Decrease in activity of smooth muscle L-type Ca2+ channels and its reversal by NF-{kappa}B inhibitors in Crohn's colitis model

Kazuya Kinoshita, Koichi Sato, Masatoshi Hori, Hiroshi Ozaki, and Hideaki Karaki

Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan

Submitted 22 January 2003 ; accepted in final form 14 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
We investigated the mechanisms of dysmotility of the colonic circular muscle of the Crohn's disease rat model. Contractions induced by KCl, carbachol, and Bay K 8644 were decreased in circular smooth muscles isolated from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat colon. However, the absolute force and Ca2+ sensitivity of contractile proteins were not affected as assessed in {alpha}-toxin permeabilized smooth muscle. The current density of the L-type Ca2+ channel in circular smooth muscle cells was significantly decreased in the TNBS-treated colonic cells. However, expressions of the L-type Ca2+ channel mRNA and protein did not differ between control and TNBS-treated preparations. Pretreatment with the NF-{kappa}B inhibitors pyrrolidinedithiocarbamate and sulfasalazine partially recovered the decreased contractility and current density of the L-type Ca2+ channel by TNBS treatment. These results suggest that the decrease in the contraction of circular smooth muscle isolated from TNBS-induced colitis rat colon, which may be related to gut dysmotility in Crohn's disease, is attributable to the decreased activity of the L-type Ca2+ channel. The dysfunction of the L-type Ca2+ channel may be mediated by NF-{kappa}B-dependent pathways.

2,4,6-trinitrobenzenesulfonic acid; rat colon; circular smooth muscle contractility; pyrrolidinedithiocarbamate; sulfasalazine; Crohn's disease


CROHN'S DISEASE AND ULCERATIVE colitis are chronic idiopathic diseases known as inflammatory bowel disease (IBD). It has been reported that intestinal motility is impaired in IBD patients (3, 31, 32, 42), and it is well recognized that alterations in motor function in the intestine are often associated with indications of inflammation (6, 18). The development of intestinal dysmotility may result in abnormal growth of intestinal flora. It is well known that the gut microflora plays a fundamental role in maintaining normal intestinal function. A disturbance of this flora has been clearly demonstrated to play a role in the pathogenesis of IBD: i.e., a failure of microflora may induce a translocation of bacteria and/or bacterial products through the impaired mucosal barrier (13). These events probably produce a vicious cycle, increasing the severity of intestinal injury.

One of the primary transcriptional factors in the regulation of inflammatory gene expression is the NF-{kappa}B family (4). Members of the NF-{kappa}B family serve as important regulators of the host immune and inflammatory response. Activation of NF-{kappa}B increases the expression of genes encoding proinflammatory mediators such as cytokines (TNF-{alpha} and IL-1{beta}, -6, and -12), cell-adhesion molecules (VCAM-1 and ICAM-1), inducible nitric oxide synthase, and cyclooxygenase-2 (28). In addition, NF-{kappa}B is activated in patients with IBD (26). Because pyrrolidinedithiocarbamate (PDTC) and sulfasalazine are potent inhibitors of NF-{kappa}B in vitro (33, 43) and in vivo (10, 14), these agents are considered to inhibit the development of acute and chronic inflammation through the inhibition of NF-{kappa}B (10, 14). Therefore, inhibitors of NF-{kappa}B such as PDTC or sulfasalazine can be used not only as therapeutic agents but also as a powerful tools to prove the causality between NF-{kappa}B-mediated inflammation and gastrointestinal dysmotility.

Investigation of the pathogenesis of Crohn's disease has made use of some established animal models, particularly the hapten 2,4,6-trinitrobenzenesulfonic acid (TNBS) (12, 24, 44). TNBS induces acute inflammation that progresses over several weeks to a chronic stage morphologically similar to Crohn's disease. Typically, mucosal injury and inflammatory cell infiltration are observed within 2 h after exposure to TNBS-ethanol (44). Features of chronic inflammation and lymphocytic cell infiltration occur at 48 h after exposure and are sustained over several weeks (24, 44).

A previous report (1) has indicated that the contractile measurement in isolated intestinal strips from patients shows a decreased contractile force generated by smooth muscle. In addition, it has been reported (11, 22, 23, 46) that the contractile properties have been altered in smooth muscle isolated from TNBS-induced colitis or ileitis. However, the molecular mechanism responsible for the decreased smooth muscle contractions in IBD has not been clarified. The aim of the present study was to determine the mechanism of gastrointestinal dysmotility in colonic smooth muscle isolated from rat colon following TNBS-induced colitis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Induction of inflammation. Experiments and animal care were carried out in compliance with guidelines outlined by the Guide to Animal Use and Care of the University of Tokyo. Male Sprague-Dawley rats (180-250 g; Charles River Japan) were anesthetized with 40 mg/kg ip pentobarbital sodium. The abdomen was opened by midline laparotomy, and the proximal colon was gently exteriorized. TNBS colitis was induced by injecting 100 mg/kg TNBS in a 50% ethanol solution into the colonic lumen 2-5 cm distal to the cecalcolonic junction. In some experiments, the NF-{kappa}B inhibitors, PDTC (100 mg/kg) and sulfasalazine (200 mg/kg), were intraperitoneally administered 2 h before and 24 h after the induction of inflammation.

Contraction studies. Rats were killed 2 or 7 days after the induction of inflammation by TNBS treatment. The abdomen was opened, and the proximal colon was removed. The colon was then cut open along the mesenteric attachment, and the mucosa and submucosa were removed. Circular strips were made and suspended along their circular axis in a tissue bath filled with normal physiological salt solution of (in mM) 136.9 NaCl, 5 KCl, 1.5 CaCl2, 1 MgCl2, 23.8 NaHCO3, 5.5 glucose, 0.01 EDTA (pH 7.4). The muscle strips were maintained at 37°C in an atmosphere of 95% O2-5% CO2. The response of the strips was measured isometrically under a resting tension of 10 mN and recorded on a multipen recorder (Yokogawa) and on a computer with the PowerLab system (ADI Instruments, Colorado Springs, CO). Calculations were performed with the PowerLab playback software.

Measurement of permeabilized colonic muscle contraction. Force production in the colonic permeabilized muscle was measured as described previously (2, 27). Isolated colonic muscle strips were cut into small pieces and permeabilized with staphylococcal {alpha}-toxin (80 µg/ml) for 30 min. Each strip was attached to a holder under a resting tension of 1 mN. Muscle tension was recorded isometrically with a force displacement transducer (Orientic, Japan).

Cell isolation. Circular smooth muscle layers from colon were isolated and cut into small pieces and placed in Ca2+-free solution of (in mM) 135 NaCl, 5 KCl, 1.2 MgCl2, 10 glucose, 10 HEPES (pH 7.4 with Tris) for 1-2 h. The Ca2+-free solution was then replaced with enzyme solution containing 2.0 mg/ml collagenase type 2 (cat. no. CLS2; Worthington), 10 U/ml papain (cat. no. P4762; Sigma, St. Louis, MO), 2.0 mg/ml trypsin inhibitor (cat. no. T9003; Sigma), and 1.0 mg/ml bovine serum albumin (cat. no. A7030; Sigma) in Ca2+-free solution. Tissues were incubated for 20-40 min at 37°C in enzyme solution. The solution was then decanted, and tissues were washed twice with Ca2+-free solution. Tissues were gently stirred by a pipette in Ca2+-free solution. Isolated cells were stored in Ca2+-free solution containing 0.1% bovine serum albumin on ice and used within 6 h for the patch-clamp experiments.

Patch-clamp recording of Ca2+-channel currents. Aliquots of vehicle- or TNBS-treated circular smooth muscle cells were transferred to a chamber mounted on the stage of an inverted microscope, and cells were allowed to adhere to the chamber floor before perfusion was initiated. Patch pipettes were made from borosilicate glass (Sutter Instruments, Novato, CA), fire-polished to a final tip resistance of 2-5 M{Omega}, and filled with solutions consisting of (in mM) 120 CsCl, 20 tetraethylammonium chloride, 5 EGTA, 5 magnesium adenosine triphosphate, 5 creatine phosphate disodium, 0.2 guanosine triphosphate, 5 HEPES (pH 7.3 with cesium hydroxide). Slight suction was used to establish a high-resistance seal (5-50 G{Omega}) between smooth muscle cell membranes and the pipette tip, and a whole cell configuration was established by further gentle suction. Ca2+-channel currents were recorded at room temperature in a bath solution containing (in mM) 135 NaCl, 5 KCl, 2.0 CaCl2, 1.2 MgCl2, 10 glucose, 10 HEPES (pH 7.4 with Tris). Current signals were amplified by an Axopach 200A patch-clamp amplifier (Axon Instruments, Foster City, CA) connected to a Digidata 1200B interface driven by pClamp 8.0 software (Axon Instruments) running a computer for data acquisition and digitization. Analog-to-digital conversion was accomplished at a sampling rate of 5 kHz after signals were passed through a filter with a cutoff frequency of 1 kHz.

After gaining the whole cell configuration, holding potential was maintained at -40 mV. Subsequently, progressive 10-mV depolarizing steps of 300-ms duration were applied from -30 to +60 mV to obtain Ca2+-channel currents. Current-voltage relations were calculated from average peak currents generated by 10-mV step potentials during 2-3 consecutive trials. In some experiments, the same protocol was repeated after the control bath solution was exchanged for the same solution containing the L-type Ca2+-channel agonist Bay K 8644 (0.3 µM) or the L-type Ca2+-channel blocker nifedipine (0.3 µM).

Quantitative real-time RT-PCR analysis. This procedure is based on the time point during cycling when amplification of the PCR product is first detected rather than by the amount of PCR product accumulated after a fixed number of cycles. The threshold cycle parameter (Ct) is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe passes a fixed threshold above the baseline. The Ca2+-channel {alpha}1c-subunit target gene copy number in unknown samples is quantified by measuring Ct and by using a standard curve to determine the starting copy number.

A standard curve was constructed for the Ca2+-channel {alpha}1c-subunit gene, as a target gene, and for the 18S ribosomal RNA (18SrRNA) gene, as an endogenous control. PCR products for the Ca2+-channel {alpha}1c-subunit and 18SrRNA were cloned into the pCR2.1 plasmid vector, and these plasmid vectors with serially diluted DNA were used as a standard template. Expression of the Ca2+-channel {alpha}1c-subunit mRNA of the unknown samples was divided by the endogenous reference (18SrRNA) amount to obtain a normalized target value. Final results are expressed as n-fold differences in Ca2+-channel {alpha}1c-subunit gene expression in TNBS-treated colon relative to that in vehicle-treated colon.

Total RNA was extracted from colonic circular smooth muscle strips by the acid-guanidinium isothiocyanate phenol chloroform method (9), and the RNA concentrations were adjusted to 1 µg/µl with RNase-free distilled water. Quantitative PCR was performed as follows. First-strand cDNA was synthesized from 1 µg total RNA by using a random 9-mers primer and AMV RT XL at 30°C for 10 min, 55°C for 30 min, 99°C for 5 min, and 4°C for 5 min. All PCR reactions were performed in an ABI Prism 7000 sequence detection system (PerkinElmer Applied Biosystems, Foster City, CA) by using the following primers: forward, 5'-CAGAGATCAATCGGAACAACAACT-3'; reverse, 5'-CAGTGGCGCACCTGAAGAG-3'; Taqman probe (5'-FAM/3'-TAMRA), 5'-CCAGACGTTCCTCAGGCTGTGC-3'. Four nanograms of cDNA, 1 µM of each primer, 425 µM FAM 6-carbosyl-fluorescein-labeled Ca2+-channel {alpha}1c-subunit Taqman probe, and VIC-labeled Taq- man ribosomsal RNA control regents were added to a 25 µl Taqman universal master mix (cat. no. 4304437; PerkinElmer Applied Biosystems). Both Taqman probes were conjugated with TAMRA (6-carboxyl-tetramethylrhodamine) as a quencher dye. Thermal cycling conditions included an initial denaturation at 94°C for 10 min, at 95°C for 20 s, and at 60°C for 1 min and lasted 40 cycles. Analysis of the 18SrRNA was also performed to normalize gene expression. The ABI sequence-detection software (version 2.0) and Microsoft Excel were used to calculate the quantities of the mRNAs.

Western immunoblotting. Membrane proteins were prepared from the circular smooth muscle from vehicle- or TNBS-treated rat colon according to the methods described by Liu et al. (20). Equal amounts (40 µg) of protein from circular muscles in rat colon were loaded onto 5.0% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membranes were incubated with rabbit anti-{alpha}1c-subunit polyclonal antibodies (1:200 dilution) and 0.5% BSA in PBS at 4°C overnight. They were then incubated at 37°C for 1 h in anti-rabbit IgG in PBS (1:1,000 dilution). The bound antibody was detected by the enhanced chemiluminescence plus kit (Amersham, Little Chalfont, UK). Intensities of the immunoreactive bands were quantified by densitometry (model LAS1000; Fuji Film).

Chemicals. Chemicals used were as follows: TNBS (Tokyo Kasei, Kogyo, Japan); carbachol, indomethacin, N{omega}-nitro-L-arginine methyl ester (L-NAME), nifedipine, PDTC, sulfasalazine, and staphylococcal {alpha}-toxin (Sigma); Bay K 8644 (RBI, Natick, MA); pentobarbital sodium (Nembutal; Abbott Laboratories, Chicago, IL); and calyculin-A (donated by Dr. N. Fusetani, University of Tokyo).

Statistical analysis. Results of the experiments are expressed as means ± SE. Statistical evaluation of the data was performed by a paired or unpaired Student's t-test for comparisons between two groups and by one-way ANOVA followed by Dunnett's test for comparisons among more than two groups. A value of P < 0.05 was taken as significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
KCl- and carbachol-induced contractions. In the preliminary experiments, we analyzed the length-tension relationship in the colonic muscles isolated from vehicle-treated and TNBS-treated rats and confirmed that the relationship was not changed after the TNBS treatment (optimal resting tension: 10 mN; data not shown).

We first examined the effects of 72.7 mM KCl and 10 µM carbachol on contractions in nontreated and vehicle (50% ethanol)-treated rat colonic smooth muscles killed at 2 and 7 days. The absolute force of contractions induced by 72.7 mM KCl in vehicle-treated colon did not differ from that of nontreated colonic smooth muscles (nontreatment, 8.90 ± 1.04 mN/mg wet wt; vehicle treatment at 2 days, 7.78 ± 1.37 mN/mg wet wt; vehicle treatment at 7 days, 6.01 ± 0.61 mN/mg wet wt; n = 4-6). In addition, 10 µM carbachol-induced contractions in the vehicle-treated rat colonic smooth muscle did not differ from the nontreated preparations (nontreatment, 9.18 ± 1.27 mN/mg wet wt; vehicle treatment at 2 days, 8.41 ± 1.12 mN/mg wet wt; vehicle treatment at 7 days, 6.00 ± 1.47 mN/mg wet wt; n = 4-6).

We then examined the alterations of KCl- and carbachol-induced contractions in vehicle or TNBS treatment. Figure 1, A and B shows the typical trace of a contraction induced by KCl (72.7 mM) and carbachol (1 and 10 µM) in vehicle- and TNBS-treated colon for 2 days. Magnitudes of the contractions induced by KCl and carbachol were significantly decreased in the TNBS-treated colon compared with vehicle-treated colon. Figure 1, C-F shows the analytical results. KCl-induced contractions were markedly inhibited in the colonic smooth muscle after the induction of inflammation induced by TNBS treatment for 2 days (maximum force at 72.7 mM: vehicle, 7.78 ± 1.37 mN/mg wet wt vs. TNBS, 2.90 ± 0.68 mN/mg wet wt; P < 0.01; n = 6-8) (Fig. 1C). Carbachol-induced contractions were also markedly decreased in the colonic smooth muscle after the induction of inflammation for 2 days (maximum force at 10 µM: vehicle, 8.41 ± 1.12 mN/mg wet wt vs. TNBS, 3.5 ± 1.00 mN/mg wet wt; P < 0.01; n = 6-8) (Fig. 1D). Neither the preincubation with indomethacin (10 µM) nor L-NAME (300 µM) for 20 min in the bath reversed the decreased contractions induced by the TNBS treatment (data not shown). We also examined the difference in contractions between oral-side and anal-side noninflamed colonic smooth muscles isolated ~15 mm apart from the inflammatory region and found no difference in the amplitude of contractions between vehicle and TNBS treatments. As demonstrated in Fig. 1E, KCl-induced contractions were markedly inhibited in the colonic smooth muscle after the induction of inflammation induced by TNBS treatment for 7 days (maximum force at 72.7 mM: vehicle, 6.01 ± 0.61 mN/mg wet wt vs. TNBS, 1.39 ± 0.26 mN/mg wet wt; P < 0.01; n = 5-6). Carbachol-induced contractions were also markedly decreased in the muscle after the induction of inflammation for 7 days (maximum force at 10 µM: vehicle, 6.00 ± 0.73 mN/mg wet wt vs. TNBS, 1.44 ± 0.46 mN/mg wet wt; P < 0.01; n = 5-6) (Fig. 1F). We also investigated the difference in contractions between oral-side and anal-side noninflamed colonic smooth muscles isolated ~15 mm apart from the inflammatory region and found no difference in the amplitude of contractions between vehicle and TNBS treatments for 7 days.



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Fig. 1. Typical trace of KCl (A; 72.7 mM)- and carbachol (B; 1 and 10 µM)-induced contractions in vehicle- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-treated rat colon. Upper traces, vehicle treatment for 2 days; lower traces, TNBS treatment for 2 days. Stimulant was applied at the arrowhead and washed away at WO. C-F: analytical results of the alterations in contractions induced by KCl (C and E) and carbachol (D and F) after treatment with TNBS for 2 (C and D) and 7 (E and F) days. KCl (5.4-72.7 mM) was added to the normal physiological salt solution with equimolar replacement of NaCl. Carbachol (1 nM-10 µM) was given cumulatively. Results are expressed as means ± SE (n = 5-8). Significantly different from results in vehicle-treated colon at *P < 0.05 and **P < 0.01.

 

Bay K 8644-induced contraction. Figure 2A shows a typical trace of a contraction induced by the L-type Ca2+-channel activator, Bay K 8644 (0.3 µM), in vehicle- and TNBS-treated colon for 2 days. In vehicle-treated colon, Bay K 8644 evoked large contractions. In contrast, Bay K 8644 induced only small contractions in TNBS-treated colon, and the magnitude of contraction induced by Bay K 8644 was significantly decreased in the TNBS-treated colon compared with vehicle-treated colon. Figure 2B shows the analytical results of Bay K 8644 (1 nM-0.3 µM)-induced contractions in a concentration-dependent manner in vehicle-treated rat colonic smooth muscle. In contrast, Bay K 8644 induced only a small contraction in TNBS-treated rat colon [maximum force at 0.3 µM: vehicle, 8.16 ± 1.18 mN/mg wet wt (n = 9) vs. TNBS, 0.57 ± 0.27 mN/mg wet wt (n = 10); P < 0.01].



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Fig. 2. A: typical trace of a Bay K 8644 (0.3 µM)-induced contraction in vehicle- and TNBS-treated colon. Upper trace, vehicle treatment for 2 days; lower trace, TNBS treatment for 2 days. B: analytical results of the alterations in contractions induced by Bay K 8644 in the vehicle- and TNBS-treated rat colon. Bay K 8644 (1 nM-0.3 µM) was added cumulatively. Results are expressed as means ± SE (n = 9-10). Significantly different from results in vehicle-treated colon at *P < 0.05 and **P < 0.01, respectively.

 

Muscle contractility in the {alpha}-toxin-permeabilized muscle. Figure 3 shows the effects of treatment with TNBS for 2 days on the pCa2+-force relationship in permeabilized colonic smooth muscle. To obtain the maximum contractile force, a myosin light chain was maximally phosphorylated by calyculin-A, a potent inhibitor of myosin phosphatase (16) at pCa2+ 4.5. The maximum force in vehicle-treated muscle (0.51 ± 0.17 mN/mg wet wt; n = 12) was not significantly different from that of the TNBS-treated muscle (0.88 ± 0.28 mN/mg wet wt; n = 12) (Fig. 3A). Ca2+ sensitivity of the contractile element also did not differ between vehicle- and TNBS-treated colonic smooth muscles (EC50 values for pCa2+: vehicle, 5.23 ± 0.16 vs. TNBS, 4.83 ± 0.17; n = 12)(Fig. 3B).



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Fig. 3. Effect of treatment with TNBS on muscle contractility in the permeabilized rat colon. A: pCa2+-absolute force. B: the pCa2+-relative force relationship. One hundred percent represents the maximum force induced by 30 µM Ca2+ with 1 µM calyculin-A. Results are expressed as means ± SE (n = 12).

 

Ca2+-channel currents in circular muscle cell. L-type Ca2+ channels play a pivotal role in smooth muscle contraction (5, 8). To evaluate the function of the Ca2+ channel, the density of the Ca2+-channel current was compared in circular smooth muscle cells isolated from the vehicle- and TNBS-treated colon. Figure 4A shows a typical inward current trace of the incremental 10-mV depolarizing steps from a holding potential of -40 mV to a test voltage as positive as +60 mV. These depolarizing steps were found to elicit inward currents in the smooth muscle cells. In both cell types, the inward currents were completely blocked by 0.3 µM nifedipine, an L-type Ca2+-channel blocker, indicating that these inward currents were L-type Ca2+-channel currents. As demonstrated in Fig. 4A, the magnitude of the L-type Ca2+-channel current was significantly decreased in the TNBS-treated circular muscle cells compared with vehicle-treated cells.



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Fig. 4. Whole cell Ca2+-channel currents in circular smooth muscle cells of rat colon. A: top trace shows Ca2+-channel currents in circular smooth muscle cells from vehicle-treated rat colon elicited by incremental 10-mV depolarizing steps from -30 to +60 mV (holding potential, -40 mV). The middle trace shows the Ca2+-channel currents in TNBS-treated cells. These currents were completely blocked by the L-type Ca2+-channel blocker nifedipine (0.3 µM). B: current-voltage relationships of peak Ca2+ current density in circular smooth muscle cells of vehicle- and TNBS-treated rat colon (n = 6-7). Significantly different from results in circular smooth muscle cell from vehicle-treated colon at *P < 0.05 and **P < 0.01. Significantly different from results in circular smooth muscle cells from sulfasalazine-pretreated colon at #P < 0.05 and ##P < 0.01. C: steady-state activation relationship (data are adopted from B).

 

Figure 4B shows the current-voltage relationships for vehicle- and TNBS-treated colonic smooth muscle cells. The average density of the Ca2+-channel current was decreased in circular smooth muscle cells of TNBS-treated tissue compared with vehicle-treated cells [peak Ca2+-channel current densities: -3.43 ± 0.35 pA/pF (10 mV) for vehicle-treated; -1.06 ± 0.19 pA/pF (0 mV) for TNBS-treated]. Results further indicate that TNBS treatment changed the reversal potential of the Ca2+-channel current by 11 mV (vehicle, +51 mV vs. TNBS, +40 mV).

To characterize the voltage dependence of Ca2+-channel current, steady-state activation relationships were examined. In Fig. 4C, peak conductance (normalized to 1.0) was plotted against test potential, which indicates that the peak activation voltage of the Ca2+ channel was not changed after the TNBS-treatment (control, 8.3 ± 1.7 vs. TNBS, 5.7 ± 2.0; n = 6-7). On the other hand, half-activation voltage of the Ca2+ channel was significantly shifted to negative potential (control, -9.9 ± 1.5 vs. TNBS, -15.5 ± 4.9 mV; P < 0.05; n = 6-7).

We further examined the effects of TNBS treatment on the L-type Ca2+-channel current activated by Bay K 8644. Figure 5A shows a typical trace of Ca2+-channel current elicited by 10-mV depolarizing steps from -40 mV to +60 mV before and after the addition of 0.3 µM Bay K 8644, indicating that Bay K 8644 amplified the magnitude of Ca2+-channel current in the vehicle-treated cells. Figure 5B shows the current-voltage relationships for vehicle-treated cells before and after the addition of Bay K 8644. In vehicle-treated cells, Bay K 8644 (0.3 µM) significantly increased the maximal peak current densities from -2.97 ± 0.35 pA/pF to -5.98 ± 0.62 pA/pF (n = 6). Figure 5, C and D shows Ca2+-channel current elicited by depolarizing steps and the current-voltage relationships before and after the addition of Bay K 8644 in the TNBS-treated colonic smooth muscle cells. Bay K 8644 (0.3 µM) did not significantly increase the Ca2+ channel current densities (control, -0.92 ± 0.46 pA/pF; Bay K 8644, -1.33 ± 0.51 pA/pF; n = 7). Membrane capacitance values did not differ significantly between vehicle- and TNBS-treated cells [56.1 ± 3.5 pF (range, 46-68 pF) in vehicle-treated cells; 55.9 ± 2.2 pF (range, 47-64 pF) in TNBS-treated cells].



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Fig. 5. A and B: Effect of Bay K 8644 on the whole cell Ca2+-channel currents in circular smooth muscle cells from vehicle-treated rat colon. A: control currents (upper trace) elicited by incremental 10-mV depolarizing steps from -30 to +60 mV (holding potential, -40 mV). Currents were increased by 0.3 µM Bay K 8644 (bottom trace). B: current-voltage relationships for the peak Ca2+ current density with or without Bay K 8644 (0.3 µM). Peak Ca2+-channel current density was increased by Bay K 8644 (n = 6). Significantly different from results before the treatment with Bay K 8644 at *P < 0.05 and **P < 0.01. C and D: effect of Bay K 8644 on the whole cell Ca2+-channel currents in circular smooth muscle cells from TNBS-treated rat colon. C: control currents (upper trace) elicited by incremental 10-mV depolarizing steps from -30 to +60 mV. The holding potential is -40 mV. Currents were slightly increased by 0.3 µM Bay K 8644. D: current-voltage relationships of peak Ca2+ current density with or without Bay K 8644. Peak Ca2+-channel current density was not significantly increased by 0.3 µM Bay K 8644 (n = 7).

 

Expression of the Ca2+-channel {alpha}1c-subunit. To determine the mechanism of the Ca2+ channel's reduced activity, we examined the effects of TNBS-induced inflammation on the mRNA levels of the Ca2+-channel {alpha}1c-subunit by using real-time RT-PCR. The relative quantity of {alpha}1c-mRNA expression seemed to be increased in the TNBS-treated rat colonic smooth muscles, but there was no significance between the vehicle- and TNBS-treated [vehicle, 1.00 ± 0.53 (n = 12) vs. TNBS, 2.97 ± 0.69 (n = 14)] (Fig. 6A).



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Fig. 6. A: real-time PCR analysis of the L-type Ca2+-channel {alpha}1c-subunit mRNA level in vehicle- and TNBS-treated rat colon. Data are expressed as fold increases in channel {alpha}1c-subunit mRNA levels in samples prepared from vehicle-treated colon. Estimations were performed in duplicate (vehicle, n = 12; TNBS, n = 14). B: Western blotting of the L-type Ca2+-channel {alpha}1c-subunit. Comparison of the expression of the channel {alpha}1c-subunit protein in circular smooth muscle membrane from vehicle- and TNBS-treated rat colon. Typical results of Western blot analysis for {alpha}1c-subunit protein are shown. No band was detected in the furthest right lane when 1 µg/µl control antigen was incubated with primary antibody.

 

We further investigated the protein expression of the Ca2+-channel {alpha}1c-subunit by using the immunoblotting method (Fig. 6B). The primary antibody corresponded to residues 818-835 of the rat {alpha}1c-subunit (pore-forming-subunit). An immunoreactive band was detected at ~200 kDa by using colonic smooth muscle membrane proteins prepared from vehicle- and TNBS-treated rat colon. The immunoreactivity was abolished by coincubation of primary antibody with control antigen (molecular weight, 2396) for 1 h at room temperature, confirming the specificity of the primary antibody for its recognition sequence on the rat {alpha}1c-subunit. Density of the immunoreactive band of the Ca2+-channel {alpha}1c-subunit did not differ between vehicle- and TNBS-treated colon (TNBS, 83.8 ± 4.9% of vehicle; n = 4).

Effect of NF-{kappa}B inhibitors, PDTC, and sulfasalazine on contractions. It has been reported (34, 37, 39) that treatment with TNBS induces many inflammatory cytokines such as TNF-{alpha}, IL-1{beta}, and IL-6. Because these cytokines are known to be expressed following the activation of NF-{kappa}B, we examined the effects of the NF-{kappa}B inhibitors PDTC and sulfasalazine (10, 14, 33, 43) on smooth muscle contractility. The maximum contractile force induced by 59.2 mM KCl decreased from 6.54 ± 1.07 to 2.25 ± 0.32 mN/mg wet wt 2 days after the induction of inflammation by TNBS (P < 0.05; n = 6-8). Similar inhibitory action was observed in the 1-µM carbachol-induced contractions [vehicle, 6.13 ± 0.62 mN/mg wet wt (n = 6); TNBS, 2.01 ± 0.78 mN/mg wet wt (n = 8); P < 0.05]. Although treatments with PDTC (100 mg/kg) or sulfasalazine (200 mg/kg) alone had no effect on the high K+- and carbachol-induced contractions, these treatments partially but significantly produced a recovery in the decreased contractions induced by the TNBS treatments [KCl: TNBS + PDTC, 4.23 ± 0.49 mN/mg wet wt (n = 9), TNBS + sulfasalazine, 4.63 ± 0.39 mN/mg wet wt (n = 10); carbachol: TNBS + PDTC, 3.61 ± 0.64 mN/mg wet wt (n = 9), TNBS + sulfasalazine, 4.31 ± 0.42 mN/mg wet wt (n = 10)] (Fig. 7, A and B).



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Fig. 7. Effect of NF-{kappa}B inhibitors pyrrolidinedithocarbamate (PDTC) and sulfasalazine (SS) on the decreased contractions after the TNBS treatment. A: 59.2 mM KCl. B: 1 µM carbachol. C: 0.1 µM Bay K 8644. Results are expressed as means ± SE (n = 9-10). Significantly different from results in vehicle-treated colon at **P < 0.01. Significantly different from results in TNBS-treated colon at #P < 0.05 and ##P < 0.01. n.s., Not significant.

 

In vehicle-treated rat colon, the absolute force induced by 0.1 µM Bay K 8644 was 7.47 ± 1.15 mN/mg wet wt (n = 9) (Fig. 7C). Bay K 8644-induced contractions were strongly decreased in the colonic smooth muscle 2 days after the induction of inflammation by TNBS (0.54 ± 0.27 mN/mg wet wt; n = 10). Contractions induced in the TNBS-treated tissue recovered, in part, in response to the pretreatment with PDTC and sulfasalazine [TNBS + PDTC, 3.75 ± 0.84 mN/mg wet wt (n = 10); TNBS + sulfasalazine, 5.27 ± 0.95 mN/mg wet wt (n = 9)].

Effect of sulfasalazine on the inhibition of Ca2+-channel current by TNBS. We finally examined whether NF-{kappa}B inhibitors restore the reduced Ca2+-channel activity. As shown in Fig. 4B, the average density of Ca2+-channel current decreased in TNBS-treated cells, and the current levels were partially but significantly restored by pretreatment with sulfasalazine [peak Ca2+-channel current densities: -1.06 ± 0.19 pA/pF (0 mV) (n = 7) in TNBS-treated cells; -1.90 ± 0.22 pA/pF (10 mV) (n = 6) in TNBS + sulfasalazine-treated cells]. Current-voltage curves indicate that the peak activation and half-activation voltage of Ca2+-channel activation in TNBS + sulfasalazine-treated cells were 10 and -13.5 mV, respectively (Fig. 4C). In contrast, membrane capacitance values were not significantly different between TNBS-treated and TNBS + sulfasalazine-treated cells (TNBS, 55.9 ± 2.2 pF, range 47-64 pF; TNBS + sulfasalazine, 52.0 ± 2.8 pF, range 42-64 pF).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Abnormal motility is induced by colonic inflammation, and this abnormality may lead to the diarrhea or constipation characteristic of Crohn's disease (1, 3, 36, 42). It has been suggested that many factors, including a downregulation of receptors or abnormal regulation of neurons, may be connected with intestinal dysmotility not only in Crohn's disease but also in experimental Crohn's disease models (6). In the present study, we examined the effects of inflammatory changes on the contractile properties of intestinal smooth muscle by using the hapten-induced Crohn's disease rat model. Our main findings are 1) contractions induced by KCl, carbachol, and Bay K 8644 are decreased by TNBS treatment, 2) Ca2+-channel current density is decreased in TNBS-treated colonic muscle cells without any change in the expression of Ca2+-channel {alpha}1c-protein, and 3) pretreatment with NF-{kappa}B inhibitors restores the suppressed contractions and Ca2+-channel activity.

Contractile activity. Treatment with TNBS for 2 days significantly decreased the KCl and carbachol-induced contractions in rat colon (Fig. 1). It has been reported (15, 17, 29, 35, 40) that prostaglandins and nitric oxide are generated during intestinal inflammation. Therefore, it is possible that these substances participate in the inhibition of contractile activity in the inflamed colonic smooth muscles. However, in the present study, neither the cyclooxygenase inhibitor indomethacin (21) nor the nitric oxide synthase inhibitor L-NAME (30) reversed the reduction of these contractions. These results imply that the decreased contractility of smooth muscle in TNBS-treated colon may be mediated by myogenic changes.

To further examine the effects of TNBS treatment on contractile properties, we investigated whether TNBS directly impairs contractile proteins by using permeabilized muscles. It was found that the absolute force and Ca2+ sensitivity in the {alpha}-toxin-permeabilized rat colonic muscles did not change after TNBS treatment (Fig. 3). This result suggests that the decrease in contractions induced by TNBS treatment may be mediated by a mechanism unrelated to the contractile apparatus.

L-type Ca2+-channel activity. Ca2+ mobilization via the L-type Ca2+ channel is the primary mechanism for the excitation-contraction coupling in gastrointestinal smooth muscles (5, 8). Data indicating that the contraction induced by Bay K 8644, an activator of the L-type Ca2+ channel, was markedly inhibited in TNBS-treated colon (Fig. 2) led us to speculate that TNBS treatment may change the L-type Ca2+-channel activity in smooth muscle cells. Therefore, we then examined the activity of the L-type Ca2+ channel by using the whole cell voltage-clamp technique and found that the L-type Ca2+-channel current density significantly decreased in the TNBS-treated cells (Fig. 4). These changes were not due to the change in cell size (membrane area), because the membrane capacitance did not change after the TNBS treatment. Calculated values for half-activation that reflect voltage-sensitivity were significantly different in vehicle- and TNBS-treated cells (i.e., the voltage sensitivity of the L-type Ca2+ channel was sensitized in the TNBS-treated cells).

We further investigated the effects of the L-type Ca2+-channel activator Bay K 8644 on Ca2+-channel currents and its modification by TNBS treatment. The Ca2+-channel currents in vehicle-treated cells responded to the Bay K 8644, and the peak amplitude of the Ca2+ channel increased approximately twofold. In contrast, Ca2+-channel currents in TNBS-treated cells did not respond to Bay K 8644 (Fig. 5).

The {alpha}1c-subunit is a voltage-gated pore of the L-type Ca2+ channel and a binding site of dihydropyridine drugs (38). It has been reported that cardiac disease such as atrial fibrillation or cardiac hypertrophy is associated with a decrease in the L-type Ca2+-channel current density, which may contribute to the dysfunction of myocardium contractions (25, 41). Moreover, it has been reported that these diseases cause reductions in the density of L-type Ca2+ channels by reducing the concentrations of mRNA encoding the pore-forming {alpha}-subunit and thereby reducing the concentrations of corresponding subunit proteins (7, 45). Liu et al. (19) also reported that the expression of Ca2+ channels decreases during inflammation induced by the injection of ethanol-acetic acid in the canine colon. However, in the present study with the TNBS-induced colitis model, the mRNA and protein of the {alpha}1c-subunit of the L-type Ca2+ channel were unchanged after TNBS treatment (Fig. 6), suggesting that the down-regulation of Ca2+-channel expression is not related to the decreased Ca2+-channel current density. These results suggest that TNBS treatment may cause structural changes in the {alpha}1c-subunit, which may contribute to the change in the binding affinity for Bay K 8644. As a result, TNBS treatment more strongly inhibited the contractions and the Ca2+-channel activation induced by Bay K 8644 in the TNBS-treated cells (see Figs. 2 and 5). The precise mechanism for the decrease in activity of the L-type Ca2+ channel was not clarified in the present study.

Effect of NF-{kappa}B inhibitors. NF-{kappa}B is a key player in the regulation of inflammatory gene expression (4). Activation of NF-{kappa}B increases the expression of genes encoding proinflammatory cytokines, and this molecule is also involved in the inflammation-related downstream pathways after cytokine production (28). Cytokines are important to gastrointestinal host defense, but their overproduction may cause excessive gut inflammation and intestinal motility disorders (6). It has also been reported (34, 37, 39) that inflammatory cytokines are overexpressed in TNBS-induced animal models. However, there have been few studies examining the causality between gastrointestinal dysmotility and inflammatory cytokines.

In the present study, we investigated the effects of NF-{kappa}B inhibitors on the decreases in stimulant-induced contractions and the Ca2+-channel current density in TNBS-treated rat colon. Pretreatment with PDTC or sulfasalazine partially but significantly attenuated the inhibition of KCl- and carbachol-induced contractions in TNBS-treated rat colon. We also found that pretreatment with PDTC or sulfasalazine resulted in a more efficient recovery of Bay K 8644-induced contractions in TNBS-treated rat colon. Moreover, in the patch-clamp experiment, pretreatment with sulfasalazine resulted in a significant recovery of the peak Ca2+-channel current density that had been reduced in the TNBS-treated cells. These results suggest that the downregulation of Ca2+-channel current densities observed in experimental colitis smooth muscle is attributable to the effect of inflammatory cytokines on smooth muscle cells and/or the changes due to the byproducts produced by the inflammatory cytokines.

In summary, the decrease in the contractility of circular smooth muscle isolated from TNBS-induced colitis rat colon is attributable to the decreased activity of the L-type Ca2+ channel. Our results also indicate that the dysfunction of L-type Ca2+ channel activity could be reversed by the therapeutic agents used for Crohn's disease that have an inhibitory action on NF-{kappa}B.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported, in part, by a Grant-in Aid for scientific research from the Japanese Ministry of Education (The Morinaga Houshi-Kai) and the Program for the Promotion of Basic Research Activities for Innovative Biosciences.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. Ozaki, Dept. of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan (E-mail: aozaki{at}mail.ecc.u-tokyo.ac.jp).

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. Section 1734 solely to indicate this fact.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
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