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Am J Physiol Gastrointest Liver Physiol 294: G88-G98, 2008. First published November 1, 2007; doi:10.1152/ajpgi.00305.2007
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NEUROREGULATION AND MOTILITY

Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle

Jason R. Kovac, Tom Chrones, and Stephen M. Sims

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada

Submitted 6 July 2007 ; accepted in final form 31 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Following smooth muscle excitation and contraction, depletion of intracellular Ca2+ stores activates capacitative Ca2+ entry (CCE) to replenish stores and sustain cytoplasmic Ca2+ (Ca2+i) elevations. The objectives of the present study were to characterize CCE and the Ca2+i dynamics underlying human colonic smooth muscle contraction by using tension recordings, fluorescent Ca2+-indicator dyes, and patch-clamp electrophysiology. The neurotransmitter acetylcholine (ACh) contracted tissue strips and, in freshly isolated colonic smooth muscle cells (SMCs), caused elevation of Ca2+i as well as activation of nonselective cation currents. To deplete Ca2+i stores, the sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid were added to a Ca2+-free bathing solution. Under these conditions, addition of extracellular Ca2+ (3 mM) elicited increased tension that was inhibited by the cation channel blockers SKF-96365 (10 µM) and lanthanum (100 µM), suggestive of CCE. In a separate series of experiments on isolated SMCs, SERCA inhibition generated a gradual and sustained inward current. When combined with high-speed Ca2+-imaging techniques, the CCE-evoked rise of Ca2+i was associated with inward currents carrying Ca2+ that were inhibited by SKF-96365. Regional specializations in Ca2+ influx and handling during CCE were observed. Distinct "hotspot" regions of Ca2+ rise and plateau were evident in 70% of cells, a feature not previously recognized in smooth muscle. We propose that store-operated Ca2+ entry occurs in hotspots contributing to localized Ca2+ elevations in human colonic smooth muscle.

REGULATION OF SMOOTH MUSCLE function is critically dependent on Ca2+ (42). Excitatory neurotransmitters initiate contraction by interacting with cell-surface receptors to generate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which binds to receptors on the sarcoplasmic reticulum to trigger Ca2+ release (29, 42). In turn, store depletion activates plasma membrane-localized, Ca2+-permeable, store-operated channels (SOCs) in a process termed capacitative Ca2+ entry (CCE), which both replenishes stores and sustains cytoplasmic Ca2+ (Ca2+i) elevations to allow contraction (42).

In colonic smooth muscles, rhythmic variations of membrane potential, called slow waves, underlie peristaltic contractions (22). Interstitial cells of Cajal initiate and propagate depolarizations and, through interaction with smooth muscle cells (SMCs), regulate contraction (22). Input from neurotransmitters to SMCs then modulates the amplitude and duration of slow wave activity (22). For example, in canine colonic SMCs, acetylcholine (ACh) and the tachykinin neurokinin A (NKA) activate an inward, nonselective cation (NSC) current (INSC) that depolarizes SMCs to cause Ca2+ influx and contraction (17). Receptor-independent activation of CCE with sarcoplasmic reticulum Ca2+-ATPase (SERCA) blockers like thapsigargin (TG) or cyclopiazonic acid (CPA) results in a sustained Ca2+ entry through INSC (10, 20, 39, 40). Multiple SOCs have been described, with unitary channel conductance ranging from 1.5 to 7 pS (5). Moreover, contractions can be evoked independent of L-type Ca2+ channel activation (25), suggesting that additional pathways contribute to the regulation of peristalsis.

Initial identification of CCE in smooth muscles was made in mouse anococcygeus (40). Evidence has since emerged for CCE and SOCs in a variety of cell types, including vascular (8), airway (2, 10), lower esophageal sphincter, and esophageal body (39) smooth muscles. Although whole cell Ca2+i elevations have been well described in colonic SMCs both in response to excitation (4, 29) and at rest in the form of Ca2+ puffs (3), the contribution of CCE and the mechanisms underlying Ca2+ signaling are still incompletely understood.

A developing view in smooth muscle physiology is that local events can contribute, or sum, to produce global Ca2+ changes (42). Indeed, the spatial and temporal integration of Ca2+i transients is a key issue because ultimately it is the pattern of global Ca2+i elevations that shapes contraction (28). Pioneering studies of neurons during the early 1990s noted that L-type Ca2+ channels clustered in subcellular domains, giving rise to Ca2+ "hotspots" (30). SERCA can be localized functionally to cellular regions where Ca2+ entry occurs (1), suggesting a possible relationship between CCE, agonist-evoked contraction, and Ca2+ hotspots.

In the present study, our objectives were to characterize CCE and Ca2+i dynamics in human colonic SMCs by recording contraction of intact tissues and responses of freshly isolated muscle cells. With the use of high-speed Ca2+-imaging techniques combined with patch-clamp electrophysiology, we correlated CCE with inward currents carrying Ca2+. In addition, we illustrated the temporal and spatial Ca2+i dynamics underlying CCE and cholinergic-evoked contractions. Store-operated Ca2+ entry occurs in hotspots and contributes to localized Ca2+ elevations, features not previously recognized in smooth muscle. Our studies provide insight into the mechanisms whereby CCE contributes to regulation of human colonic smooth muscle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue retrieval and isolation of cells. Tissue collection was carried out with approval of the University of Western Ontario Review Board for Health Sciences Research Involving Human Subjects and conformed to the Helsinki Declaration. Transverse, descending, and sigmoid colon were obtained from patients undergoing colonic resection because of cancer, with specimens selected from disease-free margins. Samples of the entire thickness of bowel were removed, placed in oxygenated Krebs solution (composition given below in Solutions), kept at 4°C, and transported to the laboratory. Circular muscle was carefully dissected on the basis of morphology and orientation and was cleaned of nerves, blood vessels, fat, and fascia.

For isolation of SMCs, segments of colon were cut into strips (~2 mm wide, 10 mm long) and placed in 2.5 ml of dissociation solution (see Solutions). Tissues were stored in dissociation solution overnight at 4°C. On the next day, tissues were warmed to room temperature for 20–60 min and placed in a gently shaking water bath at 31°C for 45–60 min, followed by dispersion of cells by trituration with fire-polished Pasteur pipettes. All freshly isolated SMCs were studied within 8 h of dispersion. In total, muscle was obtained from 22 specimens (13 female, 9 male; mean age 65 yr)

Measurement of Ca2+i. Dissociated cells were loaded with fura 2-AM (0.2 µM) or fluo 4-AM (5 µM) with 0.05% pluronic and were incubated at room temperature for 40 min as previously described (15, 16). During incubation and throughout all experiments, care was taken to minimize light exposure to preserve fluorescence intensity. Cells were allowed to settle onto a glass coverslip that comprised the bottom of a perfusion chamber (~0.75 ml volume). The chamber was mounted on a Nikon inverted microscope (Eclipse TE2000-U; Nikon, Tokyo, Japan) equipped with a water-immersion lens (x60, numerical aperture 1.2). A Na+-HEPES bathing solution (see Solutions) was perfused (1–3 ml/min, room temperature) except where otherwise noted.

To estimate changes of Ca2+ concentration, we used the ratiometric dye fura 2. The ratio of fluorescence emission at 510 nm with alternate excitation wavelengths of 345 nm and 380 nm was measured by using a Deltascan system (Photon Technology International, Birmingham, NJ), as previously described (15, 16). Ca2+i was calibrated according to the methods of Grynkiewicz and co-workers (9), with Ca2+i = [Kd(R – Rmin)/(Rmax – R)]Sf2/Sb2, where R is the ratio and Rmin and Rmax are the ratio of fluorescence intensity at 345/380 under Ca2+-free and saturated conditions, respectively, and Sf2/Sb2 is the ratio of fluorescence values for Ca2+-free/Ca2+-bound indicator measured at 380 nm. We used a dissociation constant of 225 nM for binding of Ca2+ to fura 2 (9) and a viscosity factor of 0.8. Data were corrected for background fluorescence. The calculation of Ca2+i involves a number of assumptions and factors such as homogeneity of Ca2+ within cells that may introduce uncertainty in values.

To complement the fura 2 studies, we used the single-wavelength dye fluo 4, which is more appropriate for high-speed imaging. Cells loaded with fluo 4 were illuminated with 488 nm of light from a multiline argon ion laser, and emissions were detected at 510 nm. Images were acquired at 40–65 Hz by using a wide-field digital fluorescence imaging system (Photon Technology International) with a Cascade Photometrics 650 cooled charge-coupled device camera (653 x 492 pixels; Roper Scientific, Tucson, AZ) and ImageMaster 5 Software (Photon Technology International). With the x60 lens, each pixel represented an area of 196 x 196 nm. The spatial resolution, assessed as the 10–90% edge response, was 0.5 µm. Cells selected for study were solitary, initially relaxed, and phase bright. Exposure to the excitation laser was controlled by an electronic shutter that was closed during recovery from agonists. Results are reported as a change in fluorescence ({Delta}F/Fo, %), which is a relative measure of the free Ca2+i. Image processing was performed offline, with all Ca2+ images shown presented as baseline-subtracted images. This was achieved, pixel by pixel, by using the equation {Delta}F/Fo (%) = [F – Fo]/Fo x 100, where F represents the fluorescence at each point in an experimental time course and Fo the baseline level (as determined by using the average of 20 consecutive images preceding each experimental treatment). Representative images are shown in pseudocolor, although saturation of the images limits the dynamic range compared with accompanying data traces.

Electrophysiological recordings. Dispersed cells were allowed to settle and adhere to the bottom of a perfusion chamber and were perfused with a Na+-HEPES bathing solution (see Solutions) at 1–3 ml/min. Whole cell recordings were made in the perforated-patch configuration with electrode solution containing nystatin (250 µg/ml). All currents were recorded at room temperature (21–24°C) with an Axopatch 200A amplifier (Axon instruments, Foster City, CA) filtered at 1 kHz and sampled at 5 kHz by using pClamp 9 software.

To resolve colonic SMC currents, initial experiments were performed in a KCl electrode solution, and, where indicated, a CsCl electrode solution was used to block K+ currents and characterize inward currents. Glutamate was substituted for Cl– to give the Cs+-glutamate electrode solution (see Solutions for composition). Pipette resistance before seal formation ranged from 1 to 9 M{Omega}. Whole cell recordings were initiated when access resistance stabilized at <40 M{Omega} to allow series resistance compensation of up to 80% to be used. Capacitive currents were compensated online by using amplifier circuitry, and linear leakage was corrected offline as assessed at negative potentials.

Tissue bath studies. Colonic circular muscle strips (~2 mm wide, 10 mm long) were dissected and mounted individually in tissue baths containing 10 ml Krebs solution continuously bubbled with 5% CO2-95% O2 at 37°C. With the use of silk ties, one end of the strip was attached to a Grass FT03 isometric force transducer coupled to a Grass 79E chart recorder (Grass Instruments, Quincy, MA). Output from the transducer was digitized and was sampled at 2.5 Hz. After a 1-h equilibration period, the length of each strip was adjusted to produce maximal tension on application of 10 µM ACh. For quantification, tension responses were expressed as a percentage of the largest ACh-evoked response obtained before the commencement of the experiment.

Solutions. The Krebs solution used for retrieval of tissues and contraction studies consisted of (in mM) 116 NaCl, 5 KCl, 2.5 CaCl2, 1.2 MgSO4, 2.2 NaH2PO4, 25 NaHCO3, and 10 D-glucose, equilibrated with 5% CO2-95% O2 (pH 7.4). The HEPES bathing solution used for electrophysiological recordings and fluorescence studies contained (in mM) 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose (adjusted to pH 7.4 with NaOH). The dissociation solution contained a 135 mM K+ solution (in which the NaCl of the HEPES bathing solution was replaced with KCl) plus the following: 0.4 mg/ml collagenase (Sigma blend type F), 1.8 mg/ml bovine serum albumin, 1 mg/ml papain, and 0.125 mg/ml 1,4-dithio-L-threitol. Ca2+-free solutions had the same composition as above with the omission of CaCl2 and the addition of 0.5 mM EGTA.

For patch-clamp recording, the KCl recording electrode solution contained (in mM) 140 KCl, 20 HEPES, 1 MgCl2, and 0.1 EGTA (adjusted to pH 7.2 with KOH). CsCl recording electrode solution contained (in mM) 130 CsCl, 20 HEPES, 1 MgCl2, 10 TEA-Cl, 0.4 CaCl2, and 1 EGTA (adjusted to pH 7.2 with CsOH). Cs-glutamate electrode solution contained (in mM) 40 CsCl, 100 glutamate, 20 HEPES, 1 MgCl2, 10 TEA-Cl, 0.4 CaCl2, and 0.01 EGTA (adjusted to pH 7.2 with CsOH).

Chemicals. Fura 2-AM, fluo 4-AM, pluronic, and thapsigargin were obtained from Molecular Probes (Eugene, OR). Caffeine and cyclopiazonic acid were from RBI (Natick, MA). SKF-96365 was from Tocris Cookson (Bristol, UK), and iberiotoxin was obtained from Bachem (King of Prussia, PA). All other agents were obtained from Sigma (St. Louis, MO). Test substances were prepared from stock solutions in distilled water or DMSO, diluted into the appropriate bathing solution and applied either by bath perfusion or pressure ejection from glass micropipettes (Picospritzer II, General Valve, Fairfield, NJ). Pipettes were positioned 25–75 µm from cells, with the concentration reported being that in the application pipette. Control studies carried out with vehicle alone had no effect.

Statistics. Values are means ± SE with sample sizes (n) indicating the number of cells or muscle strips studied. All traces shown are representative of at least three experiments on muscle or cells from two or more colon specimens. Comparisons were made by using the Student's paired t-tests, with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ca2+i stores and CCE in human colonic smooth muscle. We first established the importance of Ca2+i stores in human colon by analyzing contraction of smooth muscle strips. ACh evoked a reproducible increase in tension (Fig. 1, A and C), verifying the response predicted from an earlier study (29). Following removal of Ca2+ from the bathing solution, a similar transient increase in tension could be elicited. However, with successive applications of ACh in Ca2+-free solution, contractions gradually diminished until absent, consistent with the depletion of Ca2+i stores (Fig. 1A). Recovery occurred following readdition of extracellular Ca2+ (Fig. 1A, right), confirming that responses were not due to rundown, desensitization, or tissue death.


Figure 1
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  		Fig. 1. Role for intracellular Ca2+ stores and capacitative Ca2+ entry (CCE) in contraction of human colonic smooth muscle. A: acetylcholine (ACh, 10 µM, applied at closed arrowhead) evoked contraction of intact smooth muscle strips (left). On removal of Ca2+ from bathing solution, ACh continued to evoke contractions; however, successive responses were greatly diminished, consistent with a role for cytosolic Ca2+ (Ca2+i) stores in excitation of muscle. B: to initiate complete release of Ca2+ from stores, ACh and caffeine (Caff) were applied simultaneously (10 µM and 5 mM, respectively; open arrowhead, left) and evoked contractions similar to ACh alone (For summary, see C). Addition of extracellular Ca2+ (Ca2+o) to bath (3 mM, duration indicated by arrows) before store depletion evoked no contraction. However, following treatment with cyclopiazonic acid (CPA; 10 µM), stores were readily emptied, as indicated by lack of response to ACh plus caffeine. Subsequent application of Ca2+o (3 mM) in presence of CPA evoked contraction, suggestive of CCE. ACh plus caffeine responses returned following washout (right), confirming tissue viability. C: summary of responses illustrated above expressed as means ± SE. In a series of paired experiments, contractions evoked by ACh alone (n = 12) were not different from those evoked by ACh plus caffeine (n = 12). D: CCE was evaluated by using this protocol and was found to be reproducible, with responses shown as a percentage of control cholinergic-evoked rise of tension. Addition of Ca2+ alone caused only a minor rise in tension, whereas addition of Ca2+ after pretreatment with CPA caused significantly greater tension (*P < 0.05; n = 12).

 
Distinct Ca2+i stores activated by ryanodine and/or ryanodine and Ins(1,4,5)P3 are reported in some muscles (7). Accordingly, we applied ACh [10 µM, Ins(1,4,5)P3 receptors] together with caffeine (5 mM, ryanodine receptors) to fully deplete Ca2+i stores. In a series of paired experiments, the contractions evoked by ACh plus caffeine (n = 12, 0.1 ± 0.01 N) were found to be similar to those observed with ACh alone (n = 12, 0.09 ± 0.009 N; Fig. 1, B and C). After Ca2+ was eliminated from the bathing solution, minimal responses were observed to control applications of extracellular Ca2+ (Ca2+o, 3 mM) when Ca2+i stores were not yet depleted (based on Fig. 1A). Throughout these studies, 3 mM Ca2+o was used in the presence of 0.5 mM EGTA to yield an effective free Ca2+ concentration of 2.5 mM, a value similar to that seen under physiological conditions. Subsequent store depletion with the SERCA inhibitor CPA (10 µM) resulted in emptying of stores, as evidenced by the lack of response to subsequent stimulation with ACh/caffeine (Fig. 1B). A role for CCE was noted when, in the presence of CPA, addition of Ca2+o evoked contraction (Fig. 1B). The contractions produced by Ca2+o in the presence of CPA were reproducible and significantly greater than control applications of Ca2+o alone (P < 0.05, Fig. 1D). These findings were suggestive of CCE in human colonic smooth muscles and prompted us to investigate the underlying signaling mechanisms.

Cholinergic excitation of colonic SMCs and rise of Ca2+i. To confirm the viability of our freshly isolated SMCs and to validate the results seen in our tissue-strip experiments on a cellular level, we studied Ca2+i responses to a cholinergic agonist. Initially, cells ranged in length from 75 to 250 µm and appeared spindle shaped with a bright periphery (Fig. 2A). Rapid and reproducible contraction of SMCs occurred following ACh stimulation. SMCs returned to ~90% of their resting length following a 10-min washout.


Figure 2
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  		Fig. 2. ACh evokes global and regional elevations of Ca2+i and contraction in colonic smooth muscle cells (SMCs). A: A bright-field image (left) illustrates a freshly isolated colonic SMC at rest. As visualized with Ca2+ dye fluo 4, low basal Ca2+i levels (i) increased on stimulation with ACh (ii, iii; 10 µM, 5 s). Transient rise of Ca2+i was accompanied by contraction (iii). Recovery to basal Ca2+i levels was apparent following 5 min washout (iv). Images are representative of responses recorded in 12 cells. B: time course for Ca2+i rise depicted in A. Values were obtained from an "area of interest" represented by the large black box in A. ACh, applied by pressure ejection from a pipette, resulted in a rapid rise of Ca2+i that gradually returned to baseline levels following washout, consistent with a role for Ca2+i in regulation of SMC contraction. Superimposed numerals (i–iv) represent time points where images in A were obtained. To account for baseline regional variations, in these and future traces, fluorescence (F) values were expressed as {Delta}F/Fo (%) = [(F – Fo)/ Fo] x 100. Baseline fluorescence values (Fo) were obtained before agonist application as an average of 20 frames. Fluorescence images were collected at 30 to 50 frames/s. C: To evaluate regional changes in Ca2+, areas of interest (9 x 9 pixels) were selected (a, b, c), represented by small colored boxes superimposed on image in A. Initial onset of Ca2+i rise occurred in regions a and c, and after 5 s a global Ca2+i elevation was observed (see above). Spatial dynamics of regional Ca2+ "hotspots" are illustrated with regions a and c, exhibiting a more rapid Ca2+i onset compared with region b. Region b achieved a higher overall {Delta}F/Fo in spite of gradual onset. Cells were bathed in 2.5 mM bathing solutions, and Ca2+i elevations were likely a combination of Ca2+ influx and release from stores.

 
Cells were loaded with the Ca2+-sensitive dye fluo 4 to monitor regional change of Ca2+i. To capture a representative trace of the Ca2+i responses, an area of interest was selected (Fig. 2A). (This area was chosen because it maintained position during contraction.) From a low basal Ca2+i level, there was a rapid increase on stimulation with ACh followed by a gradual return to baseline levels on washout (Fig. 2, A and B, n = 33) in agreement with previous studies in colonic smooth muscle (4, 29, 35) and our studies of human esophageal muscle (16).

Regional changes in Ca2+ were examined (Fig. 2C) in chosen areas of interest (9 x 9 pixels). Initial onset of Ca2+i rise occurred in regions a and c (Fig. 2C) before the global Ca2+i elevations described above (Fig. 2, A and B). The spatial dynamics suggested that certain regions of the cell exhibited a more rapid onset of the rise of Ca2+i (e.g., a and c compared with b). In these studies the bathing solution contained Ca2+, so the ACh-evoked hotspots could have originated from store release or influx. The CCE experiments discussed below were carried out in Ca2+-free conditions and with stores depleted to allow us to explore the different sources of Ca2+.

Electrophysiological characteristics of colonic SMCs. We examined agonist-activated ion channels in human colonic SMC contraction by using the nystatin perforated-patch configuration. When currents were recorded with KCl in the electrode solution, outwardly rectifying K+ currents were evident and were blocked with TEA (5 mM, n = 4) and iberiotoxin (100 nM, n = 3, data not shown), supporting the existence of Ca2+-activated K channels (KCa channels), as we have previously characterized in human esophageal muscle (11). In human colonic SMCs, ACh enhanced K+-mediated outward currents, providing a further functional role for Ca2+i in colonic SMCs (data not shown).

While recording ionic currents, we observed ACh-evoked INSC (Fig. 3). With cells held at –60 mV, ACh (10 µM) activated a transient inward current (Fig. 3A) similar to that seen in human esophageal SMCs (15). Responses were reproducible and recovered following washout. Voltage ramp commands (–100 to 50 mV) were periodically applied to evaluate the current-voltage relationship and reversal potential of ACh-evoked current. With Cs+ in the electrode solution to block K+ currents, reversal potentials were –6 ± 5 mV (n = 5). When Cl– was replaced with glutamate, shifting the Cl– equilibrium potential from 0 to –30 mV, the reversal potential was unaltered (–1 ± 2 mV, n = 7), indicating a negligible contribution for Cl–.


Figure 3
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  		Fig. 3. Agonist-activated nonselective cation (NSC) currents (INSC) in human colonic SMCs. A: representative trace from SMC held at –60 mV using perforated-patch configuration. With Cs+ in electrode solution to block K+ currents, ACh (10 µM, applied for duration of bar) activated a transient inward current. Voltage ramp commands (–100 to 50 mV) were applied periodically to evaluate current-voltage relationship of ACh-evoked current. Cl– was replaced with glutamate (shifting the Cl– equilibrium potential from 0 to –30 mV). Subtraction of control current (Cont) from agonist-evoked current revealed the activated current ({Delta}I, right), which reversed at –1 ± 2 mV (n = 7), consistent with activation of INSC. B: similar current, with reversal potential of –3 ± 10 mV (n = 3), was evoked by tachykinin agonist neurokinin A (NKA, 1 µM).

 
Tachykinins, present in neurons of the gastrointestinal tract, induce contraction of human colonic smooth muscle strips (24) as well as contraction and INSC in human esophageal smooth muscle (15). We examined the effects of NKA on colonic SMCs. An INSC similar to that evoked by ACh was observed and found to reverse at –2.9 ± 9.5 mV (n = 3) with Cs+ in the electrode solution. In addition, we looked for evidence of purinergic regulation in human colon. The purinergic receptor agonist (BzATP, 300 µM) evoked a INSC with reversal potentials of –8.6 ± 6.0 mV (CsCl electrode solution, n = 4) and –5.5 ± 5.3 mV (Cs-glutamate electrode solution, n = 5; data not shown). These findings are consistent with the activation of an INSC in response to muscarinic, tachykinin, and purinergic agonists in human colonic SMCs. However, we did not study interactions between the agonists to explore potential synergy.

Depletion of Ca2+i stores activates CCE. We developed a protocol to reproducibly elicit CCE in isolated SMCs while measuring global changes in Ca2+i concentration with fura 2. ACh (10 µM) evoked a transient rise in [Ca2+]i, presumably through Ins(1,4,5)P3-sensitive Ca2+ stores (Fig. 4A). Perfusion of a Ca2+-free bathing solution resulted in diminished responses, consistent with a gradual, but not complete, depletion of intracellular stores. Re-addition of Ca2+ into the bathing solution restored ACh-evoked transients (Fig. 4A). These data support a role for both the release of Ca2+ from intracellular stores as well as influx, results similar to those seen in esophageal SMCs (15, 16).


Figure 4
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  		Fig. 4. Depletion of Ca2+ stores activates CCE in colonic SMCs. A: Ca2+ dye fura 2 was used to measure global Ca2+i. ACh (10 µM, applied for 10 s at closed arrowhead) evoked a transient rise in Ca2+i, presumably through inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]-sensitive Ca2+ stores. Perfusion of a Ca2+-free bathing solution resulted in diminished responses consistent with a gradual, but not complete, depletion of intracellular stores. Reperfusion with a Ca2+-containing solution resulted in partial recovery of ACh-evoked transients (right). B: concurrent administration of ACh (10 µM) and caffeine (5 mM) to deplete both Ins(1,4,5)P3- and ryanodine-sensitive stores, produced a transient rise in Ca2+i similar to that seen with ACh alone. Repetitive applications of ACh and caffeine in a Ca2+-free bathing solution resulted in abolition of Ca2+i elevations, suggesting complete depletion of intracellular stores. Once stores were emptied, 3 mM Ca2+o was applied (indicated by bar above trace). A small but consistent increase in Ca2+i was observed, suggestive of CCE. C: summary of experiments in A and B. Store depletion with ACh alone (n = 4 cells, Ca2+o applied following lowest Ca2+i peak evoked by agonists) or in combination with caffeine (n = 7 cells) was effective in depleting stores sufficiently to allow CCE to be visualized. Values represent average peak response to Ca2+o obtained following intracellular store depletion through respective protocols, shown as percentage change from basal levels. No significant difference in magnitude of evoked CCE was noted. D: In presence of a Ca2+-free bathing solution, use of sarcoplasmic reticulum Ca2+-ATPase (SERCA)-inhibiting agents (thapsigargin, TG; 2 µM, 60 s, n = 5 cells) resulted in a gradual rise of Ca2+i levels as stores were depleted.

 
Next, using a protocol similar to that in the tissue-strip experiments (Fig. 1), we concurrently applied ACh (10 µM) and caffeine (5 mM) to deplete both Ins(1,4,5)P3- and ryanodine-sensitive stores (7). A transient Ca2+i rise similar to that seen with ACh alone (Fig. 4B) was observed. Repetitive applications of ACh and caffeine in a Ca2+-free bathing solution resulted in abolition of Ca2+i elevations, suggesting complete depletion of intracellular stores (Fig. 4B). Once stores were emptied, Ca2+o (3 mM) was applied focally from a puffer pipette. A small but consistent increase in Ca2+i was observed, reflecting CCE, in response to prior applications of ACh (n = 4) or ACh plus caffeine (n = 7, Fig. 4C). No marked decline in the magnitude of responses was observed in control experiments involving repetitive applications of ACh or ACh plus caffeine in a 2.5 mM Ca2+-bathing solution (3 cells), as we have shown earlier for human esophageal SMCs (32). Depletion of intracellular stores occurred at a faster rate in isolated SMCs (Fig. 4B) compared with whole tissues (Fig. 1B), perhaps reflecting the minimal diffusion barriers in isolated cells.

Store depletion can also be induced through inhibition of SERCA pumps (2, 8, 10). To refine our CCE-induction protocol, we used SERCA inhibitors. Application of one such inhibitor, thapsigargin (TG, 2 µM) in a Ca2+-free bathing solution induced a slow rise of Ca2+i, indicating rapid store depletion (n = 5, Fig. 4D). Subsequent cholinergic stimulation failed to evoke contraction (data not shown), confirming that store depletion occurred under these conditions.

SERCA blockade induces CCE. We next used SERCA inhibition to deplete Ca2+i stores and study changes of Ca2+i in single SMCs. Cells were loaded with fluo 4 and were bathed in an extracellular medium containing 0 Ca2+, 0.5 mM EGTA, and the reversible SERCA inhibitor CPA (10 µM). Fluorescence intensity was recorded from a stationary region of interest (Fig. 5A), chosen to minimize changes arising from SMC contraction. Basal Ca2+i levels were initially low because of the Ca2+-free solution and SERCA inhibition (Figs. 5A and 4D). Following application of 3 mM Ca2+o, a prompt increase in Ca2+i was observed (Fig. 5). After a 5-min washout, Ca2+i levels returned to baseline, whereupon a second application of Ca2+o resulted in a reproducible Ca2+i elevation (Fig. 5, A and B). Application of Ca2+o for longer durations yielded a further rise of Ca2+ (Fig. 5C), suggesting that CCE did not desensitize. A separate observation was that after the second application of Ca2+o, two distinct regions of Ca2+i rise were observed (Fig. 5A, iv), a result discussed in CCE and cholinergic excitation results in localized Ca2+i elevations at hotspots.


Figure 5
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  		Fig. 5. SERCA blockade induces CCE in human colonic SMCs. A: bright-field image (left) of freshly isolated colonic SMC at rest. Cells were loaded with fluo 4 and were bathed in an extracellular medium containing 0 Ca2+, 0.5 mM EGTA, and SERCA blocker CPA (10 µM) to deplete intracellular stores. Basal Ca2+i levels (i) were initially low and increased promptly on application of 3 mM Ca2+o (ii), reflecting CCE. After a 5-min washout, Ca2+i levels returned to baseline (iii), whereupon a second application of Ca2+o resulted in a reproducible Ca2+i elevation (iv). A 3-min perfusion of NSC channel blocker SKF-96365 (SKF) had no effect on basal Ca2+i levels (v) but inhibited subsequent Ca2+i increases evoked by addition of Ca2+o (vi). B: traces reveal fluorescence intensity obtained from a 9 x 9 pixel region of interest (3.1 µm2), schematically illustrated by black box in A. Superimposed numerals (i–vi) represent time points in which panels in A were obtained. Change in fluorescence [{Delta}F/Fo (%)] was a relative measure of free Ca2+i. C: application of Ca2+o for longer durations yielded reproducible, elevated levels of Ca2+ fluorescence. D: summary of effects of NSC inhibition described in B. A 3-min perfusion of SKF-96365 resulted in a significant blockade of CCE elicited by Ca2+o applied for 15 s (*P < 0.05).

 
The nature of the Ca2+ influx was explored by treating cells with the cation-channel blocker SKF-96365, which inhibits CCE in other cell types (19), including smooth muscle (2). A 3-min perfusion of SKF-96365 had minimal effects on basal Ca2+i levels but significantly reduced CCE evoked by a 15-s application of Ca2+o (Fig. 5).

Consistent with the activation of CCE in other cell types (10), electrophysiological recordings revealed that SERCA inhibition induced a gradual and sustained inward current (Fig. 6A, n = 8). In separate recordings, voltage ramp protocols (–100 to 50 mV) run during TG application revealed a current with reversal potential at –10 ± 1 mV (n = 4, Fig. 6B). The reversal potential of this current was suggestive of an INSC, contrasting with the L-type Ca2+ current previously described in colonic (43) and human esophageal SMCs (16).


Figure 6
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  		Fig. 6. CCE-evoked inward currents in human colonic SMCs. A: application of SERCA inhibitor TG induced a gradual and sustained inward current (2 µM, applied for time indicated by bar; n = 8 cells). B: in separate recordings, voltage-ramp protocols (–100 to 50 mV) were applied during SERCA inhibition to evaluate current-voltage relationship of resulting current. Control (Cont) values were obtained before application and for each following minute. Traces shown are representative of currents obtained at 2 min with TG, revealing activation of a INSC that reversed at –10 mV (n = 4 cells).

 
Simultaneous patch-clamp recording and Ca2+i imaging during CCE. To explore the relationship between Ca2+ influx and ionic current, we used combined patch-clamp electrophysiology and Ca2+ fluorescence. Cells were loaded with fluo 4 and immersed in the Ca2+-free bathing solution designed to evoke CCE (0 Ca2+, 0.5 mM EGTA, 10 µM CPA). Cellular Ca2+i was initially very low and uniform across the cell. Following initiation of whole cell recording, addition of Ca2+o resulted in a prompt rise of Ca2+i, indicative of CCE (Fig. 7). The rise of Ca2+i was associated with the development of a net outward current (n = 9) that likely represented reduction of the inward current carried by Na+, a feature previously described in Ca2+-permeable channels (26). Both Ca2+i and current levels returned to baseline following washout of the Ca2+o (Fig. 7B). In separate cells, the current-voltage relationships were characterized by voltage ramp protocols (–100 to 50 mV; Fig. 7C). The current modulated by addition of Ca2+ reversed direction at –17 ± 2 mV (n = 3) and was apparent as a reduction of current, hence the negative slope. Together, these observations suggest that CCE occurs in SMCs through INSC activated by store depletion.


Figure 7
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  		Fig. 7. Simultaneous patch-clamp and Ca2+i imaging reveal Ca2+ influx during CCE. A: bright-field image illustrates a human colonic SMC at rest following seal formation with a patch-clamp pipette (left). Cells were immersed in a bathing solution optimized to evoke CCE (0 Ca2+ solution containing 0.5 mM EGTA and 10 µM CPA). Addition of Ca2+o (3 mM) resulted in a prompt rise of Ca2+i. B: summary trace of an area of interest as depicted by box shown above in A, i. Superimposed numerals correspond to time points in which i–iii were obtained. Ca2+o was applied for duration of arrow. Concurrent patch-clamp recording reveals that rise of Ca2+i was closely associated with development of a net outward current that represents a reduction of inward current carried by Na+. Ca2+i and current levels returned to baseline following washout of the Ca2+o. C: in a separate cell, current-voltage relationships were characterized by voltage ramp protocols (–100 to 50 mV). Current modulated by addition of Ca2+ reversed direction at –17 mV (n = 3 cells) and was apparent as a reduction of current, hence the negative slope.

 
CCE and cholinergic excitation results in localized Ca2+i elevations at hotspots. During our initial fluorescence recordings of CCE, spatial variations in Ca2+i were apparent (Fig. 5A, iv). As a result, we hypothesized that regional specialization in Ca2+ influx could occur with CCE. Although early studies have hinted at this phenomenon (23, 41), little is reported in SMCs due to limited availability of rapid Ca2+i-imaging systems. Following SERCA inhibition (described in SERCA blockade induces CCE), CCE was activated with Ca2+o, resulting in Ca2+i elevations (Fig. 8A). Prolonged Ca2+o (60 s) resulted in contraction (Fig. 8A) similar to results observed in intact muscle strips (Fig. 1). SMCs returned to resting lengths following a 10-min washout.


Figure 8
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  		Fig. 8. CCE yields localized Ca2+i elevations at distinct hotspots in human colonic SMCs. A: bright-field image (left) illustrating areas of interest (9 x 9 pixel, 3.1 µm2). Note that depicted boxes are enlarged for visibility. Intracellular stores were depleted and CCE was evoked by using SERCA inhibition. Application of Ca2+o (3 mM) resulted in focal Ca2+i elevations (middle), and prolonged Ca2+o (60 s) resulted in contraction (right) similar to results observed in muscle strips. B: time-course of experiment shown in A with traces corresponding to individual areas of interest (i–iv). Regions exhibited distinct patterns of Ca2+ rise and plateaus. Rapid rise and onset of Ca2+i influx in region iv is suggestive of a Ca2+-influx hotspot, with delayed and more subdued rise in regions i and ii possibly reflecting diffusion of Ca2+. Findings in B correspond to images in A. Boxed region (left) is shown on an expanded scale (right) to emphasize distinct onset of regional changes. We propose that CCE-evoked Ca2+ entry occurs at Ca2+ hotspot locations (i.e., region iv) within cells. Hotspots seen in response to induction of CCE occurred in 26 of 37 cells observed (70%). In other instances, no distinct hotspots were observed and Ca2+i rose globally (11 of 37 cells, 30%).

 
With high-speed fluorescence imaging, distinct regions of Ca2+i rise were evident (Fig. 8). The rapid rise and onset of Ca2+i influx in Fig. 8B (region iv) is suggestive of a Ca2+-influx hotspot. The delayed and more subdued rise in regions i and ii might reflect diffusion of Ca2+. Onset of the Ca2+i rise occurred at different rates in the varying regions (Fig. 8B). Hotspots seen in response to induction of CCE occurred in 26 of 37 cells observed (70%). In the other instances, no distinct hotspots were observed and Ca2+i rose uniformly (11 of 37 cells, 30%). No reliable pattern of hotspot distribution was noted between different cells. Within individual cells, specific regions of Ca2+ entry were consistently visualized on repeated applications of extracellular Ca2+. On the basis of these observations, we propose that CCE-evoked Ca2+ entry originates at distinct Ca2+ hotspots within human SMCs.

Regional specialization of Ca2+ was also apparent in other conditions. Our earlier data revealed both global changes in response to ACh (Fig. 2) and changes in discrete areas of interest (Fig. 2C). Indeed, when distinct regions were examined, it was clear that Ca2+i rise occurred initially in regions a and c, with region b exhibiting a delay in onset but higher overall {Delta}F/Fo (Fig. 2C), demonstrating regional specialization in Ca2+i handling following both CCE and cholinergic excitation.

CCE blockers reduce contraction in colonic smooth muscle strips. We returned to tissue-strip experiments to extend and explore the findings from isolated SMCs. Addition of Ca2+o to tissues depleted of Ca2+ resulted in a rise of tension that was inhibited by addition of the NSC channel blockers SKF-96365 (Fig. 9A, n = 30) and lanthanum (100 µM, Fig. 9B, n = 12). The blockade was significant (Fig. 9C) and supported the notion that CCE occurred in human colonic smooth muscle.


Figure 9
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  		Fig. 9. CCE-evoked contractions are inhibited by NSC channel blockade in human colonic muscle. A: control applications of ACh (10 µM, closed arrowhead) induced contraction of colonic muscle strips (left). Removal of Ca2+ from bathing solution and addition of CPA caused store depletion. Under these conditions, CCE was evoked via addition of Ca2+o (3 mM) that induced contraction. Treatment with NSC channel blocker SKF-96365 (SKF, 10 µM) inhibited Ca2+o-evoked contractions. ACh responses returned following washout of CPA and SKF along with readdition of Ca2+ to allow for store refilling (right). B: NSC channel blocker lanthanum (La, 100 µM) also reduced CCE-evoked contractions. C: summary of experiments illustrated in A and B (means ± SE) presented as percentage of control responses. Perfusion of SKF (n = 30 cells) and La (n = 12 cells) resulted in significant inhibition (*P < 0.05) of CCE-evoked tension increases.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our objective was to characterize CCE and Ca2+i dynamics in human colonic SMCs by using several complimentary approaches. Patch-clamp recordings of freshly isolated cells identified ACh-activated INSC and SOCs activated by SERCA inhibition. Under conditions where SERCA Ca2+ pumps were inhibited, CCE contributed to contraction of colonic muscle. High-speed Ca2+-imaging techniques combined with patch-clamp electrophysiology allowed us to correlate CCE with inward currents carrying Ca2+. Analysis of the temporal and spatial Ca2+i dynamics underlying CCE revealed that store-operated Ca2+ entry occurred in localized Ca2+ hotspots, a feature not previously recognized in smooth muscle. When taken together, our studies establish the existence of CCE in human colonic smooth muscle.

ACh, the primary excitatory neurotransmitter in the gastrointestinal tract, is critical to colonic smooth muscle contraction in a wide variety of species (24, 29, 35). The present study confirms the ability of ACh to contract human colonic smooth muscle and further demonstrates that removal of extracellular Ca2+ results in depletion of intracellular stores and diminished contraction. Furthermore, we provide evidence that CCE can mediate Ca2+ entry that can contribute to Ca2+ homeostasis. Indeed, CCE contributes to contraction of ileum (25) and gall bladder (20) smooth muscle strips. Given that renal, mesenteric, and coronary artery smooth muscles are reported to be functionally unaffected by CCE (33), it was important to characterize the effect in human muscles. Indeed, NSC channel inhibitors also exhibit species specificity. For example, in organ-bath studies on mouse anococcygeus, the cation channel blocker SKF-96365 inhibited CCE, whereas another blocker, lanthanum, exhibited no effect (40). In contrast, CCE-evoked in gall bladder smooth muscle was not inhibited by SKF-96365 (20). We report here that both SKF-96365 and lanthanum inhibit CCE in human colon smooth muscle.

Slow rhythmic variations in membrane potential underlie peristaltic contractions in colonic smooth muscle (22). In addition to Ca2+, regulation is dependent on various ionic currents. In agreement with our findings, previous studies on canine colonic SMCs identified KCa channels (6) inhibited by extracellular TEA (36). In canine SMCs, cholinergic excitation resulted in inhibition of KCa activity with subsequent depolarization, voltage-dependent Ca2+ channel activation, and contraction (6). This is in contrast to our studies on human colonic SMCs and previous work in tracheal SMCs (37) where KCa was acutely activated by ACh. Such activation of a voltage-dependent KCa current would limit depolarization and hasten repolarization of SMCs following contraction.

Excitatory cholinergic stimulation can depolarize SMCs via activation of INSC, as shown in the present studies as well as, among others, jejunum, gastric corpus (31), tracheal (13), and human esophageal (15) SMCs. The nonadrenergic, noncholinergic neurotransmitter NKA contracts human colonic smooth muscle strips (24) and, in agreement with our studies, activates an INSC in canine SMCs that demonstrates a reversal potential of ~0 mV (18). Given these findings, further studies into the signaling underlying excitation by tachykinins are warranted.

The membrane currents underlying CCE-evoked SOCs in smooth muscle have not been widely recorded, due in part to their small size. Indeed, single-channel conductance of 3 pS has been described in vascular SMCs, with conductances of 1.5–7 pS emerging as a distinguishing feature of SOCs (5). SOCs most generally exhibit permeability for Ca2+ as well as Na+ (26). Accordingly, our finding that addition of extracellular Ca2+ caused a reduction of inward current could reflect the blockade of Na+ entry by Ca2+, a phenomenon previously described in cation channels (26).

Although SERCA inhibitors evoke CCE, the lack of effect on SOCs in excised plasma membrane patches (where intracellular stores are absent) suggests that they do so only indirectly (5). In airway SMCs, the SERCA inhibitor CPA activated a linear current with reversal potential of 0 mV, resulting in Ca2+i elevations (2, 10) similar to CCE-evoked SOCs identified in the present study. Given the Na+ and Ca2+ permeability of SOCs, indirect effects of a Na+/Ca2+ exchanger cannot be discounted (5). However, in smooth muscles, the Na+/Ca2+ exchanger has been suggested to make only minor contributions to Ca2+i homeostasis (14) and CCE-evoked Ca2+ influx was insensitive to the selective Na+/Ca2+ exchanger inhibitor KBR-7943 (27), suggesting a negligible role in CCE.

In agreement with our studies, whole cell Ca2+i elevations in response to cholinergic excitation have been described in cultured human colonic SMCs (35) as well canine SMCs (4, 29). Localized Ca2+ puffs have been described in cells at rest (3), with widely dispersed, propagating Ca2+ waves identified in murine colonic SMCs (28). Spatially and functionally distinct Ca2+ stores have been identified in colonic SMCs (7): one with both ryanodine and Ins(1,4,5)P3 receptors and one with only Ins(1,4,5)P3 receptors (12). Depletion of the Ins(1,4,5)P3-sensitive store reduced, but did not abolish, responses to caffeine in colonic SMCs (7). In other studies, thapsigargin depleted Ca2+ stores that were functionally and structurally distinct from those depleted by caffeine (34). In our hands, depletion of stores with a combination of ACh and caffeine was more effective than ACh alone, although the magnitude of CCE was similar. Thus, whereas the existence of distinct Ca2+i stores in human colonic SMCs is suggested, this specialization does not appear to play a critical role in activation of CCE.

In pioneering studies, Williams et al. (41) provided an initial illustration of the spatial dynamics underlying Ca2+i signaling in gastric SMCs of Bufo marinus. Expanding on these observations, others described focal Ca2+i elevations that propagated throughout the cytoplasm as "Ca2+ waves" (23) or existed in the resting SMCs as highly localized Ca2+ transients ("sparks") (4). Ca2+ sparks can modulate local cellular processes but do not appear to serve as the elementary events responsible for global Ca2+ elevation and contraction (28). However, localized Ca2+ events can contribute, or even sum, to produce contraction (42). Our results suggest that in both CCE and ACh-evoked colonic SMC responses, regional Ca2+ hotspots exist and may serve as a foundation for global Ca2+ elevations or store refilling.

Clustering of channels into the specific subcellular domains needed for localized responses has been described for Ca2+ (30) and transient receptor potential (TRP) channels (1, 21). TRP proteins are hypothesized to be components of the SOCs responsible for CCE (1) and are expressed in colonic smooth muscle (38). TRP channels have been localized within caveolae and signaling microdomains (1), suggesting a possible relationship between CCE, Ca2+ hotspots, and TRP channels.

In summary, the present study reveals that CCE is present in human colonic SMCs and contributes to Ca2+ influx. We describe the temporal and spatial Ca2+i dynamics underlying CCE and cholinergic-evoked contractions and propose that store-operated Ca2+ entry occurs in localized Ca2+ hotspots. Given that Ca2+ hotspots are target-cell and contact-type specific (1, 30), it is tempting to speculate that in vivo, they could underlie the interactions between neighboring SMCs or between SMCs and interstitial cells of Cajal to aid propagation of smooth muscle contraction.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the Canadian Institutes of Health Research (CIHR; grant no. MOP10019). J. R. Kovac was supported by a CIHR M.D./Ph.D. Studentship.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. B. Taylor, C. Vinden, and C. Rajgopal for providing colectomy specimens and to Dr. S. Armstrong for help with image analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. M. Sims, Schulich School of Medicine and Dentistry, Dept. of Physiology and Pharmacology, Univ. of Western Ontario, London, ON N6A 5C1, Canada (E-mail: stephen.sims{at}schulich.uwo.ca)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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