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 (5, 18-20). Some enteric neurons that have orexin immunoreactivity display leptin immunoreactivity and share 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 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 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 M) or SB-334867 (pharmacological orexin-1 receptor antagonist, 10-5 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).

Fig. 1. Time course of the experimental schedule. Events are marked above the time axis: acetylcholine 10-5 M (ACh), electrical field stimulation (EFS): 90V, 10 s duration of three consecutive trains of pulses with frequencies of 0.5, 5, 50 Hz. Antagonists: tetrodotoxin 10-6 M, atropine 10-5 M or SB 334867 10-5 M. Orexin-A and orexin-B were given at three concentrations: 10-9, 10-8, and 10-7 M in a cumulative manner. Isoproterenol was given at a concentration of 10-5 M. All substances were added to chambers in a volume of 0.1 ml.

Drugs and solutions

Tetrodotoxin was purchased from Alomone Labs (Jerusalem, Israel), 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea hydrochloride (SB-334867) selective OX1R antagonist, from Tocris Bioscience (UK). Acetylcholine chloride, isoproterenol, atropine, 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 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 M or ATR 10-5 M significantly reduced tissue contraction responses to 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.

Fig. 2. Representative traces of response to acetylcholine of isolated rat jejunal segments in fed (A) and 24 h fasted (B) rats. Comparison of response to acetylcholine 10-5 M in both groups (C). Data expressed as mean ± SE (n= 20 and 48 for fed and fasted groups, respectively).

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 M, tetrodotoxin (TTX)10-6 M and SB334867 10-5 M. Stimulation marked as a horizontal bar.

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 difference reach statistical significance (P<0.05). Fasting had no significant effect on response to OXA or OXB.

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 (10-9-10-7M, OXA, OXB) - P<0.001. *,** significantly different at P<0.05.

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 SB-334867 on orexin stimulation

Incubation with SB-334867 10-6 M led to a marked decrease in OXA 10-7 and 10-9 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 10-9-10-7 M (Fig. 5D).

Effect of atropine on orexin stimulation

Intestinal segments from fasted animals preincubated with atropine 10-5M had reduced contractions in response to OXA 10-9 M (13.30 ± 3.36 vs. 3.18 ± 0.45, P<0.01), 10-7 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.

Effect of TTX on orexin stimulation

TTX reduced sensitivity to OXA-the contractile effects of OXA at concentrations of 10-9 M (13.30 ± 3.36 vs. 2.80 ± 0.55) and 10-8 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 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 trans-membrane sodium transport and thus nerve conductance, therefore TTX does not block Ca2+ cellular transients or Ca2+ sensitive K+ channels following OXR activation (33, 34). Other studies confirmed this mechanism showing that the excitatory effect of OXA persisted in the presence of TTX in sympathetic preganglionic neuron preparations and brain slices (35-37). These TTX-insensitive neuronal firings are blocked by a non-selective Ca2+ 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 involves both activation of neuronal (sodium-dependent) transmission and most likely Ca2+-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 action in ENS (18, 26). Surprisingly, Satoh et al. (27) have demonstrated that in the presence of atropine and guanethidine (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 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 SB-334867 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 fasting influences muscarinic receptors. We have shown in the present study that orexin-B is more potent in the induction of 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).

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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