A novel receptor for calcitonin gene-related peptide (CGRP) mediates secretion in the rat colon: implications for secretory function in colitis TUBA ESFANDYARI, WALLACE K. MACNAUGHTON, RE´MI QUIRION,* SERGE ST. PIERRE,† JEAN-LOUIS JUNIEN,‡ AND KEITH A. SHARKEY1 Neuroscience and Gastrointestinal Research Groups, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1; *Douglas Hospital Research Centre, Department of Psychiatry, McGill University, Montreal, Quebec, Canada H4H 1R3; †Department of Chemistry, University of Quebec in Montreal, Montreal, Quebec, Canada H3C 3P8; and ‡Ferring Research Institute, Paris, France ABSTRACT The receptor responsible for CGRPinduced ion transport and permeability was examined in tissues from animals treated 7 days previously with trinitrobenzenesulfonic acid to induce colitis or in controls. CGRP caused a concentrationdependent increase in short circuit current (Isc, EC50 21 nM), which was abolished in chloride-free buffer but was not blocked by CGRP8 –37 or tetrodotoxin (TTX). Amylin and adrenomedullin caused only a modest increase in Isc. The responses to the linear CGRP2 receptor agonists [Cys(Et)2,7] hCGRP␣ and [Cys(Acm)2,7] hCGRP␣ were considerably smaller than the response to CGRP. These responses were abolished in chloride-free buffer and were TTX sensitive. Atropine, doxantrazole, and indomethacin did not block the effects of CGRP or the CGRP2 agonists. The response to [Cys(Et)2,7] hCGRP␣ was not affected by prior desensitization of the CGRP receptor and vice versa. Inflamed rats had a similar secretory response to CGRP (Isc, EC50 15 nM) and [Cys(Et)2,7] hCGRP␣ as control tissues, while being hyporesponsive to carbachol. CGRP application increased electrical conductance of inflamed preparations. Taken together, these data suggest that CGRP may play an important role in the maintenance of host defense in colitis through an apparently novel CGRP receptor located on the colonic enterocyte.— Esfandyari, T., MacNaughton, W. K., Quirion, R., St. Pierre, S., Junien, J.-L., Sharkey, K. A. A novel receptor for calcitonin gene-related peptide (CGRP) mediates secretion in the rat colon: implications for secretory function in colitis. FASEB J. 14, 1439 –1446 (2000)
Key Words: TNBS 䡠 ion transport 䡠 CGRP receptors 䡠 adrenomedullin 䡠 amylin 䡠 mast cells 䡠 secretion
Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide produced in some tissues by specific alternative splicing of the calcitonin messen0892-6638/00/0014-1439/$02.25 © FASEB
ger RNA (CGRP␣) (1, 2). In other tissues, a unique gene encodes a form of CGRP (CGRP) that differs from CGRP␣ by one and three amino acids in rats and humans, respectively (3). Both forms are widely distributed in the peripheral and central nervous system and are also found in high concentrations in the enteric nervous system of the gastrointestinal tract (4 – 6). The range of actions of CGRP is extensive and include potent vasodilatory actions, direct inotropic and chronotropic activities, as well as actions in the immune system and the central and peripheral nervous systems (4, 7, 8). CGRP is a member of a family of peptides that share sequence homology and have some overlapping biological activities. The members of the family include amylin, adrenomedullin, and CGRP (9, 10). There are pharmacological and radiochemical binding data that support the existence of at least 3 distinct receptors for CGRP in different tissues. The best-characterized receptor for CGRP is the so-called CGRP1 receptor. This receptor was proposed on the basis of differential pharmacological antagonism of carboxyl-terminal fragments of CGRP to block the action of CGRP and on the basis of differential actions of certain linear analogs of CGRP (such as [Cys(Acm)2,7]human{h}CGRP␣) (4, 11). Dennis et al. (12) reported that carboxyl-terminal fragments such as hCGRP8 –37 and hCGRP9 –37 acted as potent, competitive antagonists for CGRP-induced effects in the guinea pig atria, while being much less effective in blocking the effects of CGRP in the rat vas deferens. In contrast, linear analogs, such as [Cys(Acm)2,7]hCGRP␣, were effective agonists in the rat vas deferens and were largely inactive in the 1 Correspondence: Neuroscience Research Group, Department of Physiology and Biophysics, Health Sciences Centre, Room 2113, University of Calgary, 3330 Hospital Dr. N.W., Calgary, Alberta, Canada, T2N 4N1. E-mail:
[email protected]
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guinea pig atria (11, 12). Various groups have now shown that hCGRP8 –37 is an effective antagonist of the CGRP1 receptor in vivo and in vitro (13–17). The CGRP2 receptor is activated by certain linear analogs such as the recently described [Cys(Et)2,7] hCGRP␣ and [Cys(Acm)2,7] hCGRP␣ (11, 12, 18). This receptor is antagonized by CGRP8 –37, with a 10-fold lower affinity than at the CGRP1 receptor, but unfortunately to date, no specific antagonist has been developed for this receptor. In addition to the rat vas deferens, there is evidence for this receptor in the urinary bladder (19), liver (20), and a human adenocarcinoma cell line (Col-29), which when cultured has certain properties similar to intestinal epithelial cells (16). Finally, there is evidence for a CGRP receptor in the brain (especially the nucleus accumbens) that is distinct from either the CGRP1 or CGRP2 receptor in that it shows similar high-affinity binding for salmon calcitonin, CGRP, and amylin, a property that is not shared by any of the other CGRP receptors (4, 21, 22). To date, though a number of putative CGRP receptors have been cloned (23–26), there are none that have all the characteristics of the pharmacologically characterized receptors when expressed alone. The recent cloning of receptor activity-modifying proteins (RAMPs) may help resolve this issue (27). When the accessory protein RAMP1 was coexpressed with the calcitonin receptor-like receptor (CRLR) in HEK 293T cells, a functional receptor with CGRP1like properties was produced (27). When RAMP2 was coexpressed with CRLR, an adrenomedullin-like receptor was produced (27, 28). Further studies will be required to establish whether these proteins are constitutively colocalized in tissues that have functional receptors. CGRP plays an important role in gastrointestinal pathophysiology through its secretory action on the epithelium and its vasodilator actions (29 –31). The receptor mediating the secretory effect of CGRP has not been determined, though in the rat colon CGRPinduced chloride secretion was not inhibited by hCGRP8 –37 (16). Colitis appears to render the colonic epithelium less responsive to certain agonists, including those that act intracellularly (32–35). Since intestinal secretion is an important component of mucosal defense, we investigated whether the response to CGRP was altered in tissues from animals with colitis. In this study we have examined the receptor and mechanism mediating CGRP-induced secretion in the normal and inflamed rat colon.
Service. The animals were housed under controlled environmental conditions (23–24°C, light from 7:00 to 19:00 h, food and water ad libitum) for at least 2 days before being used. The experimental protocols used in this study were approved by the University of Calgary Animal Care Committee and conform to the guidelines established by the Canadian Council on Animal Care. Induction of colitis Rats were anesthetized with halothane (2–2.5% in oxygen), which allowed a prompt return to consciousness. While anesthetized, 0.5 ml of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 60 mg/ml) dissolved in 50% (v/v) ethanol was instilled into the lumen of the colon through a polyethylene catheter inserted rectally, such that the tip was ⬃8 cm proximal to the anus (36). Control animals received 0.5 ml of physiological saline (0.9% NaCl) as described above. After recovery from anesthesia, the animals were placed in the cage and kept under the controlled environmental conditions described above. One week after treatment, animals were killed by an overdose of sodium pentobarbital (⬎60 mg/kg, i.p.) and ⬃4 – 6 cm segments of the distal colon between 6 and 12 cm from the anus were removed. Assessment of colitis The severity of colitis was assessed by macroscopic damage scoring of the affected tissue and by measurement of myeloperoxidase (MPO) activity. The criteria for scoring of gross morphological damage is similar to that previously been described by McCafferty et al. (37). Briefly, the method of macroscopic damage scoring takes into account the presence and absence of diarrhea (0 –1) and a score based on the extent of mucosal damage from normal (score of 0) to a score of 10 depending on the presence and extent of ulceration and the extent of hyperemia. MPO activity as a quantitative index of inflammation was determined using an assay first described by Krawisz et al. (38) and subsequently modified for use with a plate reader (37, 39). The plate was immediately read at 450 nm using a Molecular Devices UV Max kinetic plate reader (Molecular Devices, Sunnyvale, Calif.). Three readings were taken 30 s apart and the activity was calculated using SoftMax software (Molecular Devices). Tissue preparation
MATERIALS AND METHODS
The distal colon was excised and placed in ice-cold oxygenated Krebs’ buffer without glucose of the following composition (mM): NaCl 115.0, KH2PO4 2.0, MgCl2 2.4, CaCl2 1.3, KCl 8.0, and NaHCO3 25.0. A thin glass rod was inserted into the colon, and the muscularis externa and associated myenteric plexus were removed from the underlying submucosa by blunt dissection. The tissues were then cut open along the mesenteric border. Four adjacent pieces of mucosa/submucosal preparations were routinely obtained from each salinetreated or untreated rat. Samples from TNBS-treated animals were obtained from the colon immediately adjacent to the most seriously inflamed area. The maximally inflamed region was not used, as removal of the muscle was not possible. Two immediately adjacent (but nevertheless grossly inflamed) pieces of mucosa/submucosal preparations were routinely obtained from each TNBS-treated rat.
Animals
Electrolyte transport
Male Wistar rats (180 –200 g) were obtained from a breeding colony maintained by the University of Calgary Animal Care
Ion transport was studied in a standard Ussing-type diffusion chamber apparatus as described previously (32, 33). In some
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experiments, normal Krebs was replaced with chloride-free Krebs solution of the following composition (mM): sodium isethionate 115.0, magnesium gluconate 2.4, calcium gluconate 1.3, potassium gluconate 8.0, KH2PO4 2.0, and NaHCO3 25.0. Short circuit current (Isc, A/cm2) was measured as an indicator of net active ion transport across the tissue with a digital data acquisition system (MP100, Biopac Systems, Santa Barbara, Calif.) and analysis software (AcqKnowledge V3.03, Biopac Systems). Tissue conductance (G, mS/cm2), an indicator of epithelial resistance, was calculated from the current and potential difference values using Ohm’s law. After a 20 min equilibration period, peptides or drugs were added to the serosal bathing fluid. When different drugs or peptides were added consecutively, 10 –15 min was left between applications. In all cases, tissues were paired so that baseline conductances were within 20% of each other. One member of the pair was exposed to the drug or peptide and the other received an equivalent volume of the vehicle. Human CGRP␣, [Cys (Acm)2,7] hCGRP␣, [Cys (Et)2,7] hCGRP␣, and hCGRP8 –37 were synthesized and purified in our laboratories (S. St. Pierre) using standard solid-phase methods. Rat adrenomedullin and rat amylin were obtained from Bachem (Torrance, Calif.). Carbachol, doxantrazole [3-(1H-tetrazol-5-yl)-9H-thioxanthen-9-one 10,10-dioxide monohydrate], indomethacin, and tetrodotoxin were purchased from Sigma Chemical Co. (St. Louis, Mo.). All the peptides were dissolved in 0.25% acetic acid. Doxantrazole was dissolved in DMSO (0.1% final bath concentration) and indomethacin was dissolved in 1.25% sodium bicarbonate. Control tissues received appropriate vehicles in all cases. Statistics Data are expressed as mean ⫾ se. Multiple groups were compared using a one-way analysis of variance with post hoc analysis (Newman-Keuls test). Unpaired Student’s t tests were used to compare two different groups. Probability values of ⬍ 0.05 were considered statistically significant. EC50 was calculated from nonlinear regression analysis of the dose response data by appropriate software (GraphPad Prism, Version 2 for Windows 95/98, GraphPad Software Inc., San Diego, Calif).
RESULTS Assessment of colitis Animals treated with TNBS developed colitis as described previously (36, 37). Typical features of the inflamed colon included hemorrhage, edema, ulceration, and adhesions. Comprehensive macroscopic assessment of damage revealed significantly increased damage in all inflamed tissues and none in the saline-treated controls (0, controls [n⫽25]; 6⫾1, TNBS-treated [n⫽31]). Myeloperoxidase levels in TNBS-treated rats were significantly higher (76⫾9 mU/mg/min, n⫽31) than saline-treated controls (6⫾1 mU/mg/min, n⫽25, P⬍0.001). Examination of subgroups of these data based on the concentration of agonist the tissues received revealed no differences, suggesting that comparable tissues were used in every group studied. CGRP AND COLONIC SECRETION
Figure 1. Concentration-response curves to CGRP and [Cys(Et)2,7] hCGRP␣ in normal tissue. Each concentration of CGRP or [Cys(Et)2,7] hCGRP␣ was added as a single dose to separate tissues. Data shown represent mean ⫾ se, n ⫽ 5–9 preparations per point.
Electrolyte transport in response to CGRP and related peptides When added to the serosal side of the colonic tissues, CGRP caused a rapid increase in ISC that peaked within 5 min and then slowly returned to baseline levels within 20 min. CGRP added to the mucosal side of the preparation caused no change in ISC. CGRP induced a concentration-dependent increase in ISC in normal colonic tissues with an EC50 value of 21 ⫾ 2.5 nM (Fig. 1). In contrast to CGRP, the response to the linear CGRP2 receptor agonist [Cys (Et)2,7] hCGRP␣ was considerably smaller in magnitude and relatively linear over the range studied (Fig. 1). We also examined a single concentration (300 nM) of another putative CGRP2 receptor agonist, [Cys (Acm)2,7] hCGRP␣. At this concentration, [Cys (Acm)2,7] hCGRP␣ had a similar, but slightly lesser effect (4⫾2 A/cm2, n⫽4) to that produced by an equimolar concentration of [Cys (Et)2,7] hCGRP␣ (8⫾3 A/cm2, n⫽5). Animals treated with TNBS developed colitis. Baseline ISC was similar in normal and inflamed distal colon (⫺96⫾15 A/cm2, control; ⫺89⫾19 A/cm2, inflamed). CGRP caused a concentration-dependent increase in ISC in inflamed tissues (Fig. 2). The EC50 value of inflamed tissues was 15 ⫾ 4 nM. There were no qualitative differences in the response to CGRP between normal and inflamed animals, and the EC50 values were not significantly different. As in normal tissues, the linear CGRP2 receptor agonist [Cys (Et)2,7] hCGRP␣ caused a smaller response than CGRP (Fig. 2). In inflamed tissues, there appeared to be a shift to the right in the concentrationresponse curve to [Cys (Et)2,7] hCGRP␣, since at 10 nM there was no response, whereas controls had a near maximal response. In contrast to CGRP, addition of the cholinergic 1441
TABLE 1. The effect of indomethacin (1 M), doxantrazole (1 M), or atropine (1 M) pretreatment of colonic preparations on CGRP- or [Cys(Et)2,7] hCGRP␣-induced increases in short circuit currenta CGRP (30 nM) (A/cm2, mean⫾se)
Vehicle Indomethacin Vehicle Doxantrazole Vehicle Atropine Figure 2. Concentration-response curves to CGRP and [Cys(Et)2,7] hCGRP␣ in inflamed tissues. Each concentration of CGRP or [Cys(Et)2,7] hCGRP␣ was added as a single dose to separate tissues. Data shown represent mean ⫾ se, n ⫽ 5–9 preparations per point.
agonist carbachol (5 M) to the serosal side of the preparation caused a smaller increase in ISC in inflamed tissues compared to noninflamed tissues (154⫾14 A/cm2, n⫽29 control; 106⫾17 A/cm2, n⫽33 inflamed, P⫽0.03). Replacement of the normal buffer with chloridefree buffer almost abolished the responses to both CGRP (12⫾1 A/cm2, Krebs’ buffer; 2⫾1 A/cm2 Cl⫺ free-buffer, n⫽4 per group; P⬍0.01), and [Cys (Et)2,7] hCGRP␣ (9⫾2 A/cm2, Krebs’ buffer; 1⫾1 A/cm2 Cl⫺ free-buffer, n⫽4 per group; P⬍0.01). To assess whether other members of the CGRP family caused electrolyte secretion in the rat colon, the effects of amylin (300 nM) and adrenomedullin (300 nM) were examined in normal and inflamed rat colon. Amylin (6⫾1 A/cm2, n⫽8 control; 8⫾2 A/cm2, n⫽4 inflamed) and adrenomedullin (5⫾2 A/cm2, n⫽6 control; 6⫾1 A/cm2, n⫽4 inflamed) only caused a modest increase in ISC at high concentrations in the normal and inflamed rat colon.
13 19 8 9 7 5
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
5, 4, 4, 4, 1, 2,
n n n n n n
⫽ ⫽ ⫽ ⫽ ⫽ ⫽
6 6 6 6 6 6
[Cys(Et)2,7] hCGRP␣ (30 nM) (A/cm2, mean⫾se)
4 6 3 7 4 5
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
1, 1, 1, 2, 1, 2,
n n n n n n
⫽ ⫽ ⫽ ⫽ ⫽ ⫽
4 4 5 5 4 4
a Data are expressed as mean ⫾ sem for the number (n) of experiments indicated.
doxantrazole (1 M), and the cyclooxygenase inhibitor indomethacin (1 M) were without effect on the response of normal tissues to CGRP or [Cys (Et)2,7] hCGRP␣ (Table 1). However, the neural blocker tetrodotoxin (TTX, 1 M) significantly reduced the response to the CGRP2 agonist [Cys (Et)2,7] hCGRP␣ (30 nM), whereas it had no effect on the secretory response to CGRP (30 nM, Fig. 3). Since the action of CGRP could not be blocked by any of the approaches tested above, we tested the ability of CGRP to desensitize its own receptor as a way to further examine the site of action of CGRP agonists. Repeated application of CGRP at a high concentration (300 nM ⫻ 2) caused a significant attenuation in the response to this agonist (1st response 26⫾5 A/cm2, 2nd response 9⫾3 A/cm2, n⫽6/group, P⬍0.05). Subsequent administration of the linear CGRP2 agonist [Cys (Et)2,7] hCGRP␣ (30 nM) to desensitized tissues resulted in a response that was similar to that of control tissues not exposed to CGRP (7⫾2 A/cm2 vs. 10⫾2 A/cm2). Unlike
The effects of antagonists on CGRP agonistinduced electrolyte transport To examine the receptors involved, the effect of a CGRP1 receptor antagonist, CGRP8 –37, was assessed on CGRP-induced chloride secretion in both normal and inflamed tissues. CGRP8 –37 pretreatment (1 M, 10 –15 min) did not change the baseline ISC, nor did it significantly alter the ISC response to CGRP (10 nM) in either group (normal: 10.8⫾2.0 A/cm2 vehicle; 11.5⫾1.1 A/cm2 CGRP8 –37; n⫽5/group; inflamed: 8.7⫾2.1 A/cm2 vehicle; 10.9⫾2.2 A/ cm2 CGRP8 –37; n⫽5/group). This dose of CGRP8 –37 has previously been shown to inhibit Cl⫺ secretion by human adenocarcinoma cell line HCA-7 in the Ussing chamber (16). The cholinergic muscarinic antagonist atropine (1 M), the mast cell stabilizer 1442
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Figure 3. The effect of tetrodotoxin (TTX) or vehicle on short circuit current response of colonic preparations in response to CGRP (30 nM) or [Cys(Et)2,7] hCGRP␣, (30 nM). Tissues were pretreated with TTX (1 M) or vehicle 10 min prior to the peptide application. Each column represents mean ⫾ se, n ⫽ 5 per group and * P⬍0.001 compared to vehicle.
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CGRP, repeated administration of [Cys (Et)2,7] hCGRP␣ (300 nM) did not cause desensitization (1st response 8⫾3 A/cm2, 2nd response 10⫾2 A/ cm2); application of CGRP (30 nM) after repeated administration of [Cys (Et)2,7] hCGRP␣ was indistinguishable from vehicle-treated tissues not exposed to the [Cys (Et)2,7] hCGRP␣ (12⫾4 A/cm2 vs. 13⫾3 A/cm2). Conductance The effect of CGRP and [Cys (Et)2,7] hCGRP␣ on epithelial resistance was assessed in control and inflamed tissues. Conductance, an indicator of epithelial resistance and ionic permeability of the epithelial tight junction, was 15 ⫾ 3 mS/cm2 in control animals and 19 ⫾ 3 in TNBS-treated rats. In control tissues, addition of CGRP or [Cys (Et)2,7] hCGRP␣ (10 –300 nM) had little effect on conductance (the maximum response was observed at 300 nM, ⌬G, 0.4⫾0.05 mS/cm2), suggesting that it was not modifying epithelial permeability. In inflamed tissues, however, in 26/29 rats tested there was an increased conductance after CGRP, but not to [Cys (Et)2,7] hCGRP␣. This was not concentration dependent (10 nM, 3.1⫾1.0 mS/cm2; 30 nM, 2.6⫾1.5 mS/cm2; 100 nM, 2.2⫾0.7 mS/cm2; 300 nM, 2.0⫾0.6 mS/cm2) but was seen at all doses of CGRP above 10 nM.
DISCUSSION In the present study we have made three new observations. First, based on the pharmacological approaches used in this study, there is a novel CGRP receptor capable of eliciting chloride secretion located on the rat colonic enterocyte. Second, the secretory response to CGRP, unlike that to other agonists, was preserved in the inflamed rat colon. Third, in inflamed colonic tissue, CGRP increased tissue conductance. CGRP-induced secretion appeared to be receptor mediated, since it rapidly desensitized to repeated application. The only available CGRP antagonist did not block the secretory response to CGRP. The receptor is likely to be located on the enterocyte since a muscarinic antagonist, a neural blocker, a mast cell stabilizer, and a prostaglandin synthesis inhibitor all failed to reduce the response to CGRP. With the use of two linear CGRP analogs we tested whether the putative CGRP2 receptor was likely responsible for the secretory response to CGRP. These analogs caused only a small secretory response that was not dose dependent. The effect of [Cys (Et)2,7] hCGRP␣ was reduced by ⬃80% after treatment with TTX, suggesting the CGRP2 receptor was located on enteric nerves of the submucosal plexus. CGRP AND COLONIC SECRETION
A lack of TTX sensitivity in the response to CGRP has been noted previously (16); nevertheless, we are confident of its efficacy since it blocked the response to [Cys (Et)2,7] hCGRP␣. Other CGRP family members, amylin and adrenomedullin, were also tested in this system and had little effect, suggesting that neither the previously characterized CGRP receptor, the amylin receptor, or the adrenomedullin receptor is responsible for the secretory response to CGRP. Taken together, these data strongly support the view that there is a novel CGRP receptor located on the rat colonic enterocyte. It has previously been reported that the effects of CGRP in the rat colon were resistant to the actions of the CGRP1 receptor antagonist hCGRP8 –37 and that the CGRP2 receptor was likely responsible for the secretory actions of CGRP (16). Our results suggest that another type of CGRP receptor exists on the colonic enterocyte and is responsible for the secretory effect of CGRP. Using colonic adenocarcinoma cell lines in addition to a preparation similar to our own, Cox and colleagues (16, 40) studied both ␣ and  forms of CGRP on chloride secretion. It is interesting that one colonic adenocarcinoma cell line (Col-29) had characteristics similar to rat colon, with no sensitivity to hCGRP8 –37, but another cell line (HCA-7) was sensitive to the CGRP1 antagonist. Based on these data, it was speculated that the CGRP2 receptor was responsible for mediating colonic secretion in normal tissues and certain adenocarcinoma cell lines (16). However, our data using two linear analogs that act as CGRP2 receptor agonists suggest that this is not the case and that a novel receptor mediates the response to CGRP. The existence of a CGRP receptor other than the type 1 receptor has been proposed in the gastric mucosa. Using isolated gastric mucosal cells, Tu and Kang (41) found that CGRP conferred protection against cytotoxic injury and this response was not blocked by hCGRP8 –37. They did not examine a CGRP2 agonist in that study. Thus, it is possible that the novel receptor we propose is also located on other epithelial sites such as the gastric mucosa. Our work extends our understanding of the mechanism of CGRP-induced chloride secretion in the rat colon by ruling out an indirect effect of CGRP on secretion through nerves, mast cells, and prostaglandins. In the guinea pig colon, the CGRP-induced chloride secretion requires intact myenteric and submucosal plexuses (29, 42). CGRP appears to act through the release of acetylcholine at the level of the myenteric plexus to cause secretion indirectly via submucosal secretomotor neurons (42). The response to CGRP in the rat colon is clearly through a different mechanism and is evident only in mucosa/ submucosal preparations (16). Recent studies have suggested that other members 1443
of the CGRP family of peptides may act through common receptors (10, 43– 46). Specifically, there appears to be some cross-reactivity between the CGRP and the adrenomedullin receptor, both of which may be the calcitonin-receptor-like receptor (CRLR) modified by the presence of RAMPs (27). The RAMP family members are transmembrane proteins whose role has been proposed to transport the CRLR to the plasma membrane or be involved in the control of the glycosylation of the CRLR (27, 47). Neither RAMP1 or CRLR is a CGRP receptor in its own right, since none of them induced significant responses to CGRP when transfected alone, but expression of both produced cells that responded to CGRP by increasing intracellular cAMP levels (27, 47). In this study, neither adrenomedullin nor amylin were effective agonists, ruling out the possibility that CGRP acts via these receptors. During colitis and in tissues recovering from inflammation, the colonic epithelium appears less responsive to many agonists, including those that act intracellularly (32–35). It has recently shown that the responsiveness to both Ca2⫹-dependent and cAMPdependent secretagogues is reduced in mouse colon 1 wk after the induction of colitis (33), and in rat colon 6 wk after the induction of colitis (32). Therefore, it was an unexpected observation to find that the effects of CGRP were essentially identical in inflamed and normal tissues, particularly in light of the fact that the response to carbachol was reduced in the same preparations. The mechanism underlying the preservation of the response to CGRP is not clear and needs further investigation. It does not appear to be through the expression of a CGRP2 receptor, since the effects of the CGRP2 agonists were very similar in control and inflamed tissues. Receptors for CGRP, calcitonin, amylin, and adrenomedullin are all Gs-coupled receptors activating adenylate cyclase (7, 10, 48 –50). It is possible that inflammation-induced hyporesponsiveness is dependent on the upstream activation sequence of the adenylate cyclase pathway and that this differs in CGRP-induced adenylate cyclase activity compared to other activators. The responsiveness to CGRP may also be the result of up-regulation of CGRP receptors in the inflamed colon and therefore may reflect an adaptive phenomenon that preserves the secretory capacity of the inflamed colon. Finally, we found that CGRP induced an increase in conductance in inflamed tissues. Paracellular pathways are regulated by the tight junctions between enterocytes (51). The concept that nerves may regulate epithelial barrier function is supported by reports describing increased blood-toward-lumen transport of horseradish peroxidase through the intestinal epithelial basolateral space into the tight junctions in rats treated with intravenous carbachol 1444
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(52) and by nerve-mediated increased epithelial permeability (53). There is also evidence that neurotransmitters may regulate tight junction structure and permeability. For example, duodenal epithelial permeability is increased by neurokinin A, an effect that is decreased by vasoactive intestinal polypeptide (54). We provide evidence here to suggest that CGRP is another neuropeptide that can alter epithelial permeability, but only in inflamed intestine. The mechanism underlying this observation is not clear. It is possible that the microenvironment to which the enterocyte is exposed in the inflamed gut alters intracellular signaling pathways regulating tight junction assembly, structure or selectivity (55). It has been shown that distal colitis in the rat induced an increase of tight junction permeability at remote sites such as duodenum and ileum that was accompanied by alterations in the tight junction structural protein, occludin (56). In inflammation, therefore, activation of the CGRP receptors may result in effects on tight junction permeability not observed in normal tissue. In conclusion, we demonstrate that CGRP causes chloride secretion in the rat colon by acting at a novel receptor located directly on the colonic enterocyte. This effect is well preserved in inflamed tissues even though the epithelium is hyporesponsive to other agonists. CGRP increased electrical conductance of the inflamed tissues. Taken together, these data suggest that CGRP may play an important role in colonic mucosal secretion and permeability and that the presence of an apparently novel CGRP receptor in the gastrointestinal tract warrants further studies. These studies were supported by an unrestricted grant-inaid from the Ferring Research Institute (Paris, France) and by grants from the Medical Research Council of Canada (MRC) to R.Q., S.S.P., and K.A.S. and from the Crohn’s and Colitis Foundation of Canada to W.K.M. T.E. was the recipient of a Pharmaceutical Manufacturers of Canada/MRC Studentship. W.K.M. is an Alberta Heritage Foundation for Medical Research (AHFMR) Scholar and K.A.S. is an AHFMR Senior Scholar.
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The FASEB Journal
Received for publication August 13, 1999. Revised for publication January 17, 2000.
ESFANDYARI ET AL.
