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Intestinal Diseases Research Programme, McMaster University, Hamilton, Ontario, Canada
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(Received 8 December 2003;
; first published online 5 January 2004)
Corresponding author P. K. Rangachari: Intestinal Diseases Research Programme, McMaster University, Room HSC 3N5C, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5. E-mail: chari{at}mcmaster.ca
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Introduction |
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In the early 1970s, Overstreet et al. (Overstreet, 1992; Overstreet et al. 1979, 1995) developed the Flinders sensitive line (FSL) rats and their counterparts the Flinders resistant line (FRL). These animals were selectively bred on the basis of their responsiveness to cholinergic agonists and anticholinesterases such as diisopropyl fluorophosphate (DFP). This responsiveness was assessed in terms of body temperature, drinking and body weight. The FSL rat exhibited an increased responsiveness to cholinergic agonists when compared to the FRL rat. They were also less active, gained weight more slowly and were much more sensitive to muscarinic agonists and stress. Many studies were done to explore the functional consequences of these phenotypic differences, though a large number were concentrated primarily on the CNS and behavioural changes (Bellido et al. 2002; Einat et al. 2002; Ferreira-Nuno et al. 2002; Friedman et al. 2002; Overstreet, 2002; Shir et al. 2001; Yadid et al. 2001; Zangen et al. 2001; Zangen et al. 2002). Relatively few studies have been done to see whether the differences persisted in isolated tissues; these have been widely used to explore the role of neurotransmitters on intestinal ion transport (Brown & Ogrady, 1997; Ferraris & Carey, 2000).
We chose to test the hypothesis that these differences would persist at a peripheral level in isolated tissues removed from central influences. We used as our target system the distal colonic epithelium. Not only does this tissue respond well to cholinergic stimulation in vitro (Asfaha et al. 1999), but if differences were revealed it would permit us to dissect the underlying mechanisms. This could have some bearing given the available information that autonomic dysfunction may play a role both in functional and in inflammatory bowel disorders.
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Methods |
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A breeding colony for Flinders rats has been established at this university. All experiments were done using non-fasted male Flinders rats (FSL and FRL). The animals used in these experiments were age-matched and their weights were in the range 300–350 g.
Rats were killed by cervical dislocation, and a segment of distal colon of approximately 5 cm in length was removed. Tissues were immediately placed in warm oxygenated normal Krebs solution containing (in mM): Na+ 145; Cl– 128; PO3–4 1.2; Ca2+ 1.5; Mg2+ 1.2; HCO–3 22; K+ 4.6; and D-glucose 10 (pH = 7.4). The colon was then opened along the mesenteric border and pinned out on a silicon gel (Sylgard 184, Dow Corning, Midland, Michigan, USA) filled Petri dish with the mucosa facing downwards. Using a sharp scalpel blade, a light incision was made along the serosal surface. The outer muscle layers were then stripped using a surgical cotton swab and a pair of forceps, leaving behind the mucosal layer. The tissues were then mounted in conventional Lucite Ussing chambers (Clearwater, FL, USA) with an area of 0.6 cm2 and bathed in a normal Krebs buffer solution (12.0 ml per chamber) and oxygenated with 95% O2 and 5% CO2 at 37.5°C.
Measurement of electrical parameters in vitro
Four open-ended electrically conducting agar salt bridges were inserted into the Lucite chambers and connected to a high-impedance millivoltmeter (DVC-1000 dual voltage clamp, WPI). In order to achieve voltage clamping, sufficient current was passed across the tissue through salt-agar bridges connected to the voltage clamp apparatus by the Ag–AgCl electrodes. Appropriate corrections were made for electrode offset and fluid resistances. Short-circuit currents (Isc), representing active ion transport (measured in µA) and transmucosal conductances (Gt) were monitored using a conventional World Precision Instruments (Sarasota, FL) DVC-1000 dual voltage clamp (Keenan & Rangachari, 1991; Rangachari & Prior, 1994; Rangachari et al. 1995). Voltage clamp signals were captured and recorded using the Acknowledge MP100 data acquisition system (Biopac, Santa Barbara, CA). All tissues were allowed to equilibrate for a minimum period of 20 min or until a stable baseline was established, prior to the addition of stimulants. Pre-treatment with either indomethacin (10–6M) or TTX (10–6M) was done during this equilibration period.
Measurement of transepithelial ion fluxes
Ion fluxes were done using conventional radioisotope methods (Keenan & Rangachari, 1991; Lad et al. 1991; Rangachari & Prior, 1994). All experiments were performed under short-circuited conditions. Tissues from the same animal that had comparable Gt values (i.e. within 25% of each other) were paired to determine net fluxes. Transepithelial fluxes of sodium and chloride ions were measured using 2.0 µCi 22Na and 5.0 µCi 36Cl per chamber. Isotopes were added to either the serosal or mucosal buffer for measurement of serosal to mucosal (S
M) or mucosal to serosal (MS) unidirectional ion fluxes. Thirty minutes after addition of radioisotopes, 50 µl samples were removed from the ‘hot’ sides of each chamber for the determination of specific activity. Tissues were then monitored over three 20 min flux periods. Isotope flux was determined by taking 1 ml samples from the ‘cold’ side of the tissues at 20 min intervals. These samples were replaced with non-radioactive aliquots of the same composition and volume in order to maintain hydrostatic balance. Carbachol was added to the serosal side of every chamber following the first flux period (i.e. after 20 min). Following the completion of the three flux periods, a second 50 µl sample was collected from the ‘hot’ sides of each chamber. This sample was averaged with the first ‘hot’ sample and used to determine specific activity. All samples were first assayed in a gamma counter (1282 Compu Gamma, LKB Wallace, Stockholm, Sweden). Following this step, three ml of scintillation fluid (Formula 963 Aqueous Counting Cocktail, Du Pont, Wilmington, DE, USA) was added to each vial and mixed before being assayed in a LS counter (5801, Beckman, Irvine, CA, USA). The 22Na counts measured by the gamma counter were subtracted from the combined activity of both radioisotopes given by scintillation counting to yield the 36Cl counts (Rangachari & Prior, 1994).
Data analysis
Unidirectional 22Na and 36Cl ion fluxes were expressed in microequivalents per centimeters squared per hour and residual ion fluxes determined from the following relationship
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S flux. JClnet fluxes were also higher but only the MS flux of Cl– was found to be significantly different as compared to FRL rats. Since both JNanet and JClnet were increased and currents were similar, there was no significant difference observed in Jres between FRL and FSL rats under basal conditions.
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S flux of Na+ in first 20 min flux period following the addition of carbachol. This was accompanied by an increase in SM flux, resulting in a significant decrease in JNanet (Fig. 4B). These changes were evident in the early phase (0–20 min post carbachol) and although there appeared to be a recovery of the SM flux towards more normal values during the second 20-min period, the decrease in MS and the net flux of Na+ persisted. With the Cl– fluxes, there was a rapid and significant decrease in JClnet that persisted throughout the period of stimulation (0–40 min postcarbachol). The decrease in JClnet stemmed from a decreased MS flux that was accompanied by an increase in the SM flux of Cl– (Fig. 4C). An initial increase in Jres was observed following the addition of carbachol, though this increase did not achieve statistical significance. However in the subsequent flux period there was a rapid and significant reduction in Jres (Fig. 4D).
Similar changes in JNanet and JClnet were also seen in FSL rats. The addition of carbachol produced similar increases in Isc to those noted with FRL rats (Fig. 4A). Here, too, the changes in Na+ flux stem largely from a decrease in MS flux, though the increases in SM flux attain statistical significance in both flux periods (Fig. 4B). As with the FRL rats, the changes in JClnet were also due to a sharp decrease in MS flux accompanied by an increase in SM flux of Cl– (Fig. 4C). The pattern of residual fluxes was, however, somewhat different. Following the addition of carbachol, as was the case for FRL rats, there is an increase in Jres. In FSL rats this increase is statistically significant, whereas in FRL rats it was not. A much larger difference between the two lines is evident in the following flux period. Whereas Jres continues to remain high in FSL rats, it actually falls dramatically during this same period in the FRL rats (Fig. 4D). With all other things being equal, Cl– secretion is greater in FRL rats during this period leading to larger reduction of JClnet. Consequently, Jres falls in FRL but not FSL rats during the second 20 min flux period following carbachol addition.
Using a similar protocol, we assessed the effects of pretreatment with indomethacin (1 µM) on the changes in ion fluxes in both lines of rats. The results shown in Table 1 emphasize the consistency in responses seen. As in the previous set of experiments, carbachol stimulation produced elevations in Isc in both lines that were accompanied by decreases in JNanet and JClnet. Again the striking reduction in Jres in the second phase of the carbachol response is prominent in FRL rats. Such a reduction is not seen in FSL rats. Indomethacin pretreatment did not significantly alter the changes in JNanet and JClnet seen with either strain. However, the decrease in Jres seen in FRL rats was not observed.
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Discussion |
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TTX has been shown to affect carbachol-induced changes in Isc in different strains of rats. Using Sprague Dawley rats, Zimmerman & Binder (1983) showed that whereas TTX had no effects on bethanechol induced responses, it did reduce significantly the responses to high concentrations of carbachol. They suggested that this was due to the inhibition of the nicotinic but not the muscarinic effects of carbachol. Whether differences in nicotinic responses exist between the two strains is not clear. Preliminary studies suggest that there were marginal inhibitory effects of hexamethonium in both strains. More detailed studies need to be done.
In female Wistar rats, Diener et al. (1989) showed that TTX inhibited responses to several cholinergic agonists, acetylcholine, carbachol and bethanechol. In a later study, Strabel and Diener (1995) studied the effects of both indomethacin and TTX on responses of the distal colon from female SIVZ-50 rats. They showed that carbachol produced a biphasic increase in Isc, an initial peak followed by a long-lasting plateau. Whereas only the plateau phase was blocked by indomethacin, both phases were suppressed by the combined presence of TTX and indomethacin. They suggested that the continuous release of neurotransmitters and prostaglandins poised the tissue to respond to carbachol. Our results suggest that the release of neurotransmitter(s) may mediate part of the responses to carbachol but that in the FRL rats at least, the production of an inhibitory prostanoid serves to modulate those responses. The identities of the neurotransmitters and the prostanoid need further definition.
Although the precise identity of this neurotransmitter is not certain, a number of candidates exist. Of these, the most likely ones are VIP or Substance P. Both are present in the large intestines of different species and receptors for both have been demonstrated on transporting epithelial cells (Cooke, 1998). SP can exert its effects on NK1, NK2 or NK3 receptors. mRNAs for all three receptors have been shown in the rat colon (Tsuchida et al. 1990). Functional effects on all three receptors have also been demonstrated in the same tissue (Cox et al. 1993). Of these NK3 receptors predominate on submucosal neurones, NK2 receptors are found on epithelial cells and NK1 receptors on both neuronal and epithelial surfaces. Stimulation of all three types induces secretion. Similarly, VIP is present in the rat colon and the tissues respond to the peptide with changes in Isc (Morel et al. 2002; Schulzke et al. 1995).
The flux analysis showed that there were clear differences in the responses of the two lines. In both sets of rats, carbachol stimulation was associated with decreases in JNanet and JClnet. Thus the increases in Isc reflect both a decrease in Na absorption as well as a stimulation of Cl– secretion. The striking difference is seen in the net residual flux. In FRL rats, there is a slight increase in Jres in the early phase followed by a significant decrease. By contrast, no changes are seen in Jres in the FSL rats. These changes in Jres are abolished in the presence of indomethacin. Under such conditions, the pattern is similar to that seen in FSL rats. Our results bear comparison with those of Zimmerman et al. (1982). They studied cholinergic responses of the distal colonic epithelium from Sprague Dawley rats (the source for the Flinders lines). They also noted that changes in Isc in response to bethanechol were associated with decreases in JNanet and JClnet due largely to decreases in MS fluxes of Na and Cl–. In their experiments, however, little change occurred in SM flux of Cl. However, as the decrease in net Cl– absorption was greater than net Na+ absorption, the authors suggested that this component, though not statistically significant, could represent Cl– secretion. Their results show little evidence for residual fluxes either under control conditions or following stimulation. Whether this represents strain differences or experimental conditions is not clear.
The identity of the ion contributing to residual flux is not clear. Under basal conditions, the presence of a positive residual suggests that either a cation absorption or anion secretion is important. The two most likely candidates are K+ and HCO3–. The rat distal colon absorbs K+ through the operation of a luminal H+– K+ ATPase (Kunzelmann & Mall, 2002). The K+ absorbed leaves the basolateral surface by several different pathways including Ca2+ or cAMP activated K+ channels or K+Cl– cotransporter (KCC1). HCO3– that is secreted by the colonic epithelium can be either generated intracellularly through the activity of carbonic anhydrase or enter the basolateral surface by a Na-dependent electroneutral mechanism. Several pathways exist for the secretion of HCO3– into the luminal side including an electrogenic HCO3– efflux, a luminal Cl–/HCO3– exchanger or an SCFA/HCO3– exchanger. On stimulation with carbachol, the rapid reduction in this component suggests that either K+ absorption or HCO3– secretion is reduced. Alternatively this could also imply an enhancement of K+ secretion or HCO3– absorption. It is also possible that different ions could be contributing at different stages of stimulation. Thus whereas HCO3– secretion could be present under basal conditions, the onset of K+ secretion could serve to reduce net residual flux. The ions responsible need further definition.
Further studies using selective inhibitors or ion-substitutions are needed to define the transepithelial ion transport pathways in both strains to determine whether phenotypic differences are seen at this level as well.
Indomethacin does not alter residual fluxes under basal conditions but serves to abolish the reduction seen on cholinergic stimulation. This suggests that a prostanoid plays a significant role in this process. But the precise nature of that arachidonic acid metabolite is unclear at present.
The Flinders rats were developed over 25 years ago and have subsequently been used as experimental models for a variety of conditions ranging from depression to multiple chemical sensitivities (Overstreet & Djuric, 2001). These studies have explored behavioural differences between the sensitive and resistant lines, and relatively few in vitro studies have been done. Our studies show that even in isolated tissues, reproducible differences exist in the responses of these two lines to cholinergic stimulation. These differences involve the arachidonic acid pathway and the release of neurotransmitters. What is interesting is that these changes do not follow the classification established in vivo for these rats. If anything, FRL rats appear to be more responsive to cholinergic stimulation, at least in the presence of indomethacin, and under basal conditions, no differences were observed in the responses to carbachol between the two lines. Similar results were obtained by Janssen et al. (2000) who found that the EC50 for carbachol inducing contractile responses in tracheal smooth muscle was marginally lower in FSL rats, however, the magnitude of the responses were higher in FRL rats. The responses of the two lines to spasmogens following allergen challenge was identical, and thus the well-described airway hyper-responsiveness seen in vivo in a previous study from their lab (Djuric et al. 1998) was not transferred to the in vitro situation. The findings in these two studies, and those of our own indicate that before these rats are used as complicated models for exploring diverse disease conditions, a more careful analysis at a fundamental level needs to be done. It may well be that the differences we have observed might not be related to the cholinergic sensitivity observed in other studies.
We have not teased out the precise pathways involved in the transport of ions across the colonic epithelium. That would require careful use of selective inhibitors and ion substitution experiments. Such studies may well reveal that phenotypic differences may exist at the level of the transport pathways as well. Experimental colitis has been shown to alter secretory responses to carbachol (Asfaha et al. 1999). Whether the two lines of Flinders rats will respond differentially under such conditions remains to be seen. Such information may provide explanations as to the variability in responses not only to infection, but also stress or even commonly expressed GI side-effects (other than dyspepsia) of nonsteroidal anti-inflammatory drugs (NSAIDS).
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) and those treated with atropine are illustrated with an open square symbol (
). The data are displayed as the means ±S.E.M. from 4 animals.
