A segment of distal colon (about 2 cm) was removed from Sprague-Dawley rats (150-250 g, supplied by the animal center of Anhui Medical University, China) killed by cervical dislocation. Pieces of colon were cleaned in a Petri dish, by perfusing with Krebs solution (114.0 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgCl₂, 2.5 mmol/L CaCl₂, 1.8 mmol/L NaH₂PO₄, 11.5 mmol/L glucose, 18.0 mmol/L NaHCO₃, pH 7.4). The adipose and connective tissues were carefully stripped off with fine forceps. The endothelium of the colon was removed by gently rubbing the inner lumen of the vessels with a rough wooden stick.

Pieces of distal colon were mounted in 40-mL organ baths containing Krebs solution, maintained at 37°C and gassed with 95% O₂ and 50% CO₂, pH 7.4. One end of each piece was fixed to a hook on the bottom of the bath and the other attached to a force displacement transducer (Xinhang, Beijing, China). A resting preload of 1 g was applied to each colon, which was then allowed to equilibrate for 1 h. During this time, the Krebs solution was changed every 20 min. Tension was continuously monitored and recorded using a Powerlab 4/25T data acquisition system (Australia AD Instruments) with an ML110 bridge bioelectric physiographic amplifier (Australia AD Instruments). ACh and atropine were dissolved in the perfusate on the day of use.

A segment of the distal colon was removed and placed in oxygenated HEPES/Ringer buffer solution (126 mmol/L NaCl, 6 mmol/L KCl, 6 mmol/L HEPES, 11 mmol/L MgCl₂, 1.5 mmol/L CaCl₂), pH 7.4 with 5 mol/L NaOH. The fecal contents were removed by repeated rinsing with HEPES/Ringer solution. After the segment was cut open longitudinally and pinned out in a dissection dish. Under a microscope, longitudinal and circular muscle layers were freed of the mesentery, serosa and mucosa, and cut into small pieces (2 mm²) and rinsed in HEPES/Ringer solution for 20 min (at 37°C). Dispersion of smooth muscle cells was accomplished by three incubations at 31°C with agitation in low-Ca²⁺ HEPES/Ringer solution containing collagenase type I (0.6 mg/mL), bovine serum albumin (2 mg/mL) and soybean trypsin inhibitor (0.6 mg/mL). They were then suspended in enzyme-free low-Ca²⁺ HEPES/Ringer solution and gently agitated through the tip of a wide-bore glass pipette to liberate single smooth muscle cells. Isolated cells were collected by low-speed centrifugation and resuspended in HEPES/Ringer solution. Trypan blue exclusion was used to verify cell viability (> 90% of all cells).

Measurement of [Ca²⁺]_i in colonic smooth muscle cells using fura-2 was performed using dual excitation wavelength fluorescence microscopy. Aliquots of cell suspension in HEPES/Ringer solution (10⁶ cells/mL) were placed in Quartz cuvettes and incubated with 5 &mgr;mol/L fura-2/AM at 37°C for 45 min. After being loaded with fura-2/AM, the cells were rinsed with Ca²⁺-free solution, centrifuged (1500 r/min) and suspended in Ca²⁺-free solution for the experiments that were carried out in a 960 MC spectrofluorophotometer (Shanghai Precision & Scientific Instruments, Shanghai, China). Cells were alternately illuminated at 340 and 380 nm and the intensity of fluorescence emission was measured at 500 nm. The [Ca²⁺]_i was calculated according to Grynkiewicz’s equation: [Ca²⁺]_i = Kd [(F - F_min)/(F_max - F)]: where the dissociation constant Kd was 224 nmol/L at 37°C; F was fluorescence intensity under different experiment conditions; F_max was the maximum fluorescence intensity obtained in the presence of 0.1% Triton ×-100; and the minimum value (F_min) was obtained by using 5 mmol/L EGTA in the external medium. Before calculation, the autofluorescence measured from cells that unloaded fura-2/AM should be subtracted. All the measurements were finished in 40 min.

Reagents were procured from the following vendors: thapsigargin, DMSO, ACh, atropine, lanthanum, fura-2/AM, Triton ×-100, EGTA, collagenase type I, bovine serum albumin (BSA) and verapamil were from Sigma (USA), and soybean trypsin inhibitor was from GIBCO (USA). The solutions of thapsigargin and fura-2/AM were prepared by dilution with DMSO. The concentration of DMSO in the final solution was < 0.1% and did not affect [Ca²⁺]_i. In cell experiments, the Ca²⁺-free HEPES/Ringer solution was prepared without CaCl₂ and with addition of EGTA (0.2 mmol/L). The low-Ca²⁺ solution had the same composition, except CaCl₂ was 0.03 mmol/L.

Biostatistical analysis was performed using the SPSS 11.0 software package. All experiments were repeated at least three times. Results of multiple experiments are given as the mean ± SE. Unpaired Student’s t test was used for statistical evaluation in two-group comparisons. P < 0.05 was accepted as statistically significant.

We first established whether SOCCs were present in rat distal colon smooth muscle cells. As shown in Table 1, for the fura-2/AM loaded cells, the incubation in Ca²⁺ free solution lasted 3 min, followed by the addition of two concentrations of Ca²⁺ (1.5, 3.0 mmol/L). It can be seen that cytosolic calcium rose from 68.32 in Ca²⁺-free solution to 104.81 and 194.44 nmol/L for the two concentrations of 1.5 mmol/L and 3.0 mmol/L, respectively. The incubation of cells in Ca²⁺-free solution in the presence of thapsigargin (1 &mgr;mol/L), followed by the addition of Ca²⁺, 1.5 and 3.0 mmol/L, increased [Ca²⁺]_i that was significantly greater (455.77 and 1005.9 nmol/L) than that in the absence of thapsigargin.

To distinguish SOCC-mediated Ca²⁺ influx from that via L-type Ca²⁺ channels, we conducted experiments in a separate set of cells using 5 &mgr;mol/L verapamil. In these cells pretreated with verapamil, the [Ca²⁺]_i response to rapid reintroduction of [Ca²⁺]_o in the presence of thapsigargin (n = 7) was determined as above. We found the presence of verapamil during rapid reintroduction of [Ca²⁺]_o (1.5 mmol/L) did not significantly influence Ca²⁺ influx induced by thapsigargin. Previous studies in other tissues have found that SOCC-mediated Ca²⁺ influx is inhibited by La^3+[20,21]. We investigated the sensitivity of multivalent cations to such Ca²⁺ influx. In a separate group of cells, pretreatment with La³⁺ and Ca²⁺ influx to rapid reintroduction in the presence of thapsigargin was greatly changed (Figure 1). Taken together, these results suggest the observed influx of extracellular Ca²⁺ in both instances was not mediated via L-type Ca²⁺ channels, but capacitative Ca²⁺ entry through SOCCs.

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Figure 1 Ca²⁺ influx through SOCCs in enzymatically dissociated smooth muscle cells of rats distal colon. After depletion of the SR by thapsigargin in the absence of extracellular Ca²⁺, subsequent rapid reintroduction of extracellular Ca²⁺ resulted in activation of CCE via SOCCs. CCE were insensitive to verapamil, but blocked by La³⁺. ^bP < 0.01 vs control.

A potential confounding factor in SOCC-mediated Ca²⁺ influx is the membrane potential, which alters the driving forces for Ca²⁺ entry. Accordingly, separate experiments were performed to determine the role of membrane potential. In the presence of verapamil (5 &mgr;mol/L), and pretreatment with high KCl solution (final concentrations of 40, 60 mmol/L), cells were re-exposed to thapsigargin, and [Ca²⁺]_o was then rapidly reintroduced (final concentration, 1.5 mmol/L). Figure 2 shows the presence of 40 mmol/L KCl did not significantly influence Ca²⁺ influx induced by thapsigargin. However, in the presence of 60 mmol/L KCl, Ca²⁺ influx was decreased (P < 0.05).

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Figure 2 CCE are not mediated via L-type Ca²⁺ channels. (A) Controls; (B) 40 mmol/L K⁺; (C) 60 mmol/L K⁺. Only 60 mmol/L K⁺ significantly decreased the Ca²⁺ influx mediated by CCE. ^aP < 0.05 vs control.

In isolated rat distal colon, removal of extracellular Ca²⁺ almost abolished the contraction induced by 40 mmol/L KCl or 5 &mgr;mol/L ACh, a muscarinic receptor agonist (n = 11, Figure 3A). Chelation of extracellular Ca²⁺ with 2.78 mmol/L EGTA, which decreases free Ca²⁺ concentration to 0.5 &mgr;mol/L in Krebs solution containing 2.5 mmol/L CaCl₂, had the same inhibitory effect on contraction induced by 40 mmol/L K⁺ or 5 &mgr;mol/L ACh (n = 7, Figure 3B). Furthermore, extracellular application of the L-type VOCC blocker verapamil (5 &mgr;mol/L) almost abolished the 40 mmol/L K⁺-induced tension. The tension decreased from 2.1 to 0.52 g and the inhibitory rate was 74% (n = 7, P < 0.001). The tension induced by 5 &mgr;mol/L ACh was also inhibited, but the inhibitory rate was only 41%. The tension decreased from 1.44 to 0.85 g (n = 7, P < 0.05) (Figure 4). These observations indicated ACh induced rat distal colon contraction by activating multiple Ca²⁺ entry pathways, whereas 40 mmol/L K⁺-induced rat distal colon contraction was solely dependent on the membrane-depolarization-mediated opening of VOCCs.

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Figure 3 Role of Ca²⁺ in rat distal colon contraction. A: Representative results showing that removal of extracellular Ca²⁺ abolished contraction induced by high K⁺ and 5 &mgr;mol/L ACh. Summarized data showing that tension induced by high K⁺ and 5 &mgr;mol/L ACh before, during and after application of Ca²⁺-free Krebs solution. ^bP < 0.01, ^dP < 0.001 vs Control; B: Representative results showing that chelation of extracellular Ca²⁺ with 2.78 mmol/L EGTA abolished contraction induced by high K⁺ and 5 &mgr;mol/L ACh. Summarized data showing that tension induced by high K⁺ and 5 &mgr;mol/L ACh in the presence of EGTA ^bP < 0.01, ^fP < 0.001 vs-EG (before).

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Figure 4 Effects of the VOCC blocker verapamil on distal colon contraction induced by high K⁺ and ACh. ^aP < 0.05, ^bP < 0.001 vs control.

In the absence of extracellular Ca²⁺, ACh induced a transient contraction that was apparently due to Ca²⁺ release from the SR. The muscarinic receptor blocker atropine (10 &mgr;mol/L) was applied to the distal colon when the ACh-induced contraction returned to baseline. In the presence of atropine, restoration of extracellular Ca²⁺ caused tonic contraction (Figure 5Aa). We further showed tonic contraction was unaffected by verapamil (n = 5, Figure 5Ab). The CCE-mediated contraction in the presence of atropine was about 0.46-fold greater than that induced by SR Ca²⁺ release and the total peak contraction induced by ACh in the presence of extracellular Ca²⁺ (n = 9, Figure 5B). Taken together, these results showed that Ca²⁺ release from the SR and CCE via SOCCs both contribute to agonist-mediated distal colon contraction.

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Figure 5 Contribution of Ca²⁺ release from SR stores and Ca²⁺ influx via CCE in ACh-induced distal colon contraction. Aa: In the presence of atropine, restoration of extracellular Ca²⁺ induced contraction, most likely due to Ca²⁺ influx via CCE; Ab: VOCC blocker verapamil (5 &mgr;mol/L) negligibly affected CCE-mediated contraction in the presence of atropine; B: Summarized data showing that CCE-mediated contraction is about 0.46-fold greater than the contraction induced by SR Ca²⁺ release in ACh-induced rat distal colon contraction. ^bP < 0.001 vs Ca²⁺ release.

La³⁺ has been shown to block SOCCs in many cell types[1131]. La³⁺ (50 &mgr;mol/L) was applied to the distal colon when CCE-induced contraction was maximized, and the CCE-mediated contraction was significantly attenuated (n = 7, Figure 6A and B). These results indicated the properties of SOCCs in the distal colon are similar to those found in other tissues.

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Figure 6 Inhibitory effects of La³⁺ on CCE-mediated distal colon contraction. ^bP < 0.01 vs control. Control: CCE-mediated contraction.

Various agonists induce contraction in a variety of smooth muscle preparations, with a concomitant increase in [Ca²⁺]_i. It is suggested the sources of Ca²⁺ for phasic and tonic contraction induced by activation of muscarinic receptors differ among regions of the digestive tract and species of animals used.

Intracellular Ca²⁺ is a critical second messenger responsible for linking external stimuli to contraction, proliferation and gene expression. It has been suggested some of the motor changes associated with intestinal infection, stress and inflammation are associated with alterations in [Ca²⁺]. Further studies of pharmacological manipulation of Ca²⁺ channels may be therapeutically useful in treating colonic motility disorders.

In this study, we investigated the importance of Ca²⁺ influx in smooth muscle contraction. We also evaluated the contribution of the different Ca²⁺ influx mechanisms and the release of Ca²⁺ from the sarcoplasmic reticulum (SR) to contraction induced by activation of muscarinic receptors in rat distal colon in vitro. The results showed that Ca²⁺ necessary for contractile response in the distal colon of rat was supplied from outside the smooth muscle cells via voltage-operated Ca²⁺ channels (VOCCs), receptor-operated Ca²⁺ channels (ROCCs) and store-operated Ca²⁺ channels (SOCCs), and the SR had a minor role in the contractile response.

The results provide further evidence for the contraction of the rat distal colon, which hopefully will be of importance in developing new drugs with unique properties, such as those that inhibit capacitative Ca²⁺ entry (CCE)-mediated Ca²⁺ influx.

Thapsigargin is a potent skin irritating sesquiterpene lactone isolated from the roots of Thapsia garganica L. It also acts as a non-phorbol-ester-type tumor promoter, which discharges intracellular Ca²⁺ stores by specific inhibition of sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase (SERCA).

This paper examined the relative contributions of internal calcium release and calcium influx to phasic and tonic contraction in the distal colon, and measured calcium concentration and calcium influx in isolated colon smooth muscle cells. The work addressed an old topic but the current discovery of SOCCs in excitable cells such as smooth muscle cell (SMC) is interesting.