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Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Roles of Substance P Receptors in Human Colon Circular Muscle: Alterations in Diverticular Disease

Lu Liu, Fei Shang, Irit Markus and Elizabeth Burcher
Journal of Pharmacology and Experimental Therapeutics August 2002, 302 (2) 627-635; DOI: https://doi.org/10.1124/jpet.102.034702

Abstract

The characteristics of [125I]Bolton-Hunter[Sar9,Met(O2)11]substance P ([125I]BH-SarSP) binding were investigated in membranes of human ascending, transverse, distal, and sigmoid colon circular muscle. Binding of [125I]BH-SarSP was of high affinity (KD = 68 nM) and low capacity (Bmax = 0.31 fmol/mg of wet weight tissue), and showed no regional differences. [125I]BH-SarSP binding was inhibited by SP ≈ [Pro9]SP ≥ (2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine (CP99994) ≫ neurokinin (NK) A ≥ neuropeptide γ > [Lys5,MeLeu9,Nle10]-NKA(4–10) ≈ (S)-N-methyl-N[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl]benzamide (SR48968) ≫ senktide, suggesting binding to NK-1 sites. Most agonists seemed to bind to two sites. In autoradiographic studies, dense binding for [125I]BH-SarSP was associated with submucosal and longitudinal muscle blood vessels, and the submucosal margin of circular muscle (corresponding to interstitial cells of Cajal), with moderate binding over most of the circular muscle. In normal colon circular muscle strips, [Pro9]SP was almost ineffective, and SP caused contractions with pD2 values of 5.3 to 5.7. No regional differences were observed in potency or efficacy. Responses to SP were inhibited by the NK-2 receptor antagonist SR48968, but not by NK-1 antagonist CP99994, indicating the involvement of NK-2 rather than NK-1 receptors. Atropine significantly inhibited contractions induced by SP, indicating a minor cholinergic component. Contractile responses to SP were considerably reduced in preparations from patients with diverticular disease, and marginally reduced in ulcerative colitis compared with control. This study clearly demonstrates an NK-1 binding site on human colon circular muscle, but its role in this tissue remains unclear and may not involve contractile mechanisms. The attenuated contractility in specimens with diverticular disease may reflect disease-related alterations of the tachykinin receptor system.

The tachykinin neuropeptide family consists of a large number of mammalian and nonmammalian peptides that share a similar carboxyl-terminal sequence. Substance P (SP), neurokinin (NK) A and neurokinin B are the principal members of this family and exert a broad spectrum of actions in both the central nervous system and peripheral tissues via NK-1, NK-2, and NK-3 receptors, respectively (Harrison and Geppetti, 2001). SP, NKA, and an elongated form of NKA, neuropeptide γ (NPγ), are abundant in the gastrointestinal tract of various mammalian species, including human. SP and NKA occur primarily in intrinsic enteric neurons, where they are colocalized with Ach, although SP-like immunoreactivity also occurs in extrinsic sensory neurons, whose cell bodies are located in the dorsal root ganglia (Holzer and Holzer-Petsche, 1997a). Recently, neurokinin B was identified in a subset of SP-immunoreactive neurons in the rat ileum (Yunker et al., 1999). In the gut, tachykinins are involved in contraction of smooth muscle, neuroneuronal communication between enteric neurons, regulation of water/ion secretion, and blood flow as well as proinflammatory responses (Holzer and Holzer-Petsche, 1997a,b; Quartara and Maggi, 1998).

Much of our knowledge about tachykinins in the gastrointestinal system is derived from extensive investigation in the guinea pig, where NK-1 receptors are prominent in contractile mechanisms (Holzer and Holzer-Petsche, 1997a,b) and have recently been identified on interstitial cells of Cajal (ICC), which have a pacemaker function (Lavin et al., 1998; Southwell and Furness, 2001). In contrast, in human colon, NK-2 receptors dominate the contraction of smooth muscle by tachykinins (Giuliani et al., 1991; Kölbel et al., 1994; Croci et al., 1998; Cao et al., 2000). These receptors have been localized autoradiographically on the circular muscle and muscularis mucosae (Gates et al., 1989; Warner et al., 2000). The actions of NK-1 receptors in human colon, on the other hand, are more diverse because immunohistochemical and autoradiographic studies have shown that NK-1 receptors are expressed on a variety of cell types, including smooth muscle, neurons, immune cells, glands, and vascular endothelium (Mantyh et al., 1988; Goode et al., 2000a; Renzi et al., 2000), implying that NK-1 receptors may be involved in the control of smooth muscle excitation, immune responses, secretion, and blood flow.

There is growing evidence that imbalanced function of the tachykinin system may influence or accompany the pathophysiology of some intestinal disorders. For instance, some radioimmunoassay and autoradiographic studies demonstrated that SP and NK-1 receptors are up-regulated in the colon of patients with ulcerative colitis and Crohn's disease (Koch et al., 1987; Mantyh et al., 1988; Goldin et al., 1989, 1995), although other studies are contradictory (cited inLee et al., 2002). Studies using in situ hybridization and immunohistochemistry revealed that ulcerative colitis and Crohn's disease are associated with up-regulation of NK-1 and NK-2 receptors (Goode et al., 2000a; Renzi et al., 2000).

We have previously characterized NK-2 receptors in the circular muscle of human colon using the technique of radioligand membrane binding (Warner et al., 1999). To date, however, no study has attempted to illustrate the nature of NK-1 receptors in the human colon. This is rather surprising because NK-1 receptors obviously play an important role in both physiological and pathophysiological states. In this study, we have reported the localization and characterization of NK-1 receptor in normal human colon from different regions, using the selective NK-1 receptor radioligand [125I]Bolton-Hunter-[Sar9,Met(O2)11]SP ([125I]BH-SarSP) (Lew et al., 1990).

Recently, in vitro alterations in NK-2 receptor-mediated circular muscle contraction in ulcerative colitis, Crohn's disease, and idiopathic chronic constipation have been reported (Shang et al., 2000;Menzies et al., 2001; Mitolo-Chieppa et al., 2001). The second aim of this study, therefore, was to investigate the contractile responses of human colon circular muscle strips to SP, carried out in normal colon compared with colon from patients with ulcerative colitis and diverticular disease.

Experimental Procedures

Specimen Collection.

Specimens of ascending, transverse, descending, and sigmoid colon were collected from male and female patients, aged 28 to 77, undergoing colectomy at the St. George Hospital (Sydney, Australia). Forty-five specimens were obtained from patients with carcinoma, who had not received radiotherapy or chemotherapy treatment before surgery. Whole ring segments (4 cm in length) of macroscopically normal regions, taken 10 to 20 cm away from the tumor, were immediately placed in cold, well carbogenated (95% O2, 5% CO2) Krebs-Henseleit solution (composition 118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, and 11.7 mM d-glucose). In addition, specimens of sigmoid colon from patients undergoing operation for ulcerative colitis (n = 12) and acute diverticular disease (n = 11) were taken, from areas away from the most grossly inflamed lesions. Specimens were transported to the laboratory in ice-cold Krebs' solution, where they were dissected on the day of arrival or the next day. The serosa, mucosa, and submucosal layers were first removed, and the circular muscle was then separated from the taenia coli. The band of circular muscle (containing the myenteric plexus and a thin layer of longitudinal muscle) was used for functional studies or sectioned into 0.5-g portions, frozen in liquid nitrogen, and stored at −70°C for later homogenate binding. For autoradiographic studies, segments of colon with all layers intact were used. The procedure and experimental protocols were reviewed and approved by the University of New South Wales Human Ethics Committee (ethics 97139). Any specimens from carcinoma patients (“normal colon”) appearing inflamed or showing abnormal histological features were discarded.

Homogenate Binding Studies.

The radioiodination of [Sar9, Met(O2)11]SP followed the method described previously (Lew et al., 1990). [125I]Bolton-Hunter ([125I]BH) reagent (1 mCi, specific activity 2200 Ci/mmol) was reacted with [Sar9,Met(O2)11]SP (50 μg, dissolved in 50 μl of borate buffer, pH 8.5) at 4°C for 60 min. The product, [125I]BH-SarSP, was then purified with reverse phase high-performance liquid chromatography using a linear gradient of 18 to 48% acetonitrile (containing 0.01% trifluoroacetic acid) at a flow rate of 1.5 ml/min over 80 min.

The membrane preparation of human colon circular muscle was based on the method reported elsewhere (Lew et al., 1990). The final pellet was resuspended in incubation buffer consisting of 50 mM Tris-HCl (pH 7.4, 25°C), 3 mM MnCl2, and 0.02% bovine albumin (BSA). Saturation binding experiments were carried out by incubating membranes [2% membranes (w/v)] with 10 to 1000 pM [125I]BH-SarSP at 25°C for 60 min. Nonspecific binding was defined using [Sar9,Met(O2)11]SP (1 μM). For competition studies, the membranes were incubated with ∼100 pM [125I]BH-SarSP and different concentrations of competing ligands. The reactions were terminated by rapid filtration with a cell harvester (Brandel Inc., Gaithersburg, MD) through Whatman GF/B glass fiber filters (Whatman, Maidstone, UK) presoaked overnight at 4°C in 0.1% polyethylenimine), followed by three washes with ice-cold wash buffer containing 50 mM Tris-HCl (pH 7.4, 4°C), 3 mM MnCl2, and 0.02% BSA. Filter-bound radioligand was then quantified in a Wizard gamma counter (>78% efficiency; PerkinElmer Wallac, Gaithersburg, MD).

Autoradiographic Studies.

Full-thickness pieces of colon from eight patients were embedded in octane compound (Tissue-Tek; Sakura Finetek, Torrance, CA) and immediately frozen in liquid nitrogen and stored at −70°C. Transverse sections (10 μm) were cut on a cryostat and thaw-mounted onto gelatin-coated glass microscope slides and stored desiccated at −20°C until required. The procedure for autoradiographic experiments was similar to that described previously (Lew et al., 1990). After preincubation (3 × 5 min) in 50 mM Tris-HCl buffer (pH 7.4, 25°C) containing 0.02% BSA, sections were incubated with [125I]BH-SarSP (100 pM) in 5 ml of incubation buffer comprising 50 mM Tris-HCl buffer (pH 7.4, 25°C), 3 mM MnCl2, and 0.02% BSA for 60 min at 25°C. Nonspecific binding was determined in the presence of 1 μM [Sar9,Met(O2)11]SP. The reaction was terminated by washing sections (3 × 5 min) with ice-cold 50 mM Tris-HCl buffer (pH 7.4, 4°C) containing 3 mM MnCl2 and 0.02% BSA, followed by two brief rinses in ice-cold distilled water and rapid drying. Labeled sections were then fixed in paraformaldehyde vapor at 70°C for 30 min, dipped in molten photographic emulsion LM-1 (Amersham Biosciences, Sydney, Australia), and exposed in the dark for 10 days at 4°C before developing and staining with pyronin Y. Adjacent unlabeled sections were fixed with 4% formaldehyde and stained with Masson's trichrome for histological examination.

Functional Studies.

Circular muscle strips (3 × 10 mm) were cut along the circular axis, mounted in 2-ml organ baths containing Krebs-Henseleit solution at 37°C, and aerated with carbogen. The preparations was set at an initial tension of 1 g and allowed to equilibrate for 60 min. Muscle activity was recorded isometrically using FTO3C force transducers (Grass Instruments, Quincy, MA) and recorded using Polygraph (University of New South Wales). After equilibration, 10 mM Ach was added to each organ bath to obtain the maximal response of the muscle strip, followed by thorough washing and a further 60-min equilibration. Discrete concentration-response curves for SP and NKA were then constructed using a 30- to 60-min concentration cycle, with peptide contact time 2 to 3 min. Tachyphylaxis was not observed under these conditions.

For experiments investigating antagonists, the concentration-response curves to SP and NKA were obtained in the presence of the selective NK-1 antagonist CP99994 (incubation time 30 min) or NK-2 antagonist SR48968 (added 2 h before the addition of agonists). The effects of atropine and indomethacin on SP-induced contractions were also examined. Both atropine (1 μM) and indomethacin (1 μM) were added 30 min before the initiation of the SP concentration cycle.

Data Analysis.

Results are presented as mean ± S.E.M. For binding studies, data were fitted to a one- or two-site model using the nonlinear regression analysis program of GraphPad Prism, version 3 (GraphPad Software, San Diego, CA). The affinities of competitors for [125I]BH-SarSP binding sites were expressed as IC50 values. In functional studies, contractile responses of tachykinins were measured in grams tension. Unless otherwise indicated, these data were then expressed as a percentage of the 10 mM Ach response. The concentration-effect curves were fitted using the nonlinear regression analysis program of GraphPad Prism. Agonist potencies were expressed as pD2 (negative log of the EC50) except in preparations from disease patients, where some concentration-response curves had clearly not reached their maxima, and here the potencies were expressed as pEC30Ach. For the insurmountable antagonist SR48968, the apparent affinity (pKB) in inhibiting NKA-induced contraction was determined according to Kenakin (1993): a double-reciprocal plot of equieffective concentrations of agonist (A) in the absence (1/A) and presence (1/A′) of the antagonist (B) was constructed and pKB was derived from the equation pKB = log{(slope − 1)/[B]}.

Concentration-response curves were compared using two-way ANOVA. Comparisons of Bmax,KD, pD2, pEC30Ach, andEmax values were carried out using one-way ANOVA followed by the Bonferroni test.

Materials.

SP, NKA, NPγ, [Pro9]SP, [Sar9,Met(O2)11]SP, [Lys5, MeLeu9,Nle10]NKA(4–10), and senktide were purchased from Auspep (Melbourne, Australia). CP99994 was obtained from Dr M. Snider (Pfizer, Groton, CT) and SR48968 from Dr. X. Emonds-Alt (Sanofi-Synthélabo Recherche, Montpellier, France). The peptidase inhibitors bestatin, captopril, chymostatin, pepstatin, and phosphoramidon were purchased from Sigma (Sydney, Australia). Bacitracin (zinc salt) was from ICN (Sydney, Australia) and diprotin A from Bachem (Bubendorf, Switzerland). The [125I]BH-reagent (1 mCi/mmol) was the product of PerkinElmer Life Sciences and purchased from Geneworks (Adelaide, Australia). Stock solutions of peptides were made in 0.01 M acetic acid containing 1% β-mercaptoethanol and stored in aliquots at −20°C.

Results

Radioligand Binding

Optimization of Binding Conditions for [125I]BH-SarSP Binding.

At an incubation temperature of 25°C, specific binding of [125I]BH-SarSP to human colon circular muscle membranes reached equilibrium by 30 min and was stable up to 120 min. Specific binding was not significantly enhanced by the presence of peptidase inhibitors bestatin (10 μM), phosphoramidon (10 μM), captopril (1 μM), chymostatin (4 μg/ml), diprotin A (10 μM), and bacitracin (40 μg/ml). Binding assays were subsequently carried out for 60 min of incubation, without any peptidase inhibitors. Under these conditions, specific binding of [125I]BH-SarSP was 50 to 75%.

Saturation Studies.

Saturation studies were carried out in circular muscle homogenates of ascending, descending, and transverse colon, but mainly focused on the sigmoid colon. In all regions, specific binding of [125I]BH-SarSP was saturable and to a homogenous population of binding sites (Fig. 1). Figure 2 shows the Bmax andKD values for individual specimens from different regions, illustrating an absence of regional differences. Overall, [125I]BH-SarSP binding was of high affinity (KD = 68 ± 8.9 pM; n = 12) and low capacity (Bmax, = 0.31 ± 0.07 fmol/mg of wet weight tissue; n = 12).

Figure 1
Figure 1

Representative “hot” saturation data obtained using increasing concentrations of [125I]BH-SarSP incubated with human sigmoid colonic circular muscle membranes. A, saturation data illustrating total (■), specific (▪), and nonspecific (▵) binding. B, Scatchard plot showing a single binding site, of KD = 0.06 nM andBmax = 0.304 fmol/mg of wet weight tissue. Eight other experiments gave similar data.

Figure 2
Figure 2

KD values (A) andBmax values (B) from different regions of the human colon, showing a lack of regional differences in binding affinity and capacity.

Competition Studies.

Naturally occurring tachykinins, selective agonists, and antagonists were used in competition studies to characterize the [125I]BH-SarSP binding site (Table 1). Data analysis showed that for the majority of agonists, the slope factors were shallow and the curves were best fitted to two sites, of high and low affinity (Table 1). On the other hand, the slopes for all antagonists and for the selective NK-2 receptor agonist [Lys5,MeLeu9,Nle10]-NKA(4–10) were close to unity, indicating binding to a single site. The rank order of affinity for ligands to inhibit [125I]BH-SarSP binding was SP ≈ [Pro9]SP ≥ CP99994 ≫ NKA ≥ NPγ > [Lys5,MeLeu9,Nle10]-NKA(4–10) ≈ SR48968 ≫ senktide (selective NK-3 receptor agonist).

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Table 1

Potency of tachykinins, analogs, and antagonists as competitors against [125I]BH-SarSP in human colon circular muscle membranes

Autoradiographic Studies.

In transverse sections of normal human colon, moderate-to-dense specific binding of [125I]BH-SarSP was seen over the circular muscle and blood vessels (Figs. 3 and 4). In the circular muscle, intensity of binding was greatest near the longitudinal muscle, declining toward the submucosa, although a broken border of denser binding often occurred at the submucosal edge (Fig.3B). Negligible specific binding was visible on the myenteric ganglia (Fig. 4B) or muscularis of the longitudinal muscle (Figs. 3B and 4B).

Figure 3
Figure 3

Low-power photomicrographs of adjacent transverse sections of two different specimens of sigmoid colon, illustrating histological staining (A and D), total binding of [125I]BH-SarSP (B and E), and nonspecific binding (C and F). A to C, longitudinal muscle (lm) containing blood vessels (bv), circular muscle (cm), myenteric plexus (mp), and part of the submucosa (s). D to F, submucosa (s) with blood vessels (arrowed), and mucosa with muscularis mucosae (mm) glands (g) and lamina propria (lp). Specific binding occurred on blood vessels of the longitudinal muscle and submucosa, and on the circular muscle. Denser binding was seen on the submucosal edge of the circular muscle. Weak specific binding occurred in the lamina propria. A number of macrophages exhibiting nonspecific binding are seen in the mucosa and submucosa of E and F, and several are circled (refer to ∗, left side of D and F).

Figure 4
Figure 4

High-power photomicrographs of adjacent transverse sections of sigmoid colon, illustrating histological staining (A and D), total binding of [125I]BH-SarSP (B and E), and nonspecific binding (C and F). A to C, longitudinal muscle (lm), circular muscle (cm), and ganglia of the myenteric plexus (mp). Specific binding occurred on the circular muscle but not the longitudinal muscle or myenteric ganglia. D to F, part of the submucosa (s). Structures showing specific binding are on circular muscle, two arterioles (a) with specific binding on the endothelium, small blood vessels (bv), and a number of microvessels (∗).

Intense binding occurred to small vessels in the longitudinal muscle (Fig. 3B). No obvious blood vessels were seen in the circular muscle. Binding sites of variable intensity occurred on small and large submucosal blood vessels (Figs. 3E and 4E). Specific binding of moderate-to-intense degree was seen on the endothelium of submucosal arterioles (Fig. 4E), with some specific binding also observed on arterial smooth muscle. Submucosal veins and venules showed weaker but still obvious specific binding. Submucosal microvessels also showed specific binding (Fig. 4E).

In the mucosa, there was no specific binding on the muscularis mucosae (Fig. 3) or lymph nodes (data not shown). In the mucosa and parts of the submucosa, a number of intense “spots” were seen in both total and nonspecific sections and these were identified as macrophages (Fig.3, E and F). There was some weak specific binding on the lamina propria, although nonspecific binding was also apparent in this region.

In all patients, small areas of dense binding were seen in the submucosa, very close to the circular muscle. It was not possible to conclude with certainty the nature of the cell types expressing these binding sites. Submucosal ganglia and small blood vessels were found in these areas, in adjacent histological sections.

Functional Studies

Normal Colon.

The contractile effects of SP and NKA were examined in circular muscle strips of sigmoid, ascending, and descending colon. SP and NKA contracted the strips in a concentration-dependent manner, although NKA was approximately 2 orders of magnitude more potent than SP (Fig. 5; Table 2). There was no significant difference in maximal contractile responses (equivalent to about 60% of the 10 mM Ach) or potency (pD2) values between different regions (Table 2). In subsequent studies, data from descending and sigmoid colon strips were combined and are described as “sigmoid” below.

Figure 5
Figure 5

Concentration-response curves to SP in circular muscle strips from normal sigmoid colon. Data represent the mean ± S.E.M. A, responses to SP were significantly reduced by 100 nM SR48968 (P < 0.0001, two-way ANOVA) but not by 100 nM CP99994 (n = 7–26). B, responses were significantly reduced by 1 μM atropine (P = 0.012, two-way ANOVA) (n = 11–25). C, responses were unaffected by 1 μM indomethacin (n = 6–26).

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Table 2

Potency of SP and NKA in contracting circular muscle from different regions of human colon

In the sigmoid circular muscle, the contractions induced by SP were almost abolished by pretreating tissue with the selective NK-2 receptor antagonist SR48968 (100 nM), but were not affected by the selective NK-1 receptor antagonist CP99994 (100 nM) (Fig. 5A). In the presence of SR48968, the concentration-response curve to SP was shifted to the right with a very great reduction of the maximal response. The response to NKA was also inhibited by SR48968 (10 and 100 nM) with an apparent pKB value for SR48968 of 9.1.

Responses to the selective agonists [Pro9]SP and [Lys5,MeLeu9,Nle10]-NKA(4–10) were also investigated in sigmoid colon strips. [Lys5,MeLeu9,Nle10]-NKA(4–10) was potent with a pD2 value of 7.46 ± 0.13 and Emax value of 64.3 ± 3.6% (n = 26), no different from NKA, whereas [Pro9]SP was extremely weak, with maximum contraction (at 10 μM) of 3.0% (n = 9) of the Ach maximum.

In sigmoid colon, atropine (1 μM) caused a small but significant (P = 0.0075, two-way ANOVA) attenuation of the response to SP (Fig. 5B). The prostaglandin synthase inhibitor indomethacin (1 μM) had no effect on responses to SP (Fig. 5C).

In strips from ascending, distal and sigmoid colon, no differences in response to Ach were seen. Indomethacin (1 μM) did not alter responses to Ach in sigmoid colon, with pD2values of 3.94 (control) and 4.05 (indomethacin).

Functional Studies in Colon with Gastrointestinal Diseases.

Contractile responses were also examined in specimens of sigmoid colon from patients with ulcerative colitis and diverticular disease. The mean weights of the circular muscle strips were similar: control, 74 mg; colitis, 75 mg; and diverticular, 77 mg. There was a trend for a potentiation of responses to Ach in strips from colitis patients (maximum increase in tension 5.6 ± 1.1 g; n= 8), whereas responses to Ach were unaltered in diverticular disease (3.9 ± 0.75 g; n = 11) compared with control (4.2 ± 0.56 g; n = 23).

When the data were expressed as increase in tension (Fig.6A), there was a significant reduction (two-way ANOVA, P = 0.049) in potency to SP in muscle strips from patients with ulcerative colitis, compared with those from control patients. However, when data were expressed as percentage of Ach maximum, responses to SP were slightly but not significantly reduced (Fig. 6B; Table 3). In colitis patients, there was a trend toward a diminution of responses in the presence of atropine, compared with untreated strips, but this was not significant (Fig. 6C).

Figure 6
Figure 6

Concentration-response curves to SP in sigmoid circular muscle strips from normal, ulcerative colitis (UC) and diverticular disease (DD) colonic specimens. Data represent the mean ± S.E.M. and represent absolute increase in tension (A and D) or percentage of maximum Ach response (B, C, E, and F). A, responses to SP were reduced (P = 0.049, two-way ANOVA) in colitis compared with control (n = 12–26). B, when data from A are expressed as percentage of maximum Ach, responses were not significantly changed. C, atropine (1 μM) had no significant effect in specimens with ulcerative colitis (n = 5–12). D and E, responses to SP were very markedly reduced in diverticular disease (n = 11–26), whether expressed as increase in gram tension (D) (P = 0.0028, two-way ANOVA) or as percentage of maximum Ach (E) (P = 0.0001, two-way ANOVA). F, indomethacin (1 μM) did not influence responses in specimens with diverticular disease (n = 9–11).

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Table 3

Potency and efficacy of SP in contracting circular muscle from sigmoid colon of normal and disease colon

Responses to SP in sigmoid colon circular muscle strips from patients with diverticular disease were significantly reduced compared with those from control patients, whether expressed as increase in tension (P = 0.0028, two-way ANOVA; Fig. 6D), or as percentage of Ach maximum (P = 0.0001, two-way ANOVA; Fig. 6E). The pEC30Ach was also reduced (Table 3). In disease patients, in the presence of indomethacin (Fig. 6F), responses to lower concentrations of SP were unaltered, although there was a nonsignificant potentiation of the contractile response to the highest concentration of SP (100 μM).

Discussion

Tachykinins, especially SP, play an important role in the various functions of the gastrointestinal tract. In humans, evidence suggests that the tachykinin NK-2 receptor is the predominant tachykinin receptor in the colon circular muscle (Giuliani et al., 1991;Kölbel et al., 1994; Croci et al., 1998). Our previous study investigating NK-2 receptors in normal sigmoid colon circular muscle showed that binding of 125I-NKA was of high capacity (Bmax of 2.1 fmol/mg of wet weight tissue) (Warner et al., 1999). In this study, we have focused on SP and the NK-1 receptor, using the selective radioligand [125I]BH-SarSP. Binding of [125I]BH-SarSP was of high affinity (KD = 68 pM) and low capacity (Bmax = 0.3 fmol/mg of wet weight tissue) compared with the NK-2 receptor. There were no regional differences observed in receptor affinity and number between normal ascending, descending, transverse, and sigmoid colon. NK-1 receptor radioligand binding data from homogenates of human tissue do not seem to have been reported previously.

Our autoradiographic studies showed the presence of [125I]BH-SarSP binding sites on circular muscle, submucosal blood vessels, with a high density of silver grains on longitudinal muscle blood vessels and a low density in the mucosa. This matches the immunohistochemical localization of SP around blood vessels, circular muscle, and in the mucosa, as well as in myenteric ganglia (Wattchow et al., 1988). A similar distribution of NK-1 binding sites (Mantyh et al., 1988, 1995) and mRNA (Goode et al., 2000a; Renzi et al., 2000) is seen in other studies in human colon, with expression up-regulated on blood vessels in specimens from ulcerative colitis and Crohn's disease patients (Mantyh et al., 1988, 1995; Goode et al., 2000a). The broken border of denser binding occurring near the submucosal edge of the circular muscle (Fig. 3B) may correspond to ICC, which are prominent in this location in human colon (Rumessen et al., 1993; Vanderwinden et al., 1996). ICC are also found in throughout the circular muscle layer and around the enteric ganglia in the distal human colon (Vanderwinden et al., 1996).

The competition binding profile of [125I]BH-SarSP using tachykinins, selective tachykinin agonists, and antagonists demonstrated binding to the NK-1 receptor. The affinity of CP99994 for [125I]BH-SarSP binding sites reported herein (pIC50 = 10.48) is comparable with the pKB value of 9.9 in the human pulmonary artery (Corboz et al., 1998). Although in saturation studies this radioligand bound to a single class of sites, most tachykinin agonists seemed to bind to more than one component. This ability to resolve the competition data into two sites was a notable feature of our study. It was also of interest that the affinity of these peptides was higher than reported in binding studies with cell lines using a similar radioligand (Fong et al., 1992). Such high-affinity, multiple-site binding was also reported in homogenates from guinea pig lung (Geraghty et al., 1992). As shown in our autoradiographic studies, there were at least two populations of [125I]BH-SarSP binding sites in the preparation used in our homogenate binding studies, one on the circular muscle and the other on blood vessels located within the external longitudinal muscle. Whether these two populations represent two distinct molecular forms is unclear from this study. Alternatively, the two binding sites may represent different activation states of the same receptor protein, or represent binding to different G proteins (Maggi and Schwartz, 1997). Contraction of human colon sigmoid circular muscle in response to NK-2 receptor agonists is mediated via Gq (Cao et al., 2000).

A striking result of this study is the absence of functional evidence for the participation of NK-1 receptors in circular muscle contraction. The highly selective agonist [Pro9]SP was virtually ineffective, and SP was 100-fold less potent than NKA in contracting isolated circular muscle strips. Furthermore, responses to SP were almost completely inhibited by the NK-2 but not by the NK-1 receptor antagonist. Thus, SP seems to contract the circular muscle via NK-2 receptors, but not via NK-1 receptors. This is at variance with the clear demonstration of NK-1 binding sites on circular muscle, shown in binding and autoradiographic studies. The question is, what is the physiological function of NK-1 receptors (strictly speaking, binding sites) on human colon circular muscle? Do these sites represent the full-length NK-1 receptor or are they a short, nonfunctional form? Both short as well as full-length versions of the human NK-1 receptor have been described, in a glioblastoma cell line (Fong et al., 1992) and in human colonic mucosa (Goode et al., 2000b). Studies using in situ hybridization and quantitative polymerase chain reaction indicate that the full-length form of the NK-1 receptor is present in the circular muscle, but whether it coexists with the short isoform is unknown (Goode et al., 2000a; Renzi et al., 2000). It should also be pointed out that not only smooth muscle cells occur in “circular muscle” but also neurons, ICC, and other cell types.

Tachykinins have both direct and indirect actions to contract smooth muscle. In several tissues, including rat duodenum, there is evidence for tachykinin-prostaglandin interactions (Hallgren et al., 1998), but this did not seem to be the case in human sigmoid colon circular muscle. We found that a minor component of the contractile response was apparently due to Ach release, as in guinea pig ileum (Holzer and Lembeck, 1980), because responses to SP (but not to NKA) were slightly but significantly inhibited by atropine. A similar result was reported by Kölbel et al. (1994). Thus, although a direct interaction of SP with NK-2 receptors of the smooth muscle predominates, there is also a presence of facilitatory NK-1 receptors on cholinergic neurons. Although we and others (Mantyh et al., 1995) did not find autoradiographic evidence for NK-1 receptors on myenteric ganglia of normal colon, other techniques have demonstrated NK-1 receptor mRNA on enteric neurons of normal human ileum and colon (Goode et al., 2000a;Renzi et al., 2000).

Therefore, one plausible explanation for our data with atropine is that, first, in the normal colon, a limited number of neuronal NK-1 receptors are expressed on ganglia, autoradiographically undetectable using a low concentration of radioligand. Second, there may be some different tachykinin receptor subtype on the ganglia. A third hypothesis is that SP coreleased from cholinergic motoneurons to the circular muscle may act locally to modulate Ach release. This might occur if NK-1 receptors were localized to terminals of cholinergic motor neurons innervating circular muscle. Alternatively, because our autoradiographic studies showed denser binding at the submucosal edge of circular muscle, corresponding to ICC, it is likely that NK-1 receptors are present on human ICC, as shown in the guinea pig intestine (Lavin et al., 1998; Southwell and Furness, 2001). ICC form a link between neurons and smooth muscle cells. We do not have any direct evidence for these hypotheses, although the binding sites seen throughout the circular muscle layer may be located on motoneurons (as well as or instead of muscle fibers and ICC).

Altered colonic motility is a feature of most if not all gastrointestinal disorders. A novel finding of our study was that contractile responses to SP were significantly decreased in both potency and efficacy in colon from diverticular disease patients. Because cholinergic responses were unchanged in diverticular disease, as reported previously (Snape et al., 1991), the altered contractility to SP is rather due to a disease-related alteration in SP receptor/signaling mechanisms than to a nonspecific damage to the tissue by inflammation. Although prostaglandins are involved in inflammatory cascades, there was no evidence for their participation in responses to SP in circular muscle specimens with diverticular disease.

The attenuated contractility to SP found in diverticular disease is unlikely to be related to the change of NK-2 receptor density and affinity because our binding studies with125I-NKA showed no change inKD orBmax values in colon circular muscle from patients with diverticular disease (Shang et al., 2000). The hypocontractility in diverticular disease was, however, not seen in response to NKA (data not shown). Thus, the reduced responsiveness to SP in this disease may well be due to deficiency of the NK-1 receptor system. To date, there is little information available on the tachykinin system in diverticular disease.

Attenuations in responses to SP were also seen in specimens from patients with ulcerative colitis, but of lesser magnitude than diverticular disease. A similar result was reported recently (Menzies et al., 2001). This finding is supported by results from our binding study, where a reduced affinity of 125I-NKA was observed in the colonic circular muscle from ulcerative colitis patients (Shang et al., 2000). No changes in ganglionic or circular muscle NK-1 receptors occurred in specimens with ulcerative colitis, although there was marked up-regulation of NK-1 receptors associated with other cell types (Mantyh et al., 1995; Goode et al., 2000a). Thus, the reduced responsiveness to SP is probably related to decreased affinity of the NK-2 receptor in ulcerative colitis. The nature of NK-1 and NK-2 receptor and signaling systems in human colon from patients with diverticular disease and ulcerative colitis is certainly worthy of further investigation.

Acknowledgments

We thank Dr. D. Z. Lubowski and Dr. D. W. King for kindly providing colon specimens and Ruth Hudson and Emma Schofield for assistance with specimen collection.

Footnotes

  • DOI: 10.1124/jpet.102.034702

  • Abbreviations:
    SP
    substance P
    NK
    neurokinin
    NPγ
    neuropeptide γ
    Ach
    acetylcholine
    ICC
    interstitial cells of Cajal
    [125I]BH-SarSP
    [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P
    BSA
    bovine serum albumin
    ANOVA
    analysis of variance
    CP99994
    (2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine
    SR48968
    (S)-N-methyl-N[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl]benzamide
    • Received February 24, 2002.
    • Accepted April 15, 2002.

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Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Roles of Substance P Receptors in Human Colon Circular Muscle: Alterations in Diverticular Disease

Lu Liu, Fei Shang, Irit Markus and Elizabeth Burcher
Journal of Pharmacology and Experimental Therapeutics August 1, 2002, 302 (2) 627-635; DOI: https://doi.org/10.1124/jpet.102.034702
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