Departments of Surgery and Physiology, Medical College of Wisconsin and Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53226
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ABSTRACT |
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This study investigated whether inflammation modulates the mobilization of Ca2+ in canine colonic circular muscle cells. The contractile response of single cells from the inflamed colon was significantly suppressed in response to ACh, KCl, and BAY K8644. Methoxyverapamil and reduction in extracellular Ca2+ concentration dose-dependently blocked the response in both normal and inflamed cells. The increase in intracellular Ca2+ concentration in response to ACh and KCl was significantly reduced in the inflamed cells. However, Ca2+ efflux from the ryanodine- and inositol 1,4,5-trisphosphate (IP3)-sensitive stores, as well as the decrease of cell length in response to ryanodine and IP3, were not affected. Heparin significantly blocked Ca2+ efflux and contraction in response to ACh in both conditions. ACh-stimulated accumulation of IP3 and the binding of [3H]ryanodine to its receptors were not altered by inflammation. Ruthenium red partially inhibited the response to ACh in normal and inflamed states. We conclude that the canine colonic circular muscle cells utilize Ca2+ influx through L-type channels as well as Ca2+ release from the ryanodine- and IP3-sensitive stores to contract. Inflammation impairs Ca2+ influx through L-type channels, but it may not affect intracellular Ca2+ release. The impairment of Ca2+ influx may contribute to the suppression of circular muscle contractility in the inflamed state.
smooth muscle; motility; inflammatory bowel disease; signal transduction; calcium; acetylcholine; calcium influx; intracellular calcium stores; inositol 1,4,5-trisphosphate; ryanodine
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INTRODUCTION |
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AN INCREASE IN FREE CYTOSOLIC Ca2+ ([Ca2+]i) is a critical step in smooth muscle contraction. The force of contraction in these cells is related nonlinearly to [Ca2+] (15, 30). The two sources from which Ca2+ may be mobilized for this increase in [Ca2+]i are the extracellular medium and the rapidly exchanging intracellular stores in the sarcoplasmic reticulum. It now seems that, in the gut smooth muscle, the utilization of these two sources is organ, species, and agonist dependent. In the guinea pig small intestine, the longitudinal muscle cells utilize Ca2+ influx through L-type dihydropyridine-sensitive channels and intracellular release from ryanodine-sensitive stores to contract in response to CCK and ACh (11, 18, 25). By contrast, the small intestinal circular muscle cells from the same species utilize Ca2+ from the D-myo-inositol 1,4,5-trisphosphate (IP3)-sensitive stores (11, 25, 27). Feline esophageal circular muscle cells, on the other hand, utilize Ca2+ influx from the extracellular medium, whereas those from the lower esophageal sphincter and fundus utilize Ca2+ efflux from IP3-sensitive stores but not from the extracellular medium (4, 14). CCK-8 mobilizes Ca2+ from the IP3-sensitive stores in the feline gallbladder smooth muscle, whereas ACh can mobilize it from both the extracellular and intracellular sources (19). Substance P-induced contraction of the rabbit anal sphincter utilizes intracellular Ca2+ release, whereas that induced by bombesin utilizes extracellular Ca2+ (5). The mobilization of Ca2+ to contract canine colonic circular muscle cells is not fully understood.
Inflammation suppresses the phasic contractions and generation of tone in the circular muscle cells of the colon of several species (12, 22, 28, 40). The molecular mechanisms of this suppression of contractions are not known. However, since an increase in cytosolic Ca2+ is central to smooth muscle contraction, our hypothesis is that Ca2+ mobilization is altered during inflammation.
The aim of this study was to identify the sources of Ca2+ utilized in contracting single circular smooth muscle cells from the canine colon and how this utilization is altered during inflammation. ACh was used as the agonist in this study because it is one of the primary neurotransmitters of spontaneous colonic contractions at the neuroeffector junction (45). Intravenous or close intra-arterial administration of atropine, a nonselective muscarinic receptor antagonist, blocks all spontaneous contractions in the intact conscious state.
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EXPERIMENTAL METHODS |
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Tissue preparation and dispersion of smooth muscle cells. A 7- to 8-cm-long segment of the proximal colon was removed under general pentobarbital sodium anesthesia (30 mg/kg; Abbott Laboratories). The segment was passed over a glass tube and scored along the longitudinal axis with a blunt blade. The longitudinal muscle layer was peeled off and discarded. The remaining tissue was scored deeper. The circular muscle layer was peeled off and collected in ice-cold HEPES buffer (pH 7.4).
Smooth muscle cells were isolated by two consecutive digestions with papain and collagenase, respectively. Briefly, the circular muscle sheet was cut into 0.5 × 0.5 cm2 pieces and incubated at 37°C in 20 ml of Ca2+-free Hanks' solution (pH 7.2) for 15 min. Then they were incubated in 20 ml of Ca2+-free Hanks' solution containing 0.38 mg/ml papain and 0.3 mg 1,4-dithiothreitol until the tissue appeared loose and sticky (~10 min). The tissue was washed with HEPES buffer and further digested at 31°C with 0.2 mg/ml collagenase type II (319 U/mg) and 0.1 mg/ml soybean trypsin inhibitor for 30-40 min. The digested tissue was washed three times with enzyme-free HEPES buffer, and the muscle cells were allowed to disperse spontaneously under gentle to-and-fro motion. Cells were harvested by filtration through a 500-µm Nitex mesh and collected by centrifugation at 350 g for 5 min. Cells were resuspended in HEPES buffer, and volume was adjusted to reach 5 × 104 cells/ml for cell contraction experiments and 106 cells/ml for [Ca2+] measurements.Permeabilization of smooth muscle cells. The cells were permeabilized with saponin for use in experiments that required intracellular application of large molecules, as previously described (25, 27). The cells were incubated for 10 min in cytosol-like medium containing 35 µg/ml saponin. Saponin-treated cells were centrifuged twice at 350 g for 5 min, washed free of saponin, and resuspended in the same medium containing, in addition, antimycin (10 µM), ATP (1.5 µM), creatine phosphate (5 mM), and creatine phosphokinase (10 U/ml). The volume was adjusted according to different usage of these cells (5 × 104 cells/ml for cell contraction study and 106 cells/ml for [Ca2+] efflux study).
Measurement of free cytosolic Ca2+ in intact cells and 45Ca2+ efflux in permeabilized cells. [Ca2+]i was measured in a 2-ml cell suspension (106 cells/ml) using the Ca2+ fluorescent dye fura 2-AM (Molecular Probes, Eugene, OR) as described by Murthy et al. (26). Muscle cells were suspended in a modified HEPES buffer (26) containing (in mM) 10 HEPES, 125 NaCl, 5 KCl, 1 CaCl2, 0.5 MgSO4, 5 glucose, 20 taurine, 43 sodium pyruvate, and 5 creatine and were incubated with 2 µM fura 2-AM and 0.02% pluronic acid for 30 min at 31°C. The fura 2-AM-loaded samples were diluted, centrifuged twice, and suspended in 2 ml of solution with the same composition for immediate measurement of [Ca2+]i. Fluorescence was measured at 510 nm, with excitation wavelengths alternating between 340 and 380 nm, using an Aminco-Bowman series 2 luminescence spectrometer. Autofluorescence of unloaded cells was subtracted from the fluorescence of fura 2-loaded cells. The [Ca2+]i was calculated from the fluorescence ratio as described by Grynkiewicz et al. (13). The dissociation constant (Kd) of 224 nM was used for fura 2-AM. The maximum and minimum fluorescence were determined after adding 50 µg/ml digitonin and 4 mM Tris-EGTA (pH 8.7), respectively, in each sample.
Ca2+ efflux was measured by a modified method of Poggioli and Putney (32), described by Murthy et al. (18, 25). Briefly, the cells (106 cells/ml) were suspended in cytosol-like solution containing 45Ca2+ (10 µCi/ml) and 10 µM antimycin (to prevent mitochondrial uptake of Ca2+) at 31°C. 45Ca2+ uptake was initiated by the addition of 1.5 mM ATP and ATP regenerating system consisting of 5 mM creatine phosphate and 10 U/ml creatine phosphokinase. The net uptake of Ca2+ was determined from duplicate 100-µl samples removed at intervals for a period of 60 min. Preliminary experiments indicated that 45Ca2+ uptake reached a steady state in 60 min (data not shown). Net Ca2+ efflux in response to ACh, IP3, or ryanodine was examined at the end of 60 min of incubation and expressed as percent decrease in the steady-state 45Ca2+ cell content.Measurement of IP3 by radioreceptor assay.
IP3 was measured in circular muscle tissue using
Amersham's assay kit containing 3H-labeled
D-myo-IP3 and bovine adrenal
IP3, as previously described by Murthy and Makhlouf (27).
Circular muscle squares (2 × 2 mm2) were incubated in
Krebs solution with 10 mM LiCl for 15 min at 37°C. ACh
(104 M) was added, and the reaction was
terminated with 2 volumes of 6% TCA at 10, 20, 40, and 60 s. The
samples were homogenized and centrifuged at 4°C for 15 min at 4,000 rpm. The pellets were collected for protein determination, whereas the
supernatant was further extracted three times with 6 volumes of
water-saturated ether. The IP3 content in the aqueous phase
was measured by incubating with
[3H]IP3 and binding protein on ice
for 15 min. After centrifugation at 2,000 g for 15 min, the
supernatant was carefully removed and the pellet was dissolved with
water. The bound IP3 in the dissolved pellet was counted
with a Packard 1900 CA beta counter.
Measurement of [3H]ryanodine binding to
single cells.
Specific [3H]ryanodine binding was measured in
isolated single cells, as described previously (18). Aliquots (0.5 ml)
containing 5 × 105 cells were incubated at 31°C
with [3H]ryanodine. In the kinetic and
competition studies, 40 nM [3H]ryanodine was
used. The binding was rapid, attaining a steady state within 10 min,
and was reversible with the addition of unlabeled ryanodine
(105 M). The Kd and
maximal binding (Bmax) were determined by using [3H]ryanodine concentrations ranging from 5 to
120 nM. The incubation was stopped at 10 min with 0.75 ml of ice-cold
HEPES buffer. Nonspecific binding was determined in the presence of
10
5 M nonlabeled ryanodine. The cell
suspension was centrifuged at 12,000 g for 5 min to separate
the bound radioligand from the free radioligand. The wash and spin
cycle was repeated two times. Kd and
Bmax values were calculated using GraphPad Prism software version 2.0 (GraphPad Software, San Diego, CA).
Measurement of cell length. Cell length was measured by scanning micrometry as described previously (11, 25, 41). An aliquot (0.45 ml) of cells (5 × 104 cells/ml) was exposed to 50 µl of test agent at 31°C for different durations for time-course experiments or 40 s for ACh and 15 s for IP3 experiments. The reaction in each case was terminated by adding acrolein (final concentration, 1%). In other experiments, the cell samples were incubated with antagonists for 5 min before the addition of agonists. The lengths of 30 consecutive intact healthy cells were measured through a phase-contrast microscope (Nikon), fitted with a video camera (Javelin CCD), and connected to a Macintosh Computer. NIH Image 1.61 was used to measure the length. The contractile response was expressed as percent cell shortening from the vehicle control.
Induction of colonic inflammation and its visual assessment. Colonic inflammation was induced by mucosal exposure to ethanol and acetic acid (22, 40, 41). On day 1, the dogs were anesthetized with Telazol (150 mg im; Elkins-Sinn, Cherry Hill, NJ). The colon was cleansed by inducing defecation with neostigmine (30 µg/kg iv). An intraluminal Silastic tube with side holes in the first 5-cm length was advanced to ~50 cm from the anal margin to flush the colon with 700 ml of Colyte. The dogs were fasted overnight.
On day 2, the dogs were anesthetized again as described above. A Silastic tube with side holes in the first 5-cm length was advanced via rectum to ~50 cm, and another similar tube was advanced to ~15 cm from the anal margin. Ethanol (75 ml, 95%) was perfused through each tube at 5 ml/min. Ten minutes later, 20 ml of 60% acetic acid and 15 ml of 10% acetic acid were perfused through the proximal and the distal tubes, respectively, to induce pancolitis. After 5 min, the colon was flushed with 100 ml of 0.9% saline through each tube. Tissues were harvested 48 h later. We have reported previously that, at this time, the myeloperoxidase activity is increased (22) and the spontaneous phasic contractions and generation of tone in response to ACh are significantly suppressed (22, 40). Lu et al. (22) have also reported that, in tissue samples taken 30 min after exposure to ethanol and acetic acid, the motility changes are absent, indicating that the changes are associated with the inflammatory response rather than mucosal injury. The visual scoring system to assess mucosal injury was adapted from Bell et al. (2) as follows: 0 = normal mucosa; 1 = localized hyperemia but no erosions, ulcers, or scars; 2 = linear ulcer or scattered erosion <2 mm or ulcer scar with no significant inflammation; 3 = linear ulcer or scar with inflammation at one site >2 mm but <5 mm; 4 = two or more sites of ulceration and/or inflammation, each up to 5 mm; 5 = two or more major sites of inflammation and ulcerations >5 mm each or one major site of inflammation extending >1 cm along the length of the mucosa.Materials and solutions. Collagenase type II and soybean trypsin inhibitor were obtained from Worthington (Freehold, NJ). Papain, 1,4-dithiothreitol, ACh, IP3, low-molecular-weight heparin, methoxyverapamil (D-600), essential amino acid mixture, saponin, ATP, antimycin, creatine phosphate, and creatine phosphokinase were purchased from Sigma Chemical (St. Louis, MO), and BAY K8644 was purchased from RBI (Natick, MA). Fura 2-AM, pluronic acid F-127, and digitonin were purchased form Molecular Probes (Eugene, OR). 45Ca2+-CaCl2 and [3H]ryanodine were obtained from New England Nuclear (Boston, MA), and IP3 assay kit containing [3H]IP3 was purchased from Amersham Life Science (Arlington Heights, IL).
The composition of HEPES buffer (pH 7.4) in mM was 120 NaCl, 2.6 KH2PO4, 4 KCl, 2 CaCl2, 0.6 MgCl2, 25 HEPES, 14 glucose, and 2.1% essential amino acid mixture. The composition of cytosol-like medium in mM was 20 NaCl, 100 KCl, 5 MgSO4, 1 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 1 EGTA, and 2% BSA. The medium was bubbled with 95% O2 and 5% CO2 to maintain a pH of 7.2. Krebs solution was bubbled with 95% O2 and 5% CO2 and consisted of (in mM) 120 NaCl, 6 KCl, 14 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, and 11 glucose. The composition of Hanks' solution in mM was 135 NaCl, 5.5 KCl, 0.5 KH2PO4, 4 NaHCO3, 0.4 Na2HPO4, 0.5 MgCl2, and 5.5 glucose, pH 7.3. The composition of the Ca2+ measurement solution was the same as that described in Measurement of free cytosolic Ca2+ in intact cells and 45Ca2+ efflux in permeabilized cells.Statistical analysis. All values are expressed as means ± SE, and n represents the number of animals. Statistical analysis was performed by ANOVA with nonrepeated measures or unpaired t-test. Multiple comparisons were performed by Student-Newman-Keuls test, and P < 0.05 was considered statistically significant.
This study was approved by the Animal Studies Subcommittee of the Zablocki Veterans Affairs Medical Center. ![]() |
RESULTS |
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All dogs developed diarrhea within 24 h after the induction of inflammation. The visual morphological score was 4.4 ± 0.3 in the inflamed tissue and 0 ± 0 in the normal tissue. The dispersed cells appeared to be healthy and relaxed. The cell viability tested with trypan blue dye exclusion was 88 ± 4% and 86 ± 3% in cells from the normal and the inflamed colon, respectively (n = 3 for each). Lu et al. (22) have reported previously that, 48 h after the induction of colonic inflammation, the myeloperoxidase activity is increased significantly in the muscularis.
Contractile response to ACh.
The resting cell length of circular muscle cells from the normal colon
(98 ± 4.2 µm; n = 6)) was not different from that of cells
obtained from the inflamed colon (97 ± 4.6 µm; n = 5). The cells from both the normal and the inflamed colons contracted in a
concentration-dependent manner in response to ACh
(1011-10
5
M). The contractile response in the inflamed cells was significantly suppressed (P < 0.05). The maximum decrease in length in
normal cells (25.4 ± 2.3% at
10
5 M ACh) was significantly greater
than that in inflamed cells (14.6 ± 3.6%; Fig.
1). The EC50 in the inflamed
cells (9.9 ± 0.4 nM) was significantly greater than that in the
normal cells (1.9 ± 0.2 nM).
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The role of Ca2± influx in cell contraction.
BAY K8644
(1012-10
5
M) and KCl (25-100 mM) concentration-dependently decreased the
cell length in both normal and inflamed cells (Fig.
2). The maximal effect in the inflamed
cells was significantly suppressed in response to both compounds
(n = 4 each).
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Cytosolic Ca2+ and
Ca2+ efflux.
The basal [Ca2+]i was not different
between the normal and the inflamed cells. ACh at
105 M evoked an increase of 80 ± 10 nM
in [Ca2+]i that reached a peak in
10-15 s (Fig. 4). The increase in
[Ca2+]i in the inflamed cells (34 ± 3 nM) was significantly smaller than that in normal cells (Fig.
5A). Similarly, 50 mM KCl increased [Ca2+]i in both types of cells, but
the increase in the inflamed cells was smaller than that in the normal
cells (Fig. 5B). The increase in
[Ca2+]i by 75 mM KCl that induced
maximal cell shortening in inflamed samples was also less than that in
response to 50 mM KCl in normal cells (65 ± 10 nM vs. 103 ± 28 nM;
n = 4). The peak [Ca2+]i in
response to KCl was reached at 15-20 s.
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IP3 accumulation in response to ACh.
Direct measurement of specific
[3H]IP3 accumulation indicated that
the basal level of IP3 in the inflamed cells (1.2 ± 0.4 pmol/mg protein) was significantly smaller than that in normal cells
(3.0 ± 0.5 pmol/mg protein). However, the stimulated levels in
response to 104 M ACh were not different
between the two states of the cells (Fig.
8). The peak IP3 accumulation
at 20 s was 9.7 ± 1.4 pmol/mg protein in normal cells and 7.0 ± 1.8 pmol/mg protein in the inflamed cells (n = 4; P > 0.05 vs. normal cells).
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Binding of [3H]ryanodine to circular muscle
cells and utilization of ryanodine-sensitive stores.
In the presence of 105 M ruthenium red,
a ryanodine channel blocker, the ACh-induced
45Ca2+ efflux was reduced from 31.5 ± 3.7%
to 17.1 ± 1.9% (n = 4; P < 0.05), and contraction
in permeabilized cells was reduced from 17.1 ± 2.7% to 5.4 ± 0.7%
(n = 4; P < 0.05). These data indicated that ACh also
mobilizes Ca2+ from the ryanodine-sensitive stores.
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DISCUSSION |
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Our findings show that the canine colonic circular muscle cells may utilize Ca2+ influx through dihydropyridine-sensitive channels as well as intracellular Ca2+ release from both the ryanodine-sensitive and IP3-sensitive stores to contract. Downregulation of Ca2+ influx through L-type Ca2+ channels may contribute to the suppression of contractility to ACh during inflammation.
Several lines of evidence support the utilization of Ca2+ influx to contract canine colonic circular muscle cells. Patch-clamp recordings have demonstrated the presence of dihydropyridine-sensitive L-type Ca2+ channels in these cells (21, 35). Close intra-arterial infusion of verapamil, an L-type channel blocker, blocks the contractile response to ACh in intact conscious dogs (23). The contractile response of circular muscle strips to ACh is blocked by nicardipine, another antagonist of L-type channels (39). The blockade of L-type channels in these cells, however, did not completely inhibit the response to ACh. The residual response (~30% of the maximum response) may utilize other pathways, such as intracellular Ca2+ release and activation of protein kinase C (1, 25, 43, 47).
Ca2+ influx and intracellular Ca2+ release interact (6, 16, 24, 33, 34, 44, 46). Ca2+ influx through L-type Ca2+ channels acts as a cofactor to induce Ca2+ release [Ca2+-induced Ca2+ release (CICR)], whereas the emptying of intracellular Ca2+ stores induces capacitive entry of Ca2+ from the extracellular medium [Ca2+ release-activated current (CRAC)]. The influx by the CRAC mechanism is mostly through nonselective cation channels and is not blocked by L-type Ca2+ channel blockers. In the canine colonic circular muscle, the contractile response to ACh was blocked almost completely by D-600, indicating that the influx was primarily through the L-type channels. In addition, when the intracellular stores were depleted by treatment with thapsigargin, the normal cells still exhibited ~60% of the response in nontreated cells, indicating that the Ca2+ influx occurred independently of intracellular Ca2+ release. Ca2+ mobilization by CICR rather than by the CRAC mechanism may, therefore, predominate in these cells.
Our findings also demonstrate that Ca2+ influx may be impaired during inflammation. The concentration-response curves to both KCl, which depolarizes the membrane, and dihydropyridine BAY K8644, which activates voltage-sensitive L-type channels, were suppressed in the inflamed colon cells. A significant inhibitory effect of verapamil was noted at a concentration that was two log order greater in the inflamed than in normal cells. Similarly, a one-log order smaller concentration of extracellular Ca2+ was required to significantly reduce the response in the inflamed than that required in the normal cells. When intracellular Ca2+ was depleted by incubation with thapsigargin, the contractile response to ACh was significantly smaller in the inflamed than in normal cells. Together, these findings indicate less dependence of the contractile response on Ca2+ influx in the inflamed state. Recently, we have demonstrated also that Ca2+ currents recorded by the patch-clamp method are significantly reduced in the inflamed colon cells (21).
The utilization of extracellular Ca2+ for smooth muscle contraction may, however, vary among species and in different organs of the gut. Grider and Makhlouf (11), Murthy et al. (25), and Kuemmerle et al. (18) found that single circular muscle cells of the guinea pig small intestine do not utilize Ca2+ influx for CCK-8-induced contraction despite the presence of L-type Ca2+ channels in these cells, as demonstrated by patch-clamp recordings (7). Biancani et al. (4) reported that the feline esophageal circular muscle cells utilize Ca2+ influx for ACh-induced contraction, whereas the cells from the lower esophageal sphincter do not. The feline fundic circular muscle cells also do not seem to utilize Ca2+ influx for their contraction (14). ACh-induced, but not CCK-8-induced, contraction of the feline gallbladder smooth muscle utilizes Ca2+ influx through the L-type channels (19). Experiments on single human jejunal circular muscle cells show that they invoke Ca2+ influx and contraction in response to KCl (11), but the contractile response to ACh is not blocked by D-600 (11), suggesting that muscarinic receptor activation does not utilize Ca2+ influx through L-type channels. By contrast, Farrugia et al. (8) reported that the ACh and erythromycin-induced increase of free cytosolic Ca2+ in the human and canine jejunal circular muscle cells utilizes Ca2+ influx through L-type channels. Dihydropyridine-sensitive Ca2+ channels are present in the human colon circular muscle cells (48).
Canine colonic circular smooth muscle cells also seem to be able to mobilize Ca2+ from both the IP3-sensitive and ryanodine-sensitive stores to contract in response to ACh. IP3 accumulation in the cells increased time-dependently and peaked at ~20 s after stimulation with ACh. This peak time is shorter than the peak time for contraction in response to ACh (~40 s), indicating the expected temporal order of events for IP3 to mobilize Ca2+ for contraction. Exogenous IP3 contracted permeabilized cells and released Ca2+ from the intracellular stores. Furthermore, heparin inhibited the contraction as well as Ca2+ efflux in response to ACh. However, at the heparin concentration of 100 µg/ml, there was still a residual response of ~36% of the control, indicating the involvement of other pathways for contraction, independent of IP3 receptors.
The colonic circular muscle cells also mobilized Ca2+ from the ryanodine-sensitive stores. Concentration-dependent binding of [3H]ryanodine established the presence of ryanodine receptors in these cells. In addition, ryanodine contracted these cells and the response to ACh was partially but significantly blocked by ruthenium red, an antagonist of ryanodine receptors. Ca2+ mobilization from ryanodine-sensitive stores has also been reported in canine circular muscle strips (39) and in the intact conscious state (23).
The availability of Ca2+ from both the intracellular sarcoplasmic stores in the canine colonic circular muscle cells seems to be similar to that reported in several other cell types, including vascular smooth muscle, neurons, chromaffin cells, sea urchin eggs, atrial cells, epithelial cells, hepatocytes, and pancreatic acinar cells (3, 29). However, some cells seem to possess only one of the stores, e.g., the skeletal muscle cells have only the ryanodine-sensitive stores and Xenopus oocytes only the IP3-sensitive stores (29). The smooth muscle cells from the longitudinal and circular muscle layers of the guinea pig small intestine also show specialization in the utilization of intracellular stores (11, 18, 25, 27); the circular muscle cells mobilize primarily from the IP3-sensitive stores, whereas the longitudinal muscle cells utilize only the ryanodine-sensitive stores.
In contrast to the impairment in Ca2+ influx, Ca2+ release from the ryanodine- and IP3-sensitive stores was not affected during inflammation. The basal level of IP3 was reduced in the inflamed cells, but its peak accumulation in response to ACh was not different between the normal and the inflamed cells. Accordingly, Ca2+ efflux and decrease in cell length in response to IP3 did not differ between the two states of the cells. Contractile response and 45Ca2+ efflux to ryanodine as well as the binding of [3H]ryanodine to its receptors also did not differ between the normal and the inflamed cells.
The increase in [Ca2+]i in response to ACh in normal canine colonic circular muscle cells (80-110 nM) is similar to that reported for other smooth muscle cells in this species, e.g., jejunal (8, 9) and colonic (39) cells. However, this increase is smaller than that reported in the guinea pig small intestinal and tracheal cells (11, 43) but not the gastric smooth muscle cells (42). The decrease in the elevation of [Ca2+]i in the inflamed state is likely to be entirely due to the impairment in Ca2+ influx. Ca2+ efflux from the intracellular stores was not affected by the inflammatory response.
The experiments in single cells demonstrate the availability of specific signal transduction pathways and second messengers in response to the activation of specific receptors. However, the precise contribution or utilization of these pathways to stimulate different types of contractions in the intact conscious state can only be speculated at this time. The colonic circular muscle of several species, including dog, human, and rat, generates three distinct types of contractions: rhythmic phasic contractions, giant migrating contractions (GMCs), and tone (20, 37, 38). The amplitudes, durations, and motility functions of these contractions differ widely. The GMCs are 2-3 times larger in amplitude and 3-4 times longer in duration than the short-duration phasic contractions that occur ~4-6 times per min (17, 36-38). Because Ca2+ is a critical second messenger in contracting these cells and the amplitude and duration of cell contraction are nonlinearly correlated with [Ca2+]i, it is likely that the three types of contractions would utilize the two Ca2+ sources differently. The availability of both ryanodine-sensitive and IP3-sensitive stores in these cells may help in generating Ca2+ oscillations and waves of different characteristics to maintain different amplitudes and duration of contractions. The frequency, amplitude, and duration of Ca2+ oscillations provide a dynamic coding system that is specific to cellular function (10, 31). The impairment of Ca2+ influx but not intracellular Ca2+ release may be one of the means by which these cells exhibit stimulation of GMCs at the same time that the phasic contractions and generation of tone are suppressed during inflammation (22, 40, 45).
In conclusion, the canine colonic circular muscle cells can mobilize Ca2+ from the extracellular medium as well as the IP3- and ryanodine-sensitive stores. The contractile response of single dispersed cells from the inflamed colon is suppressed during inflammation. This suppression may in part be due to the decrease in Ca2+ influx through the L-type channels. Ca2+ release from IP3- and ryanodine-sensitive intracellular stores may not be affected by the inflammatory response. Selective modulation of Ca2+ utilization may be important in suppressing phasic contractions and tone while concurrently stimulating GMCs in the inflamed colon.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32346 and the Veterans Affairs Medical Research Service (both to S. K. Sarna)
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. K. Sarna, General Surgery, Medical College of Wisconsin FWC, 9200 West Wisconsin Ave., Milwaukee, WI 53226 (E-mail: ssarna{at}mcw.edu).
Received 11 March 1999; accepted in final form 18 October 1999.
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