Introduction

Top Abstract Introduction Methods Results Discussion References

Alpha-2 adrenoceptor agonists are well known to inhibit gastrointestinal motor and secretory activities: for example clonidine inhibits small intestinal transit in rats (Ruwart et al., 1980) and experimental diarrhea in mice (Doherty and Hancock, 1983). Alpha-2 adrenoceptor agonists also inhibit gastroduodenal motility and gastric emptying of both solid and liquid meals in dogs (Gullikson et al., 1991), and delay gastric emptying in humans (Sninsky et al., 1986). It is thought that activation of presynaptic alpha-2 adrenoceptors suppresses gastrointestinal motor activity by suppressing acetylcholine release from postganglionic cholinergic neurons (Andrejak et al., 1980; Ruwart et al., 1980; Doherty and Hancock, 1983). Alpha-2 adrenoceptors have recently been classified into four subtypes, and three alpha-2 adrenoceptor genes are described in humans (alpha-2A, alpha-2B and alpha-2C) and rats (alpha-2B, alpha-2C and alpha-2D) (Regan et al., 1988; Lomasney et al., 1990; Lanier et al., 1991; MacKinnon et al., 1994). Postsynaptic alpha-2A adrenergic receptors have been reported to mediate the contraction of circular smooth muscle of canine proximal colon (Zhang et al., 1992). In addition, the presynaptic alpha-2 adrenoceptors of the guinea pig and rat ileum have been identified as the alpha-2D subtype and are shown to suppress the release of acetylcholine (Funk et al., 1995; Liu and Coupar, 1996).

In contrast, the alpha-2 adrenoceptor antagonists, yohimbine and idazoxan, have been reported to stimulate fecal excretion and colonic transit in rats (Theodorou et al., 1989; Croci and Bianchetti, 1992). These results suggest that normal colonic propulsion in rats may be under the inhibitory control of alpha-2 adrenoceptors, and that alpha-2 adrenoceptor antagonists may stimulate colonic motor activity by facilitating acetylcholine release from enteric neurons. However, Bharucha et al. (1997) reported that yohimbine reduced the increment in colonic tone after hypocapnic hyperventilation, which was shown to increase tonic and phasic motility of the colon and perception of colonic distension in humans (Ford et al., 1995; Bharucha et al., 1996). In particular, hyperventilation is associated with stress (Thyer et al., 1984), and is used as model to evaluate control of colonic motor function. However, the effect of alpha-2 adrenoceptor antagonists on dysfunctional colonic motor activity is still unclear.

YNS-15P is a novel and selective alpha-2 adrenoceptor antagonist. YNS-15P inhibits the binding of [³H]prazosin to alpha-1 adrenoceptors with a K_i value of 0.369 µM and the binding of [³H]MK-912 to alpha-2 adrenoceptors with a K_i value of 1.87 nM in rat brain (unpublished observations). YNS-15P is therefore about 200-fold more selective for alpha-2 adrenoceptors than for alpha-1 adrenoceptors. Furthermore, YNS-15P 1 µM shows no affinity for D1, D2, 5-HT1A, 5-HT2, 5-HT3 and muscarinic binding sites (unpublished observations).

In our study, we investigated the effect of selective alpha-2 adrenoceptor antagonists, rauwolscine, RX821002 and YNS-15P on colonic propulsion stimulated by WRS, which is used as an experimental model for IBS in rats (Williams et al., 1988). Recently, several 5-HT3 receptor antagonists, a 5-HT3 and 5-HT4 receptors dual antagonist, trimebutine and diazepam were reported to inhibit fecal excretion stimulated by stress in rats (Miyata et al., 1992, 1993; Kadowaki et al., 1993). We have compared the effect of alpha-2 adrenoceptor antagonists with the effect of a 5-HT3 receptor antagonist, granisetron, a 5-HT4 receptor antagonist, GR113808, trimebutine and diazepam. We have also studied the effects of YNS-15P on normal or bethanechol-stimulated colonic propulsion and on castor-oil-induced diarrhea.

	Methods

Top Abstract Introduction Methods Results Discussion References

Animals. Male Sprague-Dawley rats (150-260 g) were kept under standard laboratory conditions (12-hr light/dark cycle), and food and water were provided ad libitum. To determine colonic propulsion, animals were anesthetized with pentobarbital sodium (50 mg/kg i.p.) and a silicone tube was implanted through the cecum into the proximal colon with its tip extending 1 cm past the cecocolonic junction. The cannula was secured to the wall of the cecum and taken out through a skin incision made between the scapulae. After the insertion of the cannula, the animals were individually housed and allowed free access to food and water. Experiments were carried out on nonfasted, conscious animals 5 to 6 days after the surgery.

WRS-stimulated fecal excretion. Animals were exposed to WRS as previously described (Williams et al., 1988). Briefly, animals were lightly anesthetized with ether, and their foreshoulders, upper forelimbs and thoracic trunks were wrapped with cloth tape to restrict their movements. One hour later, the animals were killed by cervical dislocation. Williams et al. (1988) reported that all feces were formed and dry, and restraint stress did not result in diarrhea, therefore the number and wet weight of the fecal pellets expelled by each animal during the hour were determined. Drugs or vehicle were given p.o. 1 hr before or s.c. 30 min before exposure to WRS.

Measurement of colonic transit. Colonic propulsion was assessed by measuring the colonic transit of a charcoal marker as previously described (Kishibayashi et al., 1993). Charcoal marker (a suspension of 5% charcoal and 10% gum arabic in saline; 0.2 ml) was infused through the indwelling cannula and followed by a 0.2-ml saline flush. After the animals were killed by cervical dislocation, the colon was removed and colonic transit was measured as the percentage of the total length of the colon traversed by the charcoal marker. The fecal pellets expelled by each animal during the hour were also counted and weighed.

Bethanechol-stimulated fecal excretion. Animals were lightly anesthetized with ether and injected s.c. with bethanechol (1.5 mg/kg). One hour later, the animals were killed by cervical dislocation and the number and wet weight of the fecal pellets expelled by each animal during the hour were determined. Drugs or vehicle were given s.c. 30 min before the administration of the bethanechol.

Castor-oil-induced diarrhea. Animals were fasted overnight, with free access to water. Diarrhea was induced by p.o. administration of castor oil (5 ml/kg). After the administration of the castor oil, the animals were placed in individual cages, and the occurrence of diarrhea was observed for 2 hr. Drugs or vehicle were given s.c. 30 min before the administration of the castor oil.

Drugs. Bethanechol chloride was from Sigma Chemical Co. (St Louis, MO), diazepam from Takeda Chemical Industries (Osaka, Japan), castor oil from Nacalai Tesque Inc. (Kyoto, Japan) and rauwolscine hydrochloride and RX821002 hydrochloride from Research Biochemicals Inc. (Boston, MA). YNS-15P, granisetron, GR113808 and trimebutine were synthesized by Nippon Shinyaku Co., Ltd. (Kyoto, Japan). Trimebutine and GR113808 were suspended in 0.5% methylcellulose-saline solution and the other drugs were dissolved in saline. All drugs were administered in a volume of 5 ml/kg body weight.

Analysis of data. The colonic transit of the charcoal marker and the number and weight of the fecal pellets are shown as the mean ± S.E. from the eight rats in each group. The time of onset of diarrhea is shown as the mean ± S.E. from the 12 rats in each group. Tests of statistical significance were performed with Dunnett's multiple comparison test. P < .05 or < .01 are regarded as significant.

	Results

Top Abstract Introduction Methods Results Discussion References

Effect of alpha-2 adrenoceptor antagonists on WRS-stimulated fecal excretion. A 1-hr exposure to WRS significantly increased the number and weight of fecal pellets from 0 ± 0 to 4.88 ± 0.83 and from 0 ± 0 to 0.892 ± 0.141 g, respectively (fig. 1A). Both rauwolscine and RX821002, given s.c., inhibited the increase in the number and weight of fecal pellets induced by WRS (fig. 1). YNS-15P, given p.o. or s.c., also inhibited WRS-stimulated fecal excretion in a dose-dependent manner (fig. 2). The maximal inhibition produced by s.c. administration of rauwolscine, RX821002 and YNS-15P was more than 80%.

View larger version (54K): [in this window] [in a new window]   Fig. 1.   Effect of rauwolscine (A) and RX821002 (B) on the number and weight of fecal pellets increased by WRS for 1 hr in rats. Rauwolscine, RX821002 or vehicle was given s.c. 30 min before exposure to WRS. N, No WRS treatment; C, WRS control. The data in figures 1 through 7 represent the mean ± S.E. from eight rats. ** P < .01 compared with WRS control (C).

View larger version (50K): [in this window] [in a new window]   Fig. 2.   Effect of YNS-15P on the number and weight of fecal pellets increased by WRS for 1 h in rats. YNS-15P or vehicle was given p.o. 1 hr before (A) or s.c. 30 min before (B) exposure to WRS. * P < .05, ** P < .01 compared with WRS control (C).

Effect of granisetron, GR113808, trimebutine and diazepam on WRS-stimulated fecal excretion. Subcutaneous administration of granisetron, but not GR113808, significantly inhibited WRS-stimulated fecal excretion (fig. 3). Trimebutine and diazepam also inhibited WRS-stimulated fecal excretion (fig. 4). The inhibition produced by granisetron, trimebutine and diazepam were less than the inhibition by the alpha-2 adrenoceptor antagonists.

View larger version (70K): [in this window] [in a new window]   Fig. 3.   Effect of granisetron (A) and GR113808 (B) on the number and weight of fecal pellets increased by WRS for 1 hr in rats. Granisetron, GR113808 or vehicle was given s.c. 30 min before exposure to WRS. * P < .05, ** P < .01 compared with WRS control (C).

View larger version (62K): [in this window] [in a new window]   Fig. 4.   Effect of trimebutine (A) and diazepam (B) on the number and weight of fecal pellets increased by WRS for 1 hr in rats. Trimebutine, diazepam or vehicle was given s.c. 30 min before exposure to WRS. * P < .05, ** P < .01 compared with WRS control (C).

Effect of YNS-15P on WRS-stimulated colonic transit. Rats were exposed to WRS for a period of 30 min starting 2 hr after the infusion of the charcoal marker. YNS-15P or vehicle was given p.o. 1 hr before exposure to WRS. A 30-min exposure to WRS significantly stimulated colonic transit from 66.8 ± 3.4 to 98.1 ± 1.9% and increased the weight of the fecal pellets from 0.004 ± 0.004 to 0.686 ± 0.088 g (fig. 5). YNS-15P inhibited WRS-stimulated colonic transit and fecal excretion in a dose-dependent manner.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Effect of YNS-15P on the weight of fecal pellets and colonic transit increased by WRS for 30 min in rats. The charcoal marker was infused through the colonic cannula 2 hr before exposure to WRS. YNS-15P or vehicle was given p.o. 1 hr after the marker infusion. * P < .05, ** P < .01 compared with WRS control (C).

Effect of YNS-15P on normal colonic transit. Normal colonic propulsion was determined 1 hr after the infusion of the charcoal marker in rats. YNS-15P or vehicle was given s.c. just before the infusion of the marker. Normal colonic transit was 49.0 ± 1.3% and the weight of the fecal pellets was 0.043 ± 0.043 g (fig. 6). YNS-15P had no significant effect on either of these indicators of normal colonic propulsion.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Changes in normal fecal excretion and colonic transit during a period of 1 hr after infusion of the charcoal marker in rats. YNS-15P or vehicle (C) was given s.c. just before the marker infusion.

Effect of YNS-15P on bethanechol-stimulated fecal excretion. The administration of bethanechol (1.5 mg/kg s.c.) significantly increased the number and weight of the fecal pellets from 0.38 ± 0.26 to 3.38 ± 0.57 and from 0.047 ± 0.031 to 0.568 ± 0.119 g, respectively (fig. 7). YNS-15P had no significant effect on bethanechol-stimulated fecal excretion.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Effect of YNS-15P on the number and weight of fecal pellets increased by bethanechol for 1 hr in rats. YNS-15P or vehicle was given s.c. 30 min before the administration of bethanechol (1.5 mg/kg, s.c.). * P < .05, ** P < .01 compared with bethanechol control (C).

Effect of YNS-15P on castor-oil-induced diarrhea. Castor oil (5 ml/kg p.o.) caused watery diarrhea in 83.3% of rats within 1 hr after and 100% of rats within 2 hr after the administration, and the time of onset of diarrhea was 45.6 ± 3.3 min (fig. 8). YNS-15P did not affect the time of onset of diarrhea.

View larger version (61K): [in this window] [in a new window]   Fig. 8.   Effect of YNS-15P on the time of onset of diarrhea induced by castor oil in fasted rats. YNS-15P or vehicle (C) was given s.c. 30 min before the administration of castor oil (5 ml/kg, p.o.). The data represent the mean ± S.E. from 12 rats.

	Discussion

Top Abstract Introduction Methods Results Discussion References

An alpha-2 adrenoceptor agonist, clonidine, inhibits normal colonic motility and colonic transit in rats (Sjoqvist et al., 1985; Theodorou et al., 1989). In contrast, the alpha-2 adrenoceptor antagonists such as yohimbine and idazoxan have been reported to stimulate normal fecal excretion or colonic transit in rats (Theodorou et al., 1989; Croci and Bianchetti, 1992). These results, taken together, suggest a role of alpha-2 adrenoceptors in the regulation of normal colonic propulsion in rats. However, Bharucha et al. (1997) demonstrated that yohimbine reduced the increment in colonic tone after hyperventilation in humans. Hyperventilation is known to increase colonic tonic and phasic motor activity and perception of colonic distension in humans (Ford et al., 1995; Bharucha et al., 1996). The effects of hyperventilation on colonic motor function were reported to result from activation of central autonomic pathways or from a direct effect of hypocapnia on colonic smooth muscle. In addition, hyperventilation is also associated with stress (Thyer et al., 1984). Thus, stress caused by hyperventilation may contribute to the increase in colonic tone.

In our study, we investigated the effect of alpha-2 adrenoceptor antagonists on stress-stimulated colonic propulsion in rats. We found that YNS-15P, a novel alpha-2 adrenoceptor antagonist, as well as rauwolscine and RX821002 inhibited WRS-stimulated fecal excretion. YNS-15P also inhibited WRS-stimulated colonic transit in a dose-dependent manner, and its potency in inhibiting WRS-stimulated colonic transit was comparable with that in inhibiting WRS-stimulated fecal excretion. We therefore consider that the inhibitory activity of alpha-2 adrenoceptor antagonists on WRS-stimulated fecal excretion is due to their inhibition of WRS-stimulated colonic propulsion. Several drugs, including 5-HT3 receptor antagonists such as granisetron, ondansetron and YM060 (Miyata et al., 1992; Kadowaki et al., 1993; Kishibayashi et al., 1993), a 5-HT3 and 5-HT4 receptors dual antagonist, FK1052 (Kadowaki et al., 1993), trimebutine (Miyata et al., 1993) and diazepam (Miyata et al., 1992) have been shown to inhibit stress-stimulated fecal excretion or colonic transit. In our study, granisetron, trimebutine and diazepam significantly inhibited WRS-stimulated fecal excretion, whereas, a 5-HT4 receptor antagonist, GR113808, had no significant effect. Therefore, inhibition of fecal excretion by FK1052 may be related to its inhibitory activity on the 5-HT3 receptors. However, the inhibitory efficacy of granisetron was less potent than those of the alpha-2 adrenoceptor antagonists. These findings may suggest a much greater involvement of alpha-2 adrenoceptors than 5-HT3 receptors in the colonic motor dysfunction induced by stress in rats. In this study, we also examined the effects of YNS-15P on fecal excretion stimulated by bethanechol and on diarrhea induced by castor oil. YNS-15P had no significant effect on these models, therefore, the inhibition of YNS-15P on WRS-stimulated colonic propulsion is not due to direct nonspecific inhibition on the smooth muscle or antidiarrheal activity.

In contrast to its effect on WRS-stimulated colonic propulsion, YNS-15P failed to affect normal fecal excretion and colonic transit. These results clearly indicate that YNS-15P selectively inhibit stress-stimulated colonic propulsion. Our findings are in apparent conflict with previous reports showing stimulation of normal colonic transit or fecal excretion by alpha-2 adrenoceptor antagonists (Theodorou et al., 1989; Croci and Bianchetti, 1992). This discrepancy may be due to differences in experimental design between the present and the previous studies, although the source of the discrepancy is unclear. For example, in one previous study (Theodorou et al., 1989) [⁵¹Cr] sodium chromate was infused through the colonic cannula, and feces were collected at 1-hr intervals until no radioactivity was detectable. The radioactivity present in feces was determined using a gamma counter, and the colonic transit was expressed as the mean retention time of the radioactive marker. This indirect method of measuring colonic transit contrasts with our more direct method and may help to explain the discrepancy.

Various types of physical and psychological stressful stimuli affect gastrointestinal motility in animals (Williams et al., 1988; Enck et al., 1989; Barone et al., 1990; Enck and Holtmann, 1992) and humans (Mcrae et al., 1982; Stanghellini et al., 1983; Fone et al., 1990). Exposure to stress stimulates colonic motor activity in rats, and is used as an experimental model of irritable bowel syndrome (Williams et al., 1988). The mechanisms of colonic motor dysfunction produced by stress are not yet fully understood, but they may involve neural and hormonal factors in the peripheral and central nervous systems. Corticotropin-releasing factor and thyrotropin-releasing hormone have been postulated as mediators of stress-induced colonic motor dysfunction (Holita and Carino, 1982; Holita et al., 1985; Williams et al., 1987; Lenz et al., 1988). However, exposure to stress activates adrenergic neuronal systems. Stressful stimuli and corticotropin-releasing factor have been shown to produce changes in the activity of the adrenergic neuronal system and to enhance the release of epinephrine and norepinephrine (Brown et al., 1982; Akerstedt et al., 1983; Axelrod and Reisine, 1984; Brown et al., 1985). In our study, the precise mechanism of action of alpha-2 adrenoceptor antagonists on the stress-stimulated colonic propulsion has not been determined. However, alpha-2 adrenoceptors may be involved in stress-induced colonic motor dysfunction. Further work is necessary to clarify the inhibitory mechanism of alpha-2 adrenoceptor antagonists.

In summary, we have demonstrated that alpha-2 adrenoceptor antagonists inhibit WRS-stimulated fecal excretion or colonic transit in rats. In contrast, a alpha-2 adrenoceptor antagonist YNS-15P had no significant effect on normal or bethanechol-stimulated colonic propulsion and on castor-oil-induced diarrhea. These findings imply that alpha-2 adrenoceptors are unlikely to be involved in the regulation of normal colonic propulsion in fed rats, whereas they may play important roles in stress-induced colonic motor dysfunction.