Ionizing radiation stimulates muscarinic regulation of rat intestinal mucosal function

F. Lebrun, A. Francois, M. Vergnet, L. Lebaron-Jacobs, P. Gourmelon, and 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, F-92265 Fontenay-aux-Roses Cedex, France

    ABSTRACT
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Abstract
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Materials & Methods
Results
Discussion
References

The aim of this study was to determine whether ionizing radiation modifies muscarinic regulation of intestinal mucosal function. Rats exposed to total body 8-Gy gamma -irradiation or sham irradiated were studied up to 21 days after irradiation. Basal and carbachol-stimulated short-circuit current (Isc) and transepithelial conductance (Gt) of stripped ileum were determined in Ussing chambers. Muscarinic receptor characteristics using the muscarinic antagonist [3H]quinuclidinyl benzilate and three unlabeled antagonists were measured in small intestinal plasma membranes together with two marker enzyme activities (sucrase, Na+-K+-ATPase). Enzyme activities were decreased 4 days after irradiation (day 4). Basal electrical parameters were unchanged. Maximal carbachol-induced changes in Isc and Gt were increased at day 4 (maximal Delta Isc = 195.8 ± 14.7 µA/cm2, n = 19, vs. 115.4 ± 8.2 µA/cm2, n = 63, for control rats) and unchanged at day 7. Dissociation constant was decreased at day 4 (0.73 ± 0.29 nM, n = 10, vs. 2.14 ± 0.39 nM, n = 13, for control rats) but unchanged at day 7, without change in binding site number. Thus total body irradiation induces a temporary stimulation of cholinergic regulation of mucosal intestinal function that may result in radiation-induced diarrhea.

muscarinic receptor; short-circuit current; rat ileum

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

INTESTINAL FUNCTIONS are partly controlled by the autonomic nervous system, which is composed of the sympathetic, the parasympathetic, and the enteric nervous systems. The major neurotransmitter of the parasympathetic nervous system is ACh, which is liberated both by extrinsic and intrinsic fibers and can modulate intestinal functions by activation of secretomotor and interneurons (24). In particular, ACh plays a central role in neural regulation of the epithelium because stimulation of active chloride secretion by electrical field stimulation can be partly blocked by the muscarinic antagonist atropine (3, 6). In addition, ACh can act directly on epithelial cells via the stimulation of muscarinic receptors (37). Indeed, the alteration of electrolyte transport in guinea pig ileum and rat colon by exogenously added muscarinic agonists even in the presence of a neural pathways inhibitor (tetrodotoxin) provides evidence for a direct action of muscarinic agonists on epithelial cells (3, 6, 43).

One major consequence of ionizing radiation is the appearance of severe diarrhea, the etiology of which is to date unknown. Radiation-induced diarrhea has generally been attributed to an important disruption of intestinal structure and compromised epithelial integrity. However, recent studies have reported perturbations of fluid and electrolyte transport induced by irradiation, in conditions under which no denudation of the intestinal mucosa and no disruption of the integrity of the intestinal epithelial barrier were evident (7, 15, 26). In in vitro studies, modification of both electrical parameters and electrolyte fluxes have been observed very early after irradiation. In rabbit ileum, basal short-circuit current (Isc) and net serosal-to-mucosal Cl- fluxes were increased while net Na+ flux was unchanged in less than 2 days after a 10-Gy irradiation (15). Concerning motility, several studies have reported that changes in gastrointestinal tract motility preceded the appearance of histopathological lesions (8). In particular, in both animal models and patients, small intestinal and whole gut transit was markedly accelerated within hours after irradiation (32, 33, 40, 41).

The occurrence of diarrhea may also be ascribed to radiation-induced modifications of the different systems that regulate small intestinal functions (4). The effects of ionizing radiation on some of these regulatory systems have been partly investigated during recent years. In particular, ionizing radiation has been reported to modify the blood and tissue levels of some gastrointestinal regulatory peptides, such as neurotensin and gastrin-releasing peptide (14, 22, 23), that are known to modulate intestinal blood flow, motility, and electrolyte transport. On the other hand, it can be hypothesized that a dysregulation of the autonomic nervous system may participate in the development of diarrhea induced by exposure to ionizing radiation, as has been suggested for diarrhea associated with inflammatory bowel diseases (38). In agreement with this hypothesis, the levels or effects of several neuromodulators, such as substance P and vasoactive intestinal peptide (VIP), have been reported to be modified by ionizing radiation (9, 13). Furthermore, exposure to ionizing radiation also results in altered responses to either neurally evoked electrolyte transport or to exogenously added prostaglandin E2. (10, 16, 26). Finally, Otterson et al. (30) have suggested that the abnormal contractile patterns in canine small intestine observed after irradiation may be related to impaired neural regulation or to abnormal release of gut neuropeptides.

Thus some regulatory systems have been explored, but surprisingly the effect of ionizing radiation on the classical cholinergic pathway of regulation of intestinal transport function has not been studied. Nevertheless, some indirect or direct arguments suggest that modulation of cholinergic regulation might participate in radiation-induced dysfunctions and that ionizing radiation may modulate the direct action of ACh on the enterocyte. An indirect argument was provided by experiments indicating that ionizing radiation modified levels of acetylcholinesterase (AChE), the enzyme degrading ACh (5, 11, 30). On the other hand, a direct argument was provided by the experiments of Krantis et al. (21), who reported that the contractile responses to direct smooth muscle stimulation with the muscarinic agonist carbachol was significantly increased in the duodenum and colon but not in the jejunum of the guinea pig after gamma -irradiation.

Thus the aims of this study were to examine to what extent cholinergic regulation of intestinal fluid and electrolyte transport in the rat small intestine is modified by total body gamma -irradiation. First, this effect was assessed in vitro in the isolated rat ileum by determination of carbachol-stimulated Isc and epithelium conductance (Gt) responses in Ussing chambers. Second, the determination of mucosal muscarinic receptor characteristics was performed using a radiolabeled muscarinic antagonist, [3H]quinuclidinyl benzilate (QNB). Finally, different muscarinic antagonists were used for the determination of receptor subtypes present in small intestinal mucosa after irradiation.

    MATERIALS AND METHODS
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Introduction
Materials & Methods
Results
Discussion
References

Treatment of Animals

Male Wistar rats (Laboratoire CER Janvier) weighing between 250 and 300 g were used in all experiments, allowed food and water ad libitum, and maintained in a constant light and dark environment (12:12-h light-dark cycle).

Irradiation protocol. Conscious rats were placed in Plexiglas tubes and exposed to total body irradiation. Rats received 8-Gy gamma -irradiation (60Co source), at a dose rate of 1 Gy/min. Control rats were sham irradiated during the same period. Experimental procedures were performed from 1 to 21 days postirradiation. Intestinal samples were removed under anesthesia (pentobarbital sodium, 60 mg/kg), and then animals were euthanized with an overdose of anesthetic. All experiments were conducted according to the French regulations for animal experimentation (Ministry of Agriculture, Décret no. 87-848, 19 October 1987).

Histology. Histological control of the mucosal structure of samples of jejunum and ileum was performed on another group of rats. Samples were fixed in formaldehyde and embedded in paraffin, and sections were stained with hematoxylin-eosin. Samples were observed for general morphology and organization of the villi, for the mucus state, and for the presence of inflammatory features.

Membrane Preparation

The whole ileum and jejunum were removed and rinsed with 0.9% NaCl, and all procedures were carried out on ice. The mucosal layer was scraped from the underlying muscle layers using a glass slide. For membrane preparation, tissue was homogenized in 10 volumes of sucrose buffer (250 mM sucrose, 2 mM Tris, pH = 7.4) containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF; 0.1 mM) and centrifuged at 2,500 g for 15 min and then at 20,000 g for 20 min at 4°C. The resulting supernatant was discarded, and the pellet was resuspended in 1 ml of sucrose buffer without PMSF, quickly frozen in liquid nitrogen, and stored at -80°C until being used for radioligand binding studies and determination of enzyme activities.

Enzyme Activities

Sucrase activity was determined as described by Mahmood and Alvarado (27), by measurement of D-glucose formation in the presence of glucose oxidase and peroxidase. Na+-K+-ATPase activity was estimated with the use of a ouabain-sensitive, K+-stimulated p-nitrophenyl phosphatase assay (29). Results were expressed per milligram protein, estimated using the dye-binding method of Bradford (2) with bovine serum albumin as standard.

Electrolyte Transport Studies

Segments from distal ileum and proximal jejunum were removed and rinsed with 0.9% NaCl. The segments were stripped of external muscle layer, mounted in Ussing chambers, and bathed with warmed, oxygenated (95% O2-5% CO2) Hanks' buffer (pH 7.4) containing (in mM) 127 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 1 MgCl2, 4 NaHCO3, 1 CaCl2, 5 D-glucose, 10 Na acetate, and 20 HEPES. Two samples of tissue were tested for each rat. The Isc was monitored permanently, under basal or stimulated conditions. In parallel, Gt was calculated using Ohm's law from values of the current induced when a transepithelial potential difference (PDt) of 2 mV was applied. Basal Isc, Gt, and PDt were determined after 5 min of stabilization. Tissues were then subsequently stimulated by increasing concentrations of carbachol (10-7-10-4 M) added to the serosal side of the tissue. Between each concentration of agonist, the tissue was rinsed and allowed to return to basal level. The change in Isc (Delta Isc) was determined as the difference between basal and stimulated conditions for each concentration and used for calculation of dose-response curves. The change in Gt (Delta Gt) was determined as the difference in calculated Gt between basal and stimulated conditions. Both results from Isc and Gt were also expressed as percent of maximal response for each sample to allow an estimation of EC50 values (concentration of carbachol necessary to induce half-maximal response).

Radioligand Binding Studies

About 200 µg of the membrane preparation were incubated with increasing concentrations of the nonselective muscarinic antagonist [3H]QNB (specific activity 49 Ci/mmol), ranging from 50 pM to 2 nM in PBS containing (in mM) 137 NaCl, 2.7 KCl, 0.5 MgCl2, 8 Na2HPO4, 1.5 KH2PO4, 1 CaCl2, pH = 7.2, for 75 min at 30°C. For each concentration of [3H]QNB, the determinations were performed in triplicate and nonspecific binding was determined by the addition of 50 µM atropine in the incubation buffer. Nonspecific binding represented less than 10% of the total binding at concentrations of [3H]QNB near the half-maximal concentration for saturation (dissociation constant; Kd). Bound and free ligand were separated on GF/B Whatman paper filters (preincubated overnight in 0.6% polyethylenimine), using a rapid vacuum filtration system and rinsing three times with 3 ml 10 mM cold Tris solution, pH 7.0. The experiments were performed 1, 3, 4, and 7 days after irradiation on either irradiated or sham-irradiated rats. The radioactivity was counted in a Packard liquid scintillation counter. Analysis of specific binding data was by Scatchard transformation, with the determination of values of Kd and maximal binding capacity (Bmax).

Antagonist Displacement Binding Studies

The effect of muscarinic antagonists on [3H]QNB binding were tested only 4 days after irradiation on sham-irradiated or irradiated rats. Membrane preparation was performed, pooling four rats for each experiment, and four experiments were performed for control and irradiated conditions. The labeled antagonist, [3H]QNB (2 nM), and increasing concentrations of three unlabeled muscarinic antagonists were used: atropine (0-5 × 10-3 M), pirenzepine (0-10-2 M), and methoctramine (0-5 × 10-3 M). IC50 values (concentration of antagonist necessary to induce half-maximal inhibition of [3H]QNB binding) were determined for each antagonist and for each membrane preparation.

Chemicals

Methoctramine was obtained from Research Biochemicals International, Natick, MA. Carbamylcholine chloride (carbachol), atropine, pirenzepine, and all other enzyme substrates and salts were from Sigma Chemical, Poole, UK. [3H]QNB (37 TBq/mmol) was from Amersham International, Little Chalfont, UK.

Statistical Analysis

Results are expressed as means ± SE. A one-way ANOVA was used to test populations of control rats for enzyme activities and basal electrical parameters. A one-way ANOVA Dunn's test was used for receptor binding characteristics. Mann-Whitney's rank sum test was applied to carbachol-stimulated increases in Isc and Gt. An unpaired t-test was used for inhibitory constant of muscarinic antagonists. Significance was set at P < 0.05.

    RESULTS
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Materials & Methods
Results
Discussion
References

For all experiments (determination of Isc, enzyme activities, and receptor characteristics), no significant difference was observed between the control groups of rats tested for the different time after sham irradiation. Consequently, all results for control animals were pooled. Histological examination performed on control and irradiated rats (n = 10) showed no major modification of the mucosal structure.

Determination of Enzyme Activities

Sucrase is an enzyme associated with the apical membrane and is primarily located on the top of the villi. The results presented in Table 1 show that sucrase activity was greatly decreased to 17% of control values 4 days after irradiation (P < 0.05). Nine and 21 days after irradiation, the sucrase activity returned to control values (NS). In parallel, the activity of the basolateral enzyme Na+-K+-ATPase was determined. As shown in Table 1, irradiation modified Na+-K+-ATPase activity with a time-dependent pattern similar to the pattern observed for sucrase. Na+-K+-ATPase activity fell to 40% of control levels 4 days after irradiation (P < 0.05) and returned to control values at day 9 (NS). At day 21, a second decrease in activity of 72% was observed.

                              
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Table 1.   Modification of intestinal enzyme activities by irradiation

Functionality of Muscarinic Receptors: Ussing Chamber Studies

Basal values of electrical parameters. The mean values of Isc and Gt were determined in basal conditions in ileum samples, and values obtained for control rats and irradiated rats 4 and 7 days after irradiation are reported in Table 2. The basal Isc value (103.1 ± 5.7 µA/cm2, 63 samples for control group) was unchanged by the 8-Gy irradiation whatever the time of experiment. Similar results were obtained for basal Gt (83.0 ± 3.8 mS/cm2 for control group). In parallel, the basal PDt was determined. Irradiation induced no change in PDt whatever the time after irradiation (-3.66 ± 0.18 mV for control group vs. -3.22 ± 0.34 mV for irradiated group at day 4).

                              
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Table 2.   Effect of an 8-Gy gamma -irradiation on basal and carbachol-stimulated electrical parameters in isolated stripped pieces of ileum

Carbachol-induced increase in Isc. Addition of carbachol to the serosal side of the ileal tissue induced a slow increase in Isc that reached a plateau in 4-6 min, depending on the concentration used. The increase was maintained as long as the agonist was applied. When the tissue was rinsed, the Isc returned to basal levels in 6-10 min. The dose-response curves obtained for control (63 samples) and irradiated rats at day 4 (23 samples) and day 7 (19 samples) are reported in Fig. 1. The values of maximal Delta Isc and of EC50 (estimated from curves representing increases in Isc as percent of maximal increase) are reported in Table 2. For all groups the maximal increase in Isc was obtained with a dose of 5 × 10-5 M of carbachol. Four days after irradiation, the amplitude of the maximal carbachol-induced increase in Isc was more important than for control conditions (maximal Delta Isc = 115.4 ± 8.2 µA/cm2 for control vs. 195.8 ± 14.7 µA/cm2 for irradiated rats at day 4). On the other hand, the estimated EC50 value was unchanged (see Table 2). Seven days after irradiation, the dose-response curve was again similar to control values, and no change either in maximal response or in EC50 value was observed. Similarly, no effect of irradiation on either Delta Isc or EC50 values was observed at 1 and 14 days after irradiation (results not shown).


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Fig. 1.   Effect of irradiation on carbachol-induced increases in short-circuit current (Isc) on isolated stripped preparation of rat ileum. Each ileum sample was stimulated by increasing concentrations of carbachol ranging from 10-7 to 10-4 M. Experiments were performed on control (bullet ) and irradiated rats 4 () and 7 days (black-down-triangle ) after 8-Gy gamma -irradiation. Values are expressed as change in Isc (Delta Isc) ± SE for 63, 23, and 19 pieces of intestine from 34, 12, and 10 rats for control and 4 and 7 days of irradiation, respectively. Statistical differences between pooled control group and irradiated groups were assessed for each dose of carbachol using Mann-Whitney's rank sum test. * P < 0.05.

In pieces of jejunum the Delta Isc was also measured. The maximal Delta Isc, which was also obtained with 5 × 10-5 M carbachol, was unchanged 1 day after irradiation (92.1 ± 14.9 µA/cm2, n = 9) but was increased 4 days after irradiation (114.8 ± 9.9 µA/cm2, n = 15) as compared with control values (80.7 ± 11.2 µA/cm2, n = 11).

Carbachol-induced increase in Gt. For ileum samples similar dose-response curves were obtained for change in Gt, expressed as maximal Delta Gt or as percent of maximal responses (not shown). Their profile was exactly similar to the one observed for Isc. The values of maximal Delta Gt and EC50 reported in Table 2 showed no change in EC50 but an important change in Delta Gt 4 days after irradiation (57.9 ± 3.7 mS/cm2 for control vs. 101.1 ± 8.1 for irradiated rats), with a return to control values 7 days after irradiation.

Characteristics of Muscarinic Receptors

Figure 2A shows an example of a saturation curve obtained for [3H]QNB concentrations ranging from 50 pM to 2 nM, for an irradiated rat 4 days after the 8-Gy irradiation. Analysis of the results by Scatchard analysis of the saturation binding curve fits with a one-binding site model. The values of the receptor characteristics of control rats were 2.14 ± 0.39 nM for Kd and 66.0 ± 11.0 fmol/mg protein for Bmax (n = 13). No change was observed 1 day after irradiation. The Kd was decreased at day 4 after irradiation (Kd = 0.73 ± 0.29 nM, n = 10, P < 0.05), but no significant decrease was observed at day 7 (Kd = 0.78 ± 0.09 nM, n = 4, NS). No significant change in the number of sites (Bmax) was observed whatever the time after irradiation (Bmax = 66.0 ± 11.0 fmol/mg for control rats, n = 13, vs. 42.7 ± 6.6 for irradiated rats at day 4, n = 10).


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Fig. 2.   [3H]quinuclidinyl benzilate (QNB) binding to membranes isolated from small intestine of rat. A: example of saturation curve of [3H]QNB binding to muscarinic receptors determined for a rat 4 days after 8-Gy gamma -irradiation. Increasing concentrations of [3H]QNB (5 × 10-11 to 2 × 10-9 M) were used. Specific binding (black-triangle) was defined as arithmetic difference between total binding () and nonspecific binding (open circle ) observed in presence of 50 µM atropine. B: transformation of Scatchard of saturation binding curve. Kd, dissociation constant; Bmax, maximal binding capacity. Data points in both A and B represent mean of triplicate determinations in a single experiment.

Displacement of [3H]QNB Binding by Other Muscarinic Antagonists

Figure 3 shows that [3H]QNB binding is reduced in a dose-dependent manner by increasing concentrations of the three muscarinic antagonists, atropine, methoctramine, and pirenzepine, in sham-irradiated (Fig. 3A) and 8-Gy-irradiated rats (Fig. 3B) 4 days after irradiation. Data points of the displacement curves represent the mean of four experiments for each compound ± SE. IC50 values were estimated for each experiment. No significant change in IC50 value was observed for atropine (1.21 ± 0.39 µM for control vs. 0.47 ± 0.12 µM for irradiated rats, NS) even if there was a tendency to increased sensitivity. On the other hand, IC50 values for both the other antagonists were modified after irradiation: decreased by 71% for pirenzepine (175.0 ± 36.0 µM for control vs. 50.0 ± 12.0 µM for irradiated rats, P < 0.05) but increased by 38% for methoctramine (12.7 ± 1.0 µM for control vs. 17.5 ± 1.4 µM for irradiated rats, P < 0.05) (Table 3).


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Fig. 3.   Effect of irradiation on binding of 3 muscarinic antagonists. Displacement of 2 nM [3H]QNB by muscarinic antagonists atropine (), methoctramine (bullet ), and pirenzepine (down-triangle) was performed on sham-irradiated (A) or 8-Gy gamma -irradiated rats (B) 4 days after irradiation. Data points represent mean of 4 experiments for each compound ± SE. IC50 values were estimated for each experiment and are reported in text.

                              
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Table 3.   Effect of an 8-Gy gamma -irradiation on muscarinic receptor characteristics

    DISCUSSION
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In agreement with other studies on the rat or rabbit ileum (15, 26), no change was seen in ileal basal electrical parameters whatever the time after irradiation. MacNaughton et al. (26) reported that in rat ileum after a 10-Gy gamma -irradiation basal Isc was not significantly different 2 h or 1 or 2 days after irradiation, whereas in ferret jejunum after a 5-Gy gamma -irradiation basal Isc was decreased at 2 h and increased at 2 days after irradiation (25). In their conditions, Gt was not modified on the rat ileum at day 1. Furthermore, Gunter-Smith (15) observed in the rabbit no change in either distal ileal Isc or Gt during 4 days after a 5-Gy irradiation. However, an increase in basal Isc was observed from 1 day after irradiation with higher doses (7.5 and 10 Gy). In our conditions, the absence of change in basal parameters suggests no major disturbance of the integrity of the epithelial barrier. This is in agreement with our histological analysis using light microscopy, which revealed no marked structural changes, unlike what was previously observed in rat ileum at 7 days after a 5-Gy irradiation following electron microscopic analysis of the structure (31).

In contrast to the absence of change in basal parameters, maximal carbachol-stimulated responses of both Isc and Gt were increased 4 days after irradiation and returned to basal level 7 days after irradiation. No change in EC50 was observed in our conditions. These results suggest a greater capacity of ileum to respond to cholinergic stimulation 4 days after irradiation. This increased capacity of response was also observed in the jejunum, which suggests that irradiation affects in the same way the different parts of the small intestine. Our observations are in agreement with those of Harari et al. (18), who reported an increase in the effect of stimulation following a single dose of carbachol (10-4 M) at 5 days after a 7-Gy abdominal gamma -irradiation. It should be noted that the timing of the effect of irradiation we observed is in agreement with other transport experiments performed on rat small intestine concerning D-glucose and water transport (1). Furthermore, our results are in agreement with experiments performed by Young and Levin (42), who reported that, in a rat model, progressive starvation for up to 3 days induced no change in either basal Isc or carbachol-induced increase in Isc in ileum 1 day after the induction of starvation. However, both basal and carbachol-stimulated Isc were increased 3 days after induction of starvation. Food intake is reduced by irradiation, which suggests that this element is a factor that may complicate the interpretation of ionizing radiation effects.

In our experimental conditions, the pieces of ileum placed in the Ussing chamber still contain the submucosal plexus, which may suggest that when added to the serosal side of the chamber the carbachol may act via a stimulation of some intrinsic fibers of the enteric nervous system. MacNaughton et al. (26) have tested the responsiveness of rat ileum to electrical field stimulation from 2 h to 2 days after a total body irradiation (10 Gy, gamma ). In these experiments they observed a decrease in the responsiveness to electrical field stimulation as soon as 1 day after irradiation. The fact that ionizing radiation modifies carbachol-induced responses differently compared with electrical field stimulation-induced responses suggests that ionizing radiation may affect cholinergic regulation of intestinal secretory responses not only at the neural level but also at the level of the enterocyte.

Many factors may contribute to changes in carbachol responsiveness of enterocyte, including alteration in 1) the concentration of drug that reaches the receptor, 2) the efficiency of binding of the drug to the receptor, 3) the number of receptor sites, and 4) the efficiency of coupling of receptors to effector mechanisms. In fact, in our in vitro studies the synthetic agonist used (carbachol) is not degraded by AChE, which suggests that the change in response observed 4 days after irradiation may be associated with either perturbation at the receptor level or at the intracellular transductional level rather than with a change in agonist concentration reaching the receptors.

Modification of receptor characteristics by ionizing radiation has already been reported in the gut for substance P, neurotensin, and VIP (9, 13, 23). Our data show that 4 days after irradiation, characteristics of muscarinic receptors of the small intestine are modified, with a decrease of Kd without a change in the number of binding sites. These observations in addition to the increased intensity of change in Isc induced by carbachol suggest that after irradiation the small intestine is more sensitive to muscarinic regulation. In the control rats the displacement curves indicate an homogeneous population of muscarinic receptor sites. The affinity pattern of the antagonists is consistent with the presence of M2 muscarinic receptors because the IC50 for methoctramine is 10 times smaller than the IC50 for pirenzepine. Our experiments show that after irradiation the sensitivity for the M2 muscarinic antagonist (methoctramine) is decreased (increased IC50), whereas the sensitivity for the M1 muscarinic antagonist (pirenzepine) is increased (decreased IC50) and the sensitivity for the nonselective muscarinic antagonist (atropine) is unchanged. These results show that irradiation modifies the affinity of muscarinic receptors for agonists or antagonists differently depending on the compound used, which may be due to a change in structure or access to the different binding sites.

In fact different processes may take part in modification of muscarinic receptor characteristics. A first hypothesis concerns a change in agonist level, which may induce a feedback regulation of receptor characteristics. It is conceivable that irradiation may modify the level of ACh. Indeed, irradiation has been reported to be associated with release of reactive oxygen species (ROS), interleukin-1beta , and prostanoids, which have been shown to modulate the level of intestinal parasympathetic neurotransmitter liberation (12, 28, 34) and thus may lead to a decreased amount of ACh in irradiated tissue. In this study we did not measure the level of AChE, the enzyme that degrades ACh, in intestinal tissue. However, several studies that have addressed this subject indicate that ionizing radiation modifies levels of AChE, either decreasing or increasing it depending on the irradiation procedure (total body or abdominal irradiation) and on the tissue studied (5, 11, 30). In particular, whole body irradiation was reported to induce a decrease in ileal and jejunal AChE content (5, 11). These data are consistent with our observation of increased responsiveness to carbachol.

A second hypothesis deals with a possible modification of receptor environment. Ionizing radiation directly or via the production of potent ROS may damage constituents of the cell membrane such as proteins or lipids. Such modifications were reported by Keelan et al. (20) and are in agreement with the attenuation we observed of both apical (sucrase) and basolateral (Na+-K+-ATPase) enzyme activities. Thus ionizing radiation may have a direct effect on the molecular structure of muscarinic receptors. On the other hand, modification of protein and lipid composition can lead to an increase in membrane fluidity and a modification of the receptor environment. In this new environment, the tridimensional structure of the receptor and subsequent binding site conformation may change and so favor or disfavor agonist access to sites. This may lead to modification of binding affinities, as we have observed for muscarinic receptors. In particular, Hulme et al. (19) reported that the affinity of muscarinic receptors was differently modified by solubilization with digitonin depending on the muscarinic type considered. Indeed, the affinity of M2 type receptors was altered, whereas the affinity of M3 type receptors was quite unchanged.

We did not investigate the last hypothesis concerning a possible alteration by ionizing radiation of the signal transduction process associated with muscarinic receptors. Such an alteration has been observed in experiments showing that whole body irradiation of rats with 56Fe may lead to a deficit in striatal muscarinic cholinergic receptor-G protein coupling, reflected by a decrease in GTPase activity (36). Furthermore, ionizing radiation can modulate numerous other elements participating in intracellular signaling, such as intracellular calcium (17, 35), cAMP (13), and inositol trisphosphate receptors (39). Further experiments are required to determine the relative importance of modification of muscarinic transduction system in intestinal tissue.

In conclusion, in this study we observed that total body irradiation induces an upregulation of muscarinic regulation of mucosal fluid and electrolyte transport function in rat small intestine. Our results, together with those of Krantis et al. (21), show that both intestinal motility and electrolyte transport regulated by the cholinergic parasympathetic system can be modified by ionizing radiation, which suggests that this system may be implicated in the development of radiation-induced diarrhea.

    ACKNOWLEDGEMENTS

The authors thank C. Maubert and E. Sale for technical assistance and care of animals.

    FOOTNOTES

Some of these results were presented at the European Radiation Research Meeting in Oxford, UK, in September 1997.

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: F. Lebrun, 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 6, F-92265 Fontenay-aux-Roses Cedex, France.

Received 14 April 1998; accepted in final form 10 August 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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