significant differences in the contractile
response to orexins in fed and fasted animals. In the present study, the stimulatory
effect of orexins was in part sensitive to ATR only in fasted animals, which may
indicate
that
present
study
fasting
that
influences
orexin-B
is
muscarinic
more
receptors.
potent
in
the
We
have
shown
induction
of
in
the
intestinal
contraction than OXA. Orexin-B is able to evoke the same excitatory response as
OXA in guinea-pig myenteric plexus measured by intracellular recordings (44).
Also, OXB is equally potent as OXA in short-term stimulation of insulin release
(45). The dual action of OXA and OXB on the sympathetic nerves innervating
brown
adipose
tissue
has
been
shown
in
the
study
of
Yasuda
et
al.
(13).
Intraventriculocerebral orexin-A administration decreases, while OXB, increases,
sympathetic nerve activity. It seems that OXB is involved in the regulation of
gastrointestinal functions. Additionally it was postulated that OXB acts more
peripherally while OXA primarily targets the brain (46, 47).
52
In conclusion, the present results show that motility of the GI tract is regulated
by orexins with a predominant role of OXB being the most likely. The peripheral,
prokinetic mechanism of orexin action is both neuronal and direct. Most likely, in
rats, feeding status influences orexin action at the level of the central nervous
system since the peripheral responses are insensitive to fasting.
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Received:
September 15, 2006
A c c e p t e d : October 2, 2006
Author’s address: Dr. Wojciech Korczynski, Department of Gastrointestinal Physiology, The
Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Science, 05-110
Jab³onna, Poland, tel.: +48 22 782 44 22, fax.: +48 22 774 20 38; e-mail: [email protected]