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
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ABSTRACT |
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2,4,6-trinitrobenzenesulfonic acid; rat colon; circular smooth muscle contractility; pyrrolidinedithiocarbamate; sulfasalazine; Crohn's disease
One of the primary transcriptional factors in the regulation of
inflammatory gene expression is the NF-B family
(4). Members of the NF-
B
family serve as important regulators of the host immune and inflammatory
response. Activation of NF-
B increases the expression of genes encoding
proinflammatory mediators such as cytokines (TNF-
and IL-1
, -6,
and -12), cell-adhesion molecules (VCAM-1 and ICAM-1), inducible nitric oxide
synthase, and cyclooxygenase-2
(28). In addition, NF-
B
is activated in patients with IBD
(26). Because
pyrrolidinedithiocarbamate (PDTC) and sulfasalazine are potent inhibitors of
NF-
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-
B
(10,
14). Therefore, inhibitors of
NF-
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-
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.
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MATERIALS AND METHODS |
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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
-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, 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
) 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 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
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
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
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
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
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-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-nitro-L-arginine methyl ester
(L-NAME), nifedipine, PDTC, sulfasalazine, and staphylococcal
-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.
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RESULTS |
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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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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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Muscle contractility in the -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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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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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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Expression of the
Ca2+-channel
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
1c-subunit by using
real-time RT-PCR. The relative quantity of
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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We further investigated the protein expression of the
Ca2+-channel 1c-subunit by using the
immunoblotting method (Fig.
6B). The primary antibody corresponded to residues
818-835 of the rat
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
1c-subunit. Density of the
immunoreactive band of the Ca2+-channel
1c-subunit did not differ between vehicle- and TNBS-treated
colon (TNBS, 83.8 ± 4.9% of vehicle; n = 4).
Effect of NF-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-
, IL-1
, and IL-6.
Because these cytokines are known to be expressed following the activation of
NF-
B, we examined the effects of the NF-
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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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-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).
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DISCUSSION |
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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 -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 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
-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
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
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-B inhibitors. NF-
B is a key
player in the regulation of inflammatory gene expression
(4). Activation of NF-
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-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-B.
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DISCLOSURES |
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FOOTNOTES |
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