Département de Protection de la Santé de l'Homme et de Dosimétrie, Section Autonome de Radiobiologie Appliquée à la Médecine, Institut de Protection et de Sûreté Nucléaire, 92265 Fontenay aux Roses Cedex, France
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ABSTRACT |
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Whole body exposure to high doses of ionizing radiation is associated with small intestinal and colonic dysfunction, the etiology of which remains unknown. In this study, we investigated the role of both neural and nonneural 5-hydroxytryptamine (5-HT)-mediated pathways in radiation-induced attenuation and recovery of colonic secretory function. Rats were exposed to whole body 10-Gy gamma irradiation, and distal colonic tissues were studied in Ussing chambers 1, 3, and 7 days after exposure. Tissue responses to exogenously added 5-HT (nonneural pathway) and electrical field stimulation (EFS; neural pathway) were performed, and 5-HT receptor subtypes implicated in both responses were determined using three different 5-HT receptor antagonists: methysergide (5-HT2/1C), granisetron (5-HT3), and SDZ-205,557 (5-HT4). Maximal responses to exogenously added 5-HT were decreased at 1 and 3 days and returned to control values at 7 days. Responses to exogenous 5-HT were insensitive to both 5-HT2/1C and 5-HT3 antagonists and to TTX but were totally inhibited by SDZ-205,557 in both control and irradiated tissues. Responses to EFS were decreased 1 and 3 days after exposure and returned to control values at 7 days. In control tissues and 1 and 3 days after exposure, EFS responses were insensitive to both 5-HT2/1C and 5-HT4 antagonists but reduced by granisetron in control (51%) and at 1 (64%) and 3 days (58%) after exposure. Granisetron was more effective at 7 days (73% inhibition), which was concomitant with the appearance of a 5-HT4 antagonist-sensitive pathway (40% inhibition). In conclusion, neural and nonneural 5-HT-mediated pathways involve 5-HT3 and 5-HT4 receptors, respectively, in control as well as in irradiated tissues 1 and 3 days after exposure. Conversely, the recovery of colonic transport is associated with additional 5-HT3-mediated pathways, probably in combination with 5-HT4 receptors.
5-hydroxytryptamine; ionizing radiation; short-circuit current
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INTRODUCTION |
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THE SMALL AND LARGE INTESTINE play an important role in water and electrolyte conservation. Intestinal fluid and electrolyte movements are governed by numerous factors, including hormonal, paracrine agents, and neurotransmitters. The intestinal wall is supplied with an extensive neural network: the enteric nervous system (ENS). The ENS represents a major part of the gut-associated autonomic nervous system and consists of two main plexuses, the myenteric, localized between both smooth muscle layers, and the submucosal, just beneath the intestinal mucosa. The anatomic distribution of enteric nerves suggests that they control numerous physiological functions in both the small and large intestine. Intestinal fluid and electrolyte movements may be modulated by enteric nerves, associated for the most part with the submucosal plexus as shown in tissues lacking the myenteric plexus (removed by stripping) by the use of neurotoxic agents or transmural electrical stimulation techniques (27, 28). It has become evident that the myenteric plexus may also control fluid and electrolyte transport, as shown by studies using intact tissue and, in particular, luminally active agents such as bile acids and bacterial toxins (29, 42).
The intestinal tract shows a high sensitivity to ionizing radiation due to a rapid cell turnover and is often implicated in radiation sickness. One of the most commonly observed features of total body exposure to ionizing radiation is the appearance of severe diarrhea, the etiology of which remains undetermined. First thought to be mostly due to changes in intestinal motility, diarrhea may also result from altered fluid and electrolyte transport (22). The ENS has been shown to be involved in diarrhea stemming from cholera toxin infection (8), food allergens (10), or bile acids (31) and also may be implicated in radiation-induced intestinal dysfunction as suggested over 40 years ago (11). Fluid and electrolyte losses following exposure to ionizing radiation have been mostly ascribed to the disruption of intestinal barrier and to a reduced exchange surface (22). However, the appearance of modified water and electrolyte transport in the absence of characterized morphological alterations of the intestinal epithelium suggests the implication of functional impairments such as cellular transport capacity and/or regulatory processes (21).
The colon has been for a long time considered less radiosensitive compared with the small intestine, probably due to a slower cell turnover (7). However, even if there are less drastic morphological alterations in the colon than in the small intestine (11), radiation-induced colonic transport dysfunction has been observed in the rat in vivo and in vitro (16, 18) and may thus be a determinant in the appearance of diarrhea. Moreover, studies performed in vitro on rat muscle-stripped colon and ileum (18, 33) have shown that 10-Gy gamma total body irradiation is associated with tissue hyporesponsiveness to electrical field stimulation (EFS), suggesting a possible radiation-induced impairment of neural influence, in particular of the submucosal plexus, on intestinal fluid and electrolyte transport.
Almost 80% of the total body 5-hydroxytryptamine (5-HT) is contained in the gastrointestinal tract, mostly in enterochromaffin cells and less so in enteric nerves and mucosal mast cells (37). Changes in 5-HT tissue content have been observed after total body irradiation along the entire gastrointestinal tract (39) and may contribute to the imbalanced fluid and electrolyte transport. 5-HT may be released on the luminal and/or serosal side of the epithelium, including from enteric neurons (12), and thus acts as a neurotransmitter, neuromodulator, and paracrine agent to regulate digestive functions. The variability of the biological actions of 5-HT is linked to several different receptor subtypes, influencing blood flow, intestinal motility and water, and electrolyte transport. It is now well established that, in the rat as well as in the guinea pig small intestine and colon, 5-HT can act via neural and nonneural pathways (1, 30, 42a, 45, 46). However, receptor subtypes implicated in both pathways are still controversial, especially in the rat colon. 5-HT has been shown to be implicated in diarrhea following cholera toxin and Entamoeba histolytica infections (9, 35) and irritable bowel syndrome (2) as well as following cytostatic (cisplatin) administration (43). In addition, some 5-HT antagonists are effective in the treatment of sennoside- or cholera toxin-induced diarrhea (3, 47). A role for 5-HT in radiation-induced emesis has been demonstrated, and 5-HT3 antagonists are used clinically to treat cytostatic-induced emesis (13). Whether 5-HT is involved in radiation-induced intestinal dysfunction to date remains unknown. However, MacNaughton and Leach (34) demonstrated a reversal of attenuated colonic fluid absorption in the ferret by preirradiation treatment with the 5-HT3 antagonist granisetron.
Consequently and together with the measurement of colonic 5-HT content, the aim of this study was to investigate whether modifications in neural and/or nonneural 5-HT-mediated pathways may account for the radiation-induced changes in colonic fluid and electrolyte transport. Both neural, mediated via the submucosal plexus, and nonneural pathways were investigated on muscle-stripped distal colonic tissues mounted in Ussing chambers. The use of muscle-stripped tissue, in accordance with many other workers (6, 24, 33, 38, 46), was chosen to elucidate the effect of irradiation on the regulation of epithelial electrolyte transport by the submucosal plexus and 5-HT-associated pathways. Partial stripping (mucosa-muscularis/submucosa intact) also allows a better access of metabolic substrates and agonist/antagonists to the basolateral aspect of the epithelium in addition to transepithelial electrical measurements rather than transmural ones (24). Stimulated colonic transport was measured as variations in short-circuit current (Isc). The nonneural 5-HT-mediated pathway was followed by the measurement of exogenous 5-HT-stimulated Isc. The neural 5-HT-associated pathway was assessed, in the absence and presence of different 5-HT antagonists, by the technique of EFS, inducing neural depolarization of the submucosal plexus and resulting in a neurally mediated secretory response. Different 5-HT receptor antagonists were used to determine the receptor subtypes implicated in both pathways.
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MATERIALS AND METHODS |
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Animals
Studies were performed on male Wistar rats (225-250 g, CEJR, Le Genest, St. Isle, France). Animals were housed at constant temperature (20°C) and maintained on a 12:12-h light-dark cycle with a standard rat chow diet and water ad libitum. Experiments were conducted according to the French regulations for animal experimentation (Ministry of Agriculture Act no. 87-848, 19 October 1987).Irradiation Procedures
Animals were conscious and restrained in ventilated Plexiglas tubes and rotated for homogenous whole body exposure to a 60Co source, receiving a total dose of 10-Gy gamma irradiation (1 Gy/min). Control animals were submitted to the same conditions (sham-irradiated) but were not exposed to the radiation source. Studies were carried out 1, 3, and 7 days after exposure. Ussing chamber experiments and biochemical analyses were performed on the same animals.5-HT Assay
Stripped distal colonic tissue samples (150-200 mg) prepared as for Ussing chamber experiments were homogenized (Ultra-Turrax T25, Bioblock Scientific) in 1 ml of acidified butanol (butanol containing 0.1% concentrated HCl) and centrifuged at 2,000 g for 10 min. The supernatants were transferred to glass tubes containing heptane (1 ml) and HCl (0.1 N, 1.2 ml) and mixed. To separate the two phases, samples were then centrifuged 10 min (2,000 g). Measurement of 5-HT contained in the acid layer was performed by the technique described by Curzon and Green (14), using a spectrophotofluorometer (SFM 25, Kontron Instruments, St. Quentin en Yvelines, France), with excitation and emission wavelengths of 360 and 470 nm, respectively. Results are expressed in micrograms 5-HT per gram wet weight tissue.Ussing Chamber Experiments
Animals were anesthetized with pentobarbital sodium (60 mg/kg ip; Sanofi). The whole colon was removed, rinsed with ice-cold saline (0.9% NaCl) on ice, placed on a plastic 1-ml pipette, gently stripped of the external muscle layers by hand dissection, and opened longitudinally along the mesenteric border. Mucosal-submucosal preparations, with intact submucosal plexus, were mounted in Ussing chambers with 0.64-cm2 aperture (Corning-Costar, Cambridge, MA). Tissues were bathed with a modified Krebs buffer containing (in mM) 115 NaCl, 8.0 KCl, 2.0 KH2PO4, 2.4 MgCl2, 1.3 CaCl2, 25.0 NaHCO3, and 10.0 glucose, which was maintained at 37°C and gassed with 95% O2-5% CO2. Tissue responses to 5-HT and EFS were measured by clamping the potential difference to 0 mV, under Isc conditions with a voltage-clamp apparatus (DVC-1000, World Precision Instruments, Hertfordshire, UK). Isc was recorded continuously as the indicator of net active electrolyte transport across the tissue.Dose-Response Curves to Exogenously Added 5-HT
After a 15-min equilibration period, 5-HT was added at increasing doses (10EFS Experiments
EFS (100 V, pulse duration of 500 ms, total stimulation time of 3 s, frequency of 35 Hz) was applied with a dual-impedance stimulator (Harvard Instruments, Ealing, Les Ulis, France). To allow EFS of tissue, Ussing chambers were modified with the permanent placement of metal pins through the walls of both chambers. This allows connection to the stimulator (by crocodile clips). A stimulation of 35 Hz was chosen because it is known to induce a maximal response in rat colonic tissue (18).Use of 5-HT Antagonists Against 5-HT and EFS Responses
5-HT receptor subtypes were determined in a second set of animals. Two pieces of distal colonic tissue were studied for each rat. 5-HT (5 × 10To assess the neuronal involvement, Isc responses were measured following incubation of the tissue with TTX. Tissues were allowed to equilibrate as above, and initial EFS or 5-HT responses (separate tissues from the same animal) were then measured before TTX addition. Tissues were rinsed as above, and EFS and 5-HT responses were determined following a 30-min incubation of the tissue with TTX (1 µM).
Drugs
5-HT maleate salt, methysergide maleate, SDZ-205,557, and TTX were obtained from Sigma Chemical (St. Quentin Fallavier, France). Granisetron (Kytril) was obtained from SmithKline Beecham (Nanterre, France).Statistics
All data are expressed as means ± SE for n animals, with at least six animals per group. Tests of significant difference between groups were performed using a one-tailed unpaired Student's t-test, and a P value <0.05 was considered significant. When no significant difference was observed between control groups, data were pooled to obtain a single control value. ![]() |
RESULTS |
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Mucosal-Submucosal Content of 5-HT
5-HT tissue content was measured in mucosal-submucosal preparations of the distal colon, corresponding to those mounted in Ussing chambers. As shown in Fig. 1, exposure to ionizing radiation induced a significant decrease of 5-HT tissue content 1 day after irradiation (9.94 ± 0.46 in controls vs 6.12 ± 0.59 µg/g wet weight tissue in irradiated), followed by a return to control values at 3 days (9.63 ± 0.66 µg/g) and a significant increase at 7 days (13.40 ± 0.22 µg/g).
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Basal Electrophysiological Parameters
One day after exposure, tissue basal Isc and potential difference were increased (P < 0.05), but with no change in basal tissue conductance (Table 1). At 3 days, increased basal Isc was accompanied by a significant increase in tissue conductance (P < 0.05). Basal electrophysiological parameters returned to control values 7 days after radiation exposure. These data show that modifications to the colonic epithelium occur within 24 h after irradiation and that, at 3 days, the epithelium is compromised as shown by the increase in conductance.
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Investigation of 5-HT-Mediated Pathway: Dose-Response Curves to Exogenously Added 5-HT
In all cases, addition of 5-HT on the serosal side of the colonic tissue induced a dose-dependent increase in Isc. As shown in Fig. 2, exposure to ionizing radiation resulted in decreased maximal responses at 1 and 3 days (respectively, 50% and 71%; controls = 62.9 ± 3.2 µA/cm2 vs. 31.7 ± 7.4 and 18.6 ± 4.6, respectively, at days 1 and 3); furthermore, the maximal response was attained at a slightly lower concentration of 5-HT (10
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The concentration of 5-HT that elicited 50% of the maximal 5-HT response (described in MATERIALS AND METHODS) attained on the different days after irradiation was decreased with time after exposure (4.68 ± 0.57 in control vs. 1.74 ± 0.11, 2.15 ± 0.55, and 1.16 ± 0.32 µM in irradiated tissues at 1, 3, and 7 days, respectively).
Effect of Various 5-HT Receptor Antagonists on Exogenously Added 5-HT Responses in Control and Irradiated Tissues
Figure 3 shows typical traces obtained following addition of 5 × 10
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5-HT2/1C and 5-HT3 receptor antagonists methysergide and granisetron. The serosal addition of up to 100 µM methysergide or granisetron did not change the response to exogenous 5-HT, neither in control nor in irradiated tissues whatever the experimental time point (Fig. 3, A and B). Furthermore, experiments were also carried out with ketanserin, but in this preparation of rat distal colon this antagonist was without effect (at doses from 1 to 100 µM) on Isc responses elicited by 5-HT (data not shown).
5-HT4 receptor antagonist SDZ-205,557. The 5-HT4 receptor antagonist (100 µM) totally inhibited the tissue response to exogenously added 5-HT in control as well as in irradiated tissues all over the experimental period (Fig. 3, C and D). At a concentration of 1 and 10 µM, SDZ-205,557 inhibited 5-HT-stimulated (5 µM) Isc responses by 20% and 60%, respectively.
Effect of TTX on 5-HT and EFS Responses
The effect of TTX on exogenously added 5-HT and EFS responses is shown in Fig. 4. TTX failed to significantly reduce responses to 5-HT in control as well as in irradiated tissues, despite a slight (5-10 µA/cm2) tendency to decrease in all tissues (Fig. 4A). Conversely, EFS responses were almost totally abolished in the presence of TTX (Fig. 4B).
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Investigation of the Neural 5-HT-Associated Pathway: Tissue Responses to EFS
In control as well as in irradiated tissues, EFS induced a frequency-dependent rise in Isc, the sensitivity of which was unchanged by irradiation as shown previously in rat colon (18). Exposure to ionizing radiation, however, induced a decrease in EFS maximal responses from distal colonic tissues 1 and 3 days after irradiation, with a return to control values at day 7, similar to responses obtained with 5-HT (Fig. 5).
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Effect of Various 5-HT Receptor Antagonists on EFS-Evoked Responses in Control and Irradiated Tissues
Figure 5 shows the percent inhibition of EFS responses in control and irradiated tissues in the presence of methysergide, granisetron, or SDZ-205,557 (final concentration of 100 µM). It can be seen that the serosal addition of methysergide did not modify the EFS response from either control or irradiated tissues. Additional experiments were carried out with ketanserin (1-100 µM) in both control and irradiated animals. This agent (100 µM) reduced EFS responses by some 20-30% in both control and irradiated animals at whatever time studied (data not shown).However, serosal addition of 100 µM granisetron induced a decrease in the 35-Hz EFS response, whereas doses of 5, 10, or 50 µM were ineffective. In control tissues, granisetron inhibited the EFS response by 51% (Fig. 5). At 1 and 3 days, inhibition percentages obtained in irradiated tissues were similar to controls [64 and 58%, respectively, vs. control (51%)]. Conversely, at 7 days, the granisetron-induced inhibition in EFS response was significantly increased, with an inhibition percentage of 73 ± 3% (P < 0.05 different from control values).
Whereas SDZ-205,557 had no or little effect on EFS responses in control and irradiated tissues at 1 and 3 days (Fig. 5), the tissue response 7 days after radiation exposure was inhibited by 40% (95.2 ± 9.0 vs. 57.1 ± 3.0 µA/cm2, respectively, in absence and in presence of SDZ-205,557). This inhibition is similar to that effected by SDZ-205,557 at a concentration of 10 µM on 5-HT-stimulated (5 µM) Isc responses.
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DISCUSSION |
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It has been previously reported that neurally evoked electrolyte transport is modified after irradiation in rat ileum and colon (18, 19, 33). In addition, neural innervation may be modified following radiotherapy (25). The aim of this study was to investigate both submucosal neural and nonneural 5-HT-associated pathways involved in the modulation of rat distal colonic transport and to determine if changes in these pathways may account for both the attenuation and recovery of colonic secretory capacity following exposure to ionizing radiation. To perform these studies and to study particularly radiation-induced changes in regulation by the submucosal plexus, a stripped preparation of rat distal colon was utilized. The stripping process used in this study keeps the submucosal plexus intact and facilitates basolateral access to the site of action of agonists and antagonists. It is clear, however, that the influence of the myenteric plexus, which has been shown to influence intestinal fluid and electrolyte transport (29, 31, 42, 42a), has not been addressed in this study. Nevertheless, this approach does allow perhaps a more simple assessment of the effect of irradiation on the regulation of colonic electrolyte transport by the submucosal plexus and associated nerves. The presence of neural structures in the mucosal-submucosal preparations was confirmed by EFS-stimulated electrolyte transport and abolition of the response by prevention of neural conduction with TTX. It is notable that basal electrical parameters (Isc; 46.6 µA/cm2) of this preparation are in agreement with those reported by Bunce et al. (51.8 µA/cm2; Ref. 6) as well as 5-HT EC50 values (both ~4 µM).
The receptor subtypes implicated in the nonneural 5-HT-mediated colonic epithelial response remain controversial. In 1993, Siriwardena and Kellum (46) confirmed their previous results (44) concerning involvement of 5-HT2 receptors in the nonneural pathway of 5-HT-induced electrolyte transport in the rat distal colon. Other studies have shown a role for 5-HT4 receptors (1, 5, 6) in 5-HT-stimulated Isc responses together with an insensitivity to 5-HT1, 5-HT2, and 5-HT3 receptor antagonists (6). Our study supports evidence in favor of the involvement of 5-HT4 receptors, as proposed by Bunce et al. (6) and as shown by Budhoo et al. (5) and more recently by Albuquerque (1). Indeed, colonic epithelial responses to 5-HT were totally abolished by the 5-HT4 receptor antagonist SDZ-205,557 and were insensitive to TTX, thus confirming a 5-HT4-mediated response of nonneural origin. Zimmerman and Binder (49) have also shown responses to exogenous 5-HT to be insensitive to TTX in rat distal colon. Similar data have also been reported for intact mouse colon (ENS intact), in which TTX had no effect on 5-HT-stimulated Isc responses (23). One explanation for such data may be that 5-HT receptors are on TTX-insensitive nerves. In the light of such findings, further experiments using other inhibitors of neurally mediated responses such as hexamethonium, capsaicin, or lidocaine in both stripped and intact tissues are necessary.
In irradiated tissues, the present data suggest that responses to exogenous 5-HT still occur via the same pathway, involving 5-HT4 receptors. However, implication of the same receptor subtype does not rule out the fact that the characteristics of these receptors may be modified following radiation exposure. The decreased 5-HT EC50 values, compared with those obtained in sham-treated animals, observed in the present study could be due to changes in mucosal-submucosal tissue 5-HT content. However, these altered EC50 values are not correlated with the epithelial response capacity, since, at 3 days, the most impaired tissue response is associated with normal 5-HT tissue content. This suggests that other factors may be implicated in the impairment of colonic responses to 5-HT, such as intracellular pathways and/or receptor coupling. 5-HT4 receptors are coupled to adenylate cyclase (Gs) to increase cAMP (1). However forskolin-stimulated Isc responses in rat colon were also attenuated by irradiation (16), which suggests a more general action on adenylate cyclase activity. In fact, very recent work reported by Freeman and MacNaughton (20) has shown that attenuated EFS and forskolin responses in irradiated mice may be reversed by addition of an inhibitor of inducible nitric oxide synthase to the serosal side. Such data are exciting and may aid in the explanation of why secretory responses are reduced following irradiation.
In the rabbit ileum and colon, 5-HT has been shown to cause net intestinal water and electrolyte secretion by inhibition of neutral sodium chloride absorption (15) and, in the rat colon, also by stimulation of electrogenic chloride secretion (49). Ionizing radiation is known to affect cell turnover and multiplication (40) and could impair the acquisition of specific transporters by enterocytes. Ionic transport systems have not been followed in this study, but a modification in number and/or function may be implicated in the decreased epithelial response to 5-HT. For example, the activity of the rat colonic Na+-K+-ATPase, implicated in the establishment of driving forces necessary for ion transport, has been shown to be reduced after radiation exposure (8-Gy gamma whole body exposure; data not shown). The return to normal secretory responses at 7 days could thus be explained by the delay necessary for renewal of the epithelium from surviving stem cells (11, 40).
The investigation of the neural 5-HT-associated secretory pathways was performed using the EFS technique of the submucosal plexus. The 5-HT-associated neural pathway is mediated exclusively by 5-HT3 receptors, which are predominantly localized on neural structures (41, 45), illustrated by the 51% inhibition in the presence of the 5-HT3 antagonist granisetron and the insensitivity to both methysergide (5-HT2/1C receptor antagonist) and SDZ-205,557 (5-HT4 receptor antagonist) observed in control tissues.
One and three days after radiation exposure, the neural 5-HT-sensitive pathway still involves only 5-HT3 receptors. Conversely, at 7 days, the percent inhibition by granisetron is increased (73%) and the 5-HT4 antagonist attenuates EFS responses by some 40%. These modifications may result from changes in submucosal plexus innervation density or in 5-HT3 receptor numbers and/or effector cell characteristics. It should be noted that changes in enteric innervation patterns and/or density have been observed following radiotherapy of the abdominopelvic area (25, 26).
In the present experimental conditions, SDZ-205,557 may be considered as a strict 5-HT4 antagonist in the rat colon, since SDZ-205,557 was ineffective in inhibiting EFS responses in control tissues, although it has been reported to interact with both 5-HT3 and 5-HT4 receptors (17). The explanation for this attenuation of EFS responses by SDZ-205,557 at 7 days remains unclear. One possibility is that eventual radiation-induced changes in 5-HT3 receptor characteristics may result in the fixation of SDZ-205,557 to these receptors, so reducing the EFS response. However, like 5-HT3 receptors, 5-HT4 receptors have been shown to be localized in the gut on the presynaptic nerve terminals of cholinergic and secretomotor neurons and may consequently account for one part of the neural response. (4). Moreover, Nagakura et al. (36) have shown in the mouse that colocalized 5-HT3 and 5-HT4 receptors may have combined actions to elicit 5-HT-stimulated colonic fluid secretion. Thus it is possible that such a combined action may contribute to the recovery of EFS-induced secretory responses at 7 days after radiation exposure. These 5-HT-sensitive pathways may be explained in part by release of 5-HT by enteric interneurons, which can stimulate cholinergic and noncholinergic secretomotor neurons (12). Moreover, EFS may release 5-HT from enterochromaffin cells also via cholinergic mechanisms. In this case, released 5-HT would increase Isc via a paracrine, 5-HT4-mediated action on the epithelium. However, it would be expected in this case that the 5-HT4 antagonist would be effective in control tissues to inhibit EFS responses. This was not the case; thus the appearance of this component is associated with the recovery period after irradiation concomitant with increased tissue 5-HT content. Interestingly, atropine-sensitive EFS-evoked Isc increases were similar in control and irradiated animals at all times after irradiation (~30% inhibition, data not shown). Nevertheless, an increased sensitivity of muscle-stripped rat ileum to the muscarinic cholinergic agonist, carbachol, has been demonstrated after irradiation (32). Further work using a combination of 5-HT and cholinergic antagonists may provide additional information on this question, in addition to experiments using intact tissue.
In conclusion, these results suggest that radiation-induced impaired colonic fluid and electrolyte transport at 1 and 3 days, despite the altered EC50 values to 5-HT, do not seem to be due to changes in the 5-HT receptors implicated either in the submucosal neural or in the nonneural epithelial pathway but more probably to reduced ability of colonic epithelium to respond to the activation of these receptors. As indicated earlier, this may also be a result of release of inflammatory mediators such as nitric oxide, which are known to affect electrolyte transport (20). At 7 days postirradiation, when EFS responses had returned to control values, there was an increase in the 5-HT3-sensitive component along with the appearance of a 5-HT4-sensitive associated response. At this time, the tissue appeared more sensitive to exogenous 5-HT, and tissue levels were greater than those in controls. Thus additional submucosal serotoninergic innervation and/or the appearance of a 5-HT4 receptor-mediated pathway may participate, possibly via a combined action with 5-HT3 receptors, in the recovery of colonic transport 7 days after radiation exposure.
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ACKNOWLEDGEMENTS |
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We thank Dr. W. K. MacNaughton, Univ. of Calgary, for advice and for continued help and intellectual support. We are grateful to Q. Chau and F. Trompier for irradiation parameters and dosimetry and to C. Maubert for care of animals.
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FOOTNOTES |
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This work was supported in part by a Grant from Electricité de France to A. François.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. M. Griffiths, Institut de Protection et de Sûreté Nucléaire, Département de Protection de la Santé de l'Homme et de Dosimétrie, Section Autonome de Radiobiologie Appliquée à la Médecine, BP no. 6, 92265 Fontenay aux Roses Cedex, France (E-mail: nina.griffiths{at}ipsn.fr).
Received 10 December 1998; accepted in final form 23 September 1999.
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