Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the murine colon

Abstract
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Abstract

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca²⁺ signaling in ICC-SM and the relationship between ICC-SM Ca²⁺ transients and activation of smooth muscle cells (SMCs) using optogenetic tools. ICC-SM displayed rhythmic firing of Ca²⁺transients ~ 15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca²⁺ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca²⁺ entry through voltage-dependent Ca²⁺ conductances, L- and T-type Ca²⁺ channels. Removal of Ca²⁺ from the external solution abolished CTCs. Ca²⁺ release mechanisms reduced the duration and amplitude of Ca²⁺ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca^2+-firing patterns and drive smooth muscle activity and overall colonic contractions.

Introduction

Interstitial cells of Cajal (ICC) serve several important functions in the gastrointestinal tract, including generation of pacemaker activity (Langton et al., 1989; Ward et al., 1994; Huizinga et al., 1995), neurotransduction (Burns et al., 1996; Ward et al., 2000a; Sanders et al., 2010), and responses to stretch (Won et al., 2005). Electrical activity in ICC is transmitted to other cells in the tunica muscularis via gap junctions (Komuro, 2006). Smooth muscle cells (SMCs), ICC and another class of platelet-derived growth factor receptor alpha (PDGFRα)-positive interstitial cells are linked together in a coupled network known as the Smooth muscle cells, ICC and PDGFRα⁺ cells (SIP) syncytium (Sanders et al., 2012). The pacemaker function of ICC was deduced from morphological studies (Faussone Pellegrini et al., 1977; Thuneberg, 1982), dissection of pacemaker regions experiments (Smith et al., 1987a; Hara et al., 1986; Serio et al., 1991), studies on isolated ICC and SMCs (Langton et al., 1989; Sanders et al., 2014a; Zhu et al., 2009), studies of muscles from animals with loss-of-function mutations in c-Kit signaling (Ward et al., 1994; Ward et al., 1995; Huizinga et al., 1995) and simultaneous impalements of ICC and SMCs (Cousins et al., 2003). While these experiments were strongly indicative of the obligatory role of ICC as pacemakers in GI smooth muscles and essential role in normal electrical and contractile patterns, no experiments to date have measured pacemaker activity in ICC and responses of SMCs in terms of Ca²⁺ signaling and contraction simultaneously.

The anatomy and distribution of ICC varies from place to place throughout the GI tract. Some areas have only an intramuscular type of ICC (ICC-IM) that are closely aligned with and transduce inputs from excitatory and inhibitory enteric motor neurons (Burns et al., 1996; Beckett et al., 2004; Lies et al., 2014; Sanders et al., 2014b). Other regions contain ICC-IM and pacemaker types of ICC, that exist as a network in the myenteric plexus region of most areas of the gut (ICC-MY) (Rumessen and Thuneberg, 1982; Faussone-Pellegrini, 1992; Komuro et al., 1996; Burns et al., 1997; Komuro, 1999). The colon is more complex in that there are at least four types of ICC, distinguished by their anatomical locations and functions (Ishikawa and Komuro, 1996; Vanderwinden et al., 2000; Vanderwinden et al., 1996; Rumessen et al., 2013). One class of colonic ICC lies along the submucosal surface of the circular muscle (CM) layer (ICC-SM). These cells are known to provide pacemaker activity in colonic muscles, and their activity is integrated with a second frequency of pacemaker activity generated by ICC-MY (Smith et al., 1987a; Smith et al., 1987b; Durdle et al., 1983; Huizinga et al., 1988; Yoneda et al., 2002; Plujà et al., 2001). Pacemaker activities generated by ICC-SM and ICC-MY cause depolarization of SMCs, generation of Ca²⁺ action potentials, and excitation-contraction coupling (Yoneda et al., 2004).

ICC-SM generate slow waves in the canine colon (Smith et al., 1987a; Berezin et al., 1988). These are large amplitude and long-duration events that produce phasic contractions (Keef et al., 1992). The integrity of the ICC-SM network is required for regenerative propagation of slow waves, and disruption of the network causes passive decay of slow waves within a few millimeters (Sanders et al., 1990). Electrical coupling of ICC-SM in a network is an important feature allowing the pacemaker activity to coordinate the electrical activation of SMCs. ICC-SM in proximal colons of rodents also display pacemaker function; however, the frequency of the slow waves is higher (10–22 min⁻¹, mean 14.8. min⁻¹) (Yoneda et al., 2002). Slow waves, in this region of the GI tract, consist of a rapid upstroke phase, 148 mVs⁻¹, that settles to a plateau phase lasting approximately 2 s. The slow waves are coupled to low-amplitude CM contractions (Yoneda et al., 2004). Colonic slow waves have been reported to depend upon both Ca²⁺ entry and intracellular Ca²⁺ release mechanisms; however, Ca²⁺ signaling in colonic pacemaker cells and the coupling of Ca²⁺ events to the electrical responses were not clarified.

Previous studies have shown that all classes of ICC in the GI tract express Ca²⁺-activated Cl^- channels encoded by Ano1 (Gomez-Pinilla et al., 2009; Hwang et al., 2009). This conductance is required for slow wave activity (Hwang et al., 2009), and therefore Ca²⁺ dynamics in ICC are of fundamental importance in understanding pacemaker activity and electrical and mechanical rhythmicity in GI muscles. In the present study, we tested the hypothesis that Ca²⁺ transients in ICC-SM are linked to mechanical activation of the CM and that propagation of activity in ICC-SM is related to and controlled by Ca²⁺ entry via voltage-dependent Ca²⁺ conductances. Experiments were performed on tissues containing ICC-SM taken from mice with cell-specific expression of GCaMP6f in ICC, and changes in intracellular Ca²⁺ were monitored by confocal microscopy and digital video imaging.

Results

ICC-SM distribution within the submucosal plexus

We optimized a preparation in which the submucosal layer was separated from the tunica muscularis. We found that ICC-SM were adherent to the submucosal tissues, so this preparation allowed very clear high resolution of Ca²⁺ transients in ICC-SM in the complete absence of motion artifacts due to muscle contractions. We confirmed the presence and maintenance of ICC-SM networks, which occur in intact muscles, in these preparations.

Kit immunoreactivity revealed a dense network of Kit-positive cells in the submucosal layer of the proximal colon (Figure 1A). The network consisted of ICC-SM interconnected with branching processes (Figure 1B). The average density of cell bodies was 312 ± 33 cells mm^–2 (n = 6), and the average minimum separation between cell bodies was 49.3 ± 2.8 μm (Figure 1A&B; n = 6). Most of the ICC-SM network appeared to be adherent to the submucosal layer of the proximal colon. Isolation of the submucosa by sharp dissection showed that few ICC-SM (1.6 ± 2.1 cells mm^–2; Figure 1C; n = 6) remained adherent to the muscularis in the uppermost region (1–2 μm) of the proximal colon.

Figure 1

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Distribution of Kit⁺ submucosal interstitial cells of Cajal (ICC-SM) in the murine colon.

(A and B) Images of Kit⁺ (ICC-SM) in isolated submucosal layer of wild-type animals at ×20 and×40 magnifications (n = 6). Scale bars are 100 μm and 40 μm respectively. (C) Absence of ICC-SM on the submucosal surface of the *tunica muscularis* of the proximal colon after removing the submucosa. ICC-SM networks are intact in preparations of submucosal tissues removed from the muscle. (D and E) ICC-SM were present in Kit^+/copGFP mice at ×20 and×60 magnifications (n = 6). Scale bars are 100 μm and 40 μm respectively. (F) Absence of Kit⁺ (ICC-SM) on the submucosal surface of the *tunica muscularis* after removing the submucosa from Kit^+/copGFP proximal colon muscles (n = 6). Scale bars are 40 μm in both C and F. All image parameters were analyzed using Image J software.

Colonic muscles from Kit^+/copGFP mice expressing the copGFP exclusively in ICC were also used to confirm the distribution of ICC-SM. copGFP-positive cells in the submucosal region were present at an average density of 284 ± 27 cells mm^–2 and the average minimum separation between cell bodies was 52.7 ± 2.9 μm (Figure 1D and E; n = 6). ICC-SM were not resolved at the surface of the muscle layer after removing the submucosal layer (Figure 1F; n = 6). Colonic muscles expressing GCaMP6f exclusively in ICC were used to monitor Ca²⁺ signaling in ICC-SM. GCaMP6f-positive cells in the submucosal region were present at an average density of 291 ± 36 cells mm^–2 (n = 10), and the average minimum separation between cell bodies was 50.6 ± 3.8 μm. Representative images of the GCaMP6f-expressing cells in ICC-SM are shown below in the Ca²⁺-imaging experiments (Figure 2A; Figure 3A and Figure 6A).

Figure 2

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Temporal sequence of Ca²⁺ transients firing in submucosal interstitial cells of Cajal (ICC-SM) and smooth muscle cells (SMCs).

Representative dual-color imaging of ICC-SM (green; A) and SMCs (red; B) Ca²⁺ transients were recorded simultaneously from proximal colonic muscles of *Kit-iCre-GCaMP6f/Acta2-RCaMP1.07* mice (see Materials and methods for details). **C and D** Spatiotemporal maps (STMaps) of Ca²⁺ signals in ICC-SM and SMCs during a recording period (gray boxes in **A and B** denote cell locations; see Video 1). The STMaps show coordinated firing of Ca²⁺ transients in both types of cells. Ca²⁺ transient traces are plotted in E (ICC-SM-green, SMCs-red). Tissue displacement was also monitored and plotted (black trace; E). The black dotted arrow depicts the sequence of activation firing of Ca²⁺ transients in ICC-SM, to activation of Ca²⁺ events in SMCs, and tissue displacement. (F) A comparison of latencies (ms) from the start of the initiation of Ca²⁺ transients in ICC-SM to SMC activation and tissue displacement (n = 8). (G) Correlation analysis of latencies between ICC-SM and SMC Ca²⁺ transients and tissue displacement (R² = 0.82). All data graphed as mean ± SEM.

Figure 3

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Propagating Ca²⁺ waves in submucosal interstitial cells of Cajal (ICC-SM) network.

(A) An image of ICC-SM network from the proximal colon of a Kit-iCre-GCaMP6f mouse visualized at 10x (low resolution) magnification. Scale bar is 100 μm in A and pertains to all images in **B-F**. **A-F** Representative montage of the propagation of a Ca²⁺ wave throughout the ICC-SM network. The yellow arrow in panel C indicates the direction of Ca²⁺ transient propagation. (G) Spatiotemporal map (STMap) of Ca²⁺ signal in single ICC from the movie in panel A showing rhythmic firing of Ca²⁺ waves. Ca²⁺ activity is color coded with warm areas (red, orange) representing bright areas of Ca²⁺ fluorescence and cold colors (blue, black) representing dim areas of Ca²⁺ fluorescence. Summary data of multiple ICC-SM Ca²⁺-firing parameters (n = 15): frequency H, duration I, intensity J, spatial spread K and area of Ca²⁺ transients L. All data graphed as mean ± SEM.

Pacemaker activity of ICC-SM drives responses in SMCs

We developed a new mouse strain (Kit-iCre-GCaMP6f/Acta2-RCaMP1.07) that allowed simultaneous optical measurements of Ca²⁺ transients in ICC-SM and SMCs and tissue displacement (i.e. an optical means of monitoring muscle contractions). The dual-color mouse allowed simultaneous imaging of the two optogenetic Ca²⁺ sensors with different fluorescence characteristics (ensuring minimal spectral overlap). The sensors were expressed in a cell-specific manner in ICC and SMCs to characterize the coordination of signaling between ICC and SMCs (Figure 2 and Video 1). Ca²⁺ imaging of colonic muscles with attached submucosa showed a strong correlation between Ca²⁺ transients in the ICC-SM network and SMCs (Figure 2A–E; n = 8). Ca²⁺ transients recordings were obtained from ICC-SM and SMC at the exact coordinates in the pixel matched images (indicated by the gray box; Figure 2A&B) The sequence of activation began in ICC-SM and spread to SMCs with a latency of 56 ± 14 ms (Figure 2E&F; n = 8). Muscle displacement, an indicator of muscle contraction, was also measured and displayed latencies of 120 ± 17 ms between the rise of Ca²⁺ in SMCs and resolvable displacement (Figure 2E&F; n = 8) and 180 ± 15 ms between activation of Ca²⁺ transients in ICC-SM and contractile displacement (Figure 2E&F; n = 8). In a few instances, the time delay between ICC-SM and SMCs was not readily resolved. This could have resulted from a shift in the site of dominant pacemaker activity to a region outside the field of view (FOV). There was close correlation between the temporal latencies of ICC-SM and SMCs to tissue displacement (Figure 2G; R² = 0.82), indicating the pacemaker nature of ICC-SM, the coupling between ICC-SM and SMCs and the management of Ca²⁺ signaling and contractions in SMCs by the pacemaker activity of ICC-SM.

Video 1

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posterframe for video — Simultaneous dual-color imaging of submucosal interstitial cells of Cajal (ICC-SM) and smooth muscle cells (SMCs) in the colon.

A video of propagating Ca²⁺ waves through an ICC-SM network in the proximal colon of *Kit-iCre-GCaMP6f/Acta2-RCaMP1.07* strain imaged with a ×20 objective. simultaneous imaging of two optogenetic Ca²⁺ sensors: *GCaMP6f* in ICC-SM (left field of view [FOV]; green) and *RCaMP1.07* in SMCs (right FOV; red) with different fluorescence characteristics (ensuring minimal spectral overlap). The signal coordination between ICC and SMCs showing the correlation between Ca²⁺ transients in the ICC-SM network and activation of SMCs adjacent to ICC-SM. ICC-SM transients (green trace) preceded Ca²⁺ signals in SMCs (red trace). The scale bar (yellow) is 25 µm.

Global Ca²⁺-firing patterns in ICC-SM

Movement generated by muscle contractions is always an issue when imaging cells in muscle preparations in situ. Therefore, we used preparations of ICC-SM adherent to submucosa that were separated from muscle strips in most experiments. At low-magnification (×10), rhythmic Ca²⁺ waves occurred and spread through ICC-SM networks in isolated submucosal preparations. The Ca²⁺ waves occurred at 8–22 cycle min⁻¹ (Figure 3A–G) and averaged 14.9 ± 1.9 cycle min⁻¹ (Figure 3H; n = 15; c = 120). Similar behavior occurred at similar frequencies (i.e. 16.2 ± 1.4 cycle min⁻¹; n = 6) in ICC-SM attached to muscle tissues. Ca²⁺ waves propagated through the isolated ICC-SM networks with velocities of 219 ± 19 mm/s. Ca²⁺ transients imaged at ×10 appeared to be global and had average durations of 2.1 ± 0.4 s (Figure 3I; n = 15; c = 120) and amplitudes of 1.2 ± 0.3 ΔF/F₀ (Figure 3J; n = 15; c = 120). The spatial spread of Ca²⁺ transients was 36.8 ± 0.4 µm (Figure 3K; n = 15; c = 120). The average area occupied by Ca²⁺ transients in the spatiotemporal maps was 51.6 ± 1.6 µm*s (Figure 3L; n = 15; c = 120).

Cell-to-cell propagation of Ca²⁺ transients in ICC‐SM is shown in Figure 3 and Figure 4. Firing of global Ca²⁺ transients appeared to be sequential in nature (Figure 4A–G; n = 10; c = 60), as there was a strong correlation between the occurrence of global Ca²⁺ transients in multiple ICC-SM cells (Figure 4B; n = 10; c = 60). Overlays of STMaps of Ca²⁺ activity in adjacent cells running parallel to each other (Figure 4F&H) showed that the Ca²⁺ transients overlapped (65.4% of total Ca²⁺transients overlapped in the FOV during a 30-s recording period) (Figure 4G). Comparison between intervals of Ca²⁺ firing of multiple ICC-SM showed strong sequential firing as each cell demonstrated very close temporal firing intervals (Figure 4H&I; n = 6; c = 18). These results suggest that Ca²⁺ firing in ICC‐SM networks are entrained and propagate cell‐to‐cell.

Figure 4

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Submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ signaling activity is entrained.

(A) Raw image of multiple colonic ICC-SM in a field of view (FOV). Ten cells were color coded, and their Ca²⁺ fluorescence activity traces were plotted in B. Three ICC-SM (coded as red, green and blue regions of interest; ROIs in A) were selected and STMaps of the average Ca²⁺ fluorescence intensity across the diameter of the cell during a 30-s recording were constructed **C–E**. STMaps from each cell were color coded to correspond to the red, green, and blue cells and merged into a summed STMap in F. Percentage of fluorescence area overlap of intracellular Ca²⁺transients between ICC-SM cells is plotted in G and cell location is identified in H (n = 10). The durations of Ca²⁺ waves were such that there was a significant overlap of the Ca²⁺ events in individual cells across the FOVs at this magnification. Thus, each cell within the FOVs demonstrated similar temporal firing intervals (calculated as peak to peak intervals, n = 6) as shown in I.

ICC-SM Ca²⁺ signals composed of multiple Ca²⁺-firing sites

Low-power imaging suggested global changes in cell Ca²⁺, however, imaging of ICC-SM at higher power (×60–×100) allowed a more detailed visualization of the subcellular nature of the Ca²⁺ transients and revealed complex firing patterns. Imaging of ICC-SM networks with high spatial resolution showed that subcellular Ca²⁺ transients originated from distinct firing sites (Figure 5; n = 25). STMaps constructed from Ca²⁺ transients in single ICC-SM during 30 s of imaging (Figure 5A&B) identified the positions of all frequent firing sites within ICC-SM. Activity plots of individual firing sites showed they can fire once or multiple times during single Ca²⁺ waves (Figure 5B&C). The number of firing sites in a single ICC-SM ranged from 5 to 12 sites with an overall average of 8.2 ± 2 sites/cell (Figure 5D; n = 25).

Figure 5

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Ca²⁺ transients in submucosal interstitial cells of Cajal (ICC-SM) arise from multiple firing sites.

(A) Spatiotemporal map (STMap) of Ca²⁺-firing sites from a single ICC-SM during four consecutive firing cycles. Ca²⁺ activity is color coded with warm areas (red, orange) representing bright areas of Ca²⁺ fluorescence and cold colors (blue, black) representing dim areas of Ca²⁺ fluorescence. Nine distinct firing sites were detected in this cell and are marked as color squares on the right of the STMap. (B) Traces of the Ca²⁺ transients at each of the nine Ca²⁺-firing sites shown on the STMap in panel A. (C) 3-D surface plots showing the Ca²⁺ activity at the Ca²⁺-firing sites shown on the STMap in panel A over four consecutive Ca²⁺-firing cycles. (D) Average number of the firing sites per cell (n = 25). Data graphed as mean ± SEM.

We employed Ca²⁺ particle analysis (Drumm et al., 2017) to identify and quantify Ca²⁺-firing sites in ICC-SM. Ca²⁺-firing sites were distinguished based on their spatial and temporal characteristics (Figure 6A–D and Video 2). The firing sites were color coded, as shown in the example in Figure 6D, to visualize and quantify all active sites firing Ca²⁺ transients. The activities of firing sites were plotted as occurrence maps (Figure 6E). Occurrence maps of all firing sites in a region of ICC-SM network demonstrated that the global Ca²⁺ waves resolved at low power resulted from summation of localized Ca²⁺ transients from a multitude of firing sites (Figure 6D–J). Ca²⁺ transients were clustered temporally as Ca²⁺ waves swept through ICC-SM networks (Figure 6E&J). Also apparent from the occurrence maps was that the firing sequence of sites changed from Ca²⁺ wave to Ca²⁺ wave. Not all firing sites discharged Ca²⁺ transients during each wave cycle, and some sites fired more than once (Figure 6E&J). From particle analysis the average particle area/frame of Ca²⁺ transients averaged 3.2 ± 0.4 µm² (Figure 6H; n = 25) and particle count/frame averaged 0.27 ± 0.1 (Figure 6I; n = 25).

Figure 6

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Submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transient initiation sites.

(A) Representative image of an ICC-SM network from proximal colon of a Kit-iCre-GCaMP6f mouse at ×60 magnification. (B) Heat map of total Ca²⁺ PTCLs generated from the video shown in A (see Video 2). (C) Particles were thresholded temporally to generate a heat map indicating Ca²⁺-firing sites in the ICC-SM. (D) Image showing individually color-coded Ca²⁺-firing sites in the field of view (FOV) shown in C. (E) The temporal characteristics of each individual, color-coded firing site is displayed as an occurrence map, with each ‘lane’ representing the occurrence of firing PTCLs within each firing site. Activity traces for PTCLs for the duration of recording from the entire FOV are shown in **F and G**. Traces for PTCL area (F; red) and PTCL count (G; blue) are shown. **H and I** Summary graphs show average PTCL areas and counts for Ca²⁺-firing sites in ICC-SM (n = 25). (J) One Ca²⁺ wave in ICC-SM (white trace) is expanded and the numerous Ca²⁺ initiation sites that fired during this wave are superimposed. (K) Distribution plot showing average percentages of firing sites during a Ca²⁺ wave. Values are calculated for 1 s and plotted in 85 ms bins (n = 25). Data graphed as mean ± SEM.

Video 2

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Particle analysis also showed that Ca²⁺-firing sites were most active during the first ~256 ms of a Ca²⁺ wave, and activity decayed with time. This point was further illustrated by distribution plots showing the average percentage of firing sites discharging at various times during Ca²⁺ waves (Figure 6K; n = 25). Initially high firing and decay as a function of time suggest that Ca²⁺ entry mechanisms may be important for: (i) initiating Ca²⁺ transients and (ii) organizing the occurrence of Ca²⁺ transients into clusters. It is also possible that Ca²⁺ stores, loading during the diastolic period between Ca²⁺ waves, are more excitable at the onset of each Ca²⁺ wave.

Ca²⁺ influx mechanisms are required for initiation of clustered Ca²⁺ transients in ICC-SM

The effects of reducing extracellular Ca²⁺ ([Ca²⁺]_o) on Ca²⁺ transients was investigated to evaluate the importance of Ca²⁺ influx for the patterning of Ca²⁺ signaling in ICC-SM. Removal of [Ca²⁺]_o abolished Ca²⁺ transients in ICC-SM within 10 min (Figure 7E–L; n = 6). Stepwise reduction in [Ca²⁺]_o (from 2.5 mM to 0 mM) showed that Ca²⁺ transients decreased in a concentration-dependent manner (Figure 7; n = 6). ICC-SM Ca²⁺ PTCL area and count of firing sites were reduced in response to lowering [Ca²⁺] _o (Figure 7F–J; n = 6). Reducing [Ca²⁺]_o from 2.5 mM (control conditions) to 2 mM caused a reduction in the average PTCL area by 19.6 ± 4.5% (Figure 7K; n = 6) and PTCL count by 25.4 ± 2.9% (Figure 7L; n = 6) with no significant change in the duration of PTCLs (Figure 7M; n = 7). Further reduction of [Ca²⁺] _o to 1 mM reduced Ca²⁺ transient parameters by 47.2 ± 3.5% PTCL area (Figure 7K; n = 6) and 45.1 ± 4.4% PTCL count (Figure 7L; n = 6) and showed a significant change in the duration of PTCLs (Figure 7M; n = 7). Lowering [Ca²⁺] _o to 0.5 mM also reduced Ca²⁺ PTCL area by 73.9 ± 3% (Figure 7K; n = 6) and PTCL count by 73.2 ± 2% (Figure 7L; n = 6) with no significant change in the duration of PTCLs (Figure 7M; n = 7). Removal of Ca²⁺ from the extracellular solution (Ca²⁺‐free KRB solution containing 0.5 mM EGTA) abolished Ca²⁺ signals 8–10 min after solution replacement. The Ca²⁺ transient PTCL area was reduced by 99.4 ± 0.6% (Figure 7K; n = 6) and PTCL count by 99.3 ± 0.7% (Figure 7L; n = 6) with no significant change in the duration of PTCLs (Figure 7M; n = 7). We also noted that the highly organized CTCs occurring in the presence of 2.5 mM [Ca²⁺] _o became less organized as [Ca²⁺] _o was reduced (e.g. compare the tight clusters in Figure 7A with the more diffuse clusters in Figure 7C). These experiments highlight the importance of Ca²⁺ entry mechanisms in Ca²⁺ signaling within ICC-SM.

Figure 7

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The effect of lowering [Ca²⁺]_o on Ca²⁺ transients in submucosal interstitial cells of Cajal (ICC-SM).

(A) ICC-SM Ca²⁺ transients and Ca²⁺-firing sites were color coded and plotted as an occurrence map under control conditions with [Ca²⁺]_o = 2.5 mM. (**B–E**) showing the effects of reducing [Ca²⁺]_o to 2 mM B; 1 mM C; 0.5 mM D and after Ca²⁺ removal of [Ca²⁺]_o (0 mM added and final solution buffered with 0.5 mM EGTA) E. **F-J** Traces of Ca²⁺ PTCL activity in ICC-SM (PTCL area, blue and PTCL count, green) under control conditions F and after reducing [Ca²⁺]_o to 2 mM G; 1 mM H; 0.5 mM I and after removal of [Ca²⁺]_o as shown in J. Summary graphs of Ca²⁺ PTCLs in ICC-SM under control conditions and with reduced [Ca²⁺]_o in K (PTCL area); L (PTCL count) and M (PTCL duration). Data were normalized to controls and expressed as percentages (%). Significance was determined using one-way ANOVA, * = p<0.1, ** = p<0.01, **** = p<0.0001, n = 6. All data graphed as mean ± SEM.

Molecular expression of Ca²⁺entry channels in ICC-SM

The apparent dependence on the Ca²⁺ gradient to maintain pacemaker function in ICC-SM suggests that Ca²⁺ entry mechanisms are critical for initiation and organization of CTCs. Therefore, we examined expression of several Ca²⁺ channels that might be responsible for Ca²⁺ entry in ICC-SM (Figure 8). After isolation of the submucosal layer from the proximal colon of Kit^+/copGFP mice and subsequent cell dispersion, we sorted copGFP-positive ICC-SM with fluorescence activated cell-sorting (FACS) and evaluated the expression of voltage-dependent and voltage-independent Ca²⁺ channels by qPCR (Figure 8A&B). First, we confirmed the purity of sorted ICC-SM with cell-specific markers (Figure 8A). Kit receptors and ANO1 channels are signatures of ICC throughout the GI tract and enrichment of Kit and Ano1 expression was observed in sorted ICC-SM compared to unsorted cells (Kit expression was 0.21 ± 0.014; Ano1 expression was 0.14 ± 0.02 relative to Gapdh). The expression levels of Myh11 an SMC marker and Uch11 a pan neuronal marker encoding PGP9.5 were minimal (Myh11 expression was 0.03 ± 0.002; Uch11 expression was 0.002 ± 0.0001 relative to Gapdh), confirming the purity of ICC-SM sorted by FACS.

Figure 8

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Molecular expression of genes related to Ca²⁺ entry channels.

(A) Relative expression of cellular-specific biomarker genes in submucosal interstitial cells of Cajal (ICC-SM; sorted to purity by FACS) and compared with unsorted cells dispersed from submucosal tissues obtained from Kit^+/copGFP mice. Relative expression was determined by qPCR and normalized to *Gapdh* expression. Genes examined were *Kit* (tyrosine kinase receptor, found in ICC), *Ano1* (Ca²⁺-activated Cl^- channel), *Uch11* (neural marker encoding PGP 9.5), *Myh11* (smooth muscle myosin). (B) Relative expression of major Ca²⁺ entry channels considered most likely to be expressed in colonic ICC from RNAseq of total ICC in murine colon (Lee et al., 2017). L-Type Ca²⁺ channels (*Cacna1c* and *Cacna1d*) T-type Ca²⁺ channels (*Cacna1g* and *Cacna1h*) and Store‐operated Ca²⁺ entry (SOCE) channels (*Orai1*, *Orai2* and *Orai3*) were evaluated. All data graphed as mean ± SEM (n = 4).

ICC-SM expressed L-type voltage-dependent Ca²⁺ channels encoded by Cacna1c and Cacna1d abundantly (Ca_V 1.2 and Ca_V 1.3 channels, respectively). Cacna1c showed a 0.012 ± 0.0005 and Cacna1d showed 0.066 ± 0.008 relative to Gapdh (Figure 8B; n = 4). ICC-SM also expressed Cacna1h (Ca_V 3.2) and to a lesser extent Cacna1g (Ca_V 3.1), both T-type voltage-dependent Ca²⁺ channels (Figure 8B; n = 4). Cacna1h expression was abundant in ICC-SM (0.07 ± 0.003 relative to Gapdh). Cacna1g expression was less than Cacna1h 0.0014 ± 0.0001 relative to Gapdh (Figure 8B; n = 4).

Maintenance and refilling of cellular Ca²⁺ stores may also be important for mediation and shaping of Ca²⁺ signals in ICC-SM. Store‐operated Ca²⁺ entry (SOCE) has been established as a mechanism for filling stores upon depletion (Putney, 2018; Gibson et al., 1998). Contributions from SOCE via STIM and Orai interactions are essential for maintenance of Ca²⁺ stores in other types of ICC (Zheng et al., 2018; Youm et al., 2019; Drumm et al., 2020a; Drumm et al., 2020b). Colonic ICC-SM showed enrichment in Orai1 and Orai2 but Orai3 was not resolved (Figure 8B; n = 4). Orai1 expression was 0.02 ± 0.001 relative to Gapdh and Orai2 was 0.074 ± 0.008 relative to Gapdh (Figure 8B; n = 4).

L-type Ca²⁺ channels are important for organization of Ca²⁺ transients in ICC-SM

L-type Ca²⁺ channels (Ca_V 1.3, Ca_V 1.2) were expressed in ICC-SM, and Ca²⁺ transients showed dependence on the Ca²⁺ gradient. Previous studies have reported that L-type channel antagonists inhibit slow waves in the colon (Yoneda et al., 2002; Keef et al., 2002). Therefore, we evaluated the contributions of Ca²⁺ entry via L-type channels to Ca²⁺ transients in ICC-SM. Nicardipine (1 μM) abolished Ca²⁺ transients in ICC-SM (Figure 9A&B; n = 8). Firing site occurrence maps (Figure 9C&D; n = 8) describe the inhibitory effects of nicardipine on Ca²⁺ transients. Ca²⁺ PTCL area was reduced to 10.5 ± 4.7% (Figure 9E–G; n = 8) and PTCL count was reduced to 12.3 ± 4.8% (Figure 9H; n = 8). The number of firing sites also decreased to 8.4 ± 3% in the presence of nicardipine (Figure 9I; n = 8). Isradipine inhibits Ca_V 1.2 and Ca_V 1.3 equally (Rüegg and Hof, 1990; Koschak et al., 2001). Isradipine (1 μM) also reduced Ca²⁺ transients in ICC-SM (Figure 9—figure supplement 1A&B; n = 7). ICC-SM Ca²⁺-firing was reduced in the presence of isradipine as shown in the firing sites occurrence maps (Figure 9—figure supplement 1C&D). PTCL area was reduced to 18 ± 5% (Figure 9—figure supplement 1E–G; n = 7), and PTCL count was reduced to 19.5 ± 6% (Figure 9—figure supplement 1H; n = 7). The number of firing sites was inhibited by isradipine to 21.5 ± 6% (Figure 9—figure supplement 1I; n = 7). These data show that ICC-SM Ca²⁺ transients depend upon Ca²⁺ influx via L-type Ca²⁺ channels, and since isradipine had nearly the same or a lesser effect on Ca²⁺ transients than nicardipine, Ca_V 1.2 appear dominant as the Ca²⁺ entry mechanism.

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L-type Ca²⁺ channel antagonist, nicardipine effects on submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transients.

**A and B** Representative heat-map images of an ICC-SM network from the proximal colon of a Kit-iCre-GCaMP6f mouse showing active Ca²⁺ PTCLs under control conditions and in the presence of nicardipine (1 μM). Ca²⁺ activity is color coded with warm areas (white, red) representing bright areas of Ca²⁺ fluorescence and cold colors (purple, black) representing dim areas of Ca²⁺ fluorescence. Scale bar is 10 μm in both **A and B**. **C and D** Firing sites showing Ca²⁺ activity in ICC-SM. Firing sites were color coded and plotted as an occurrence map under control conditions and in the presence of nicardipine (1 μM). Traces of firing sites showing PTCL area (E; blue) and PTCL count (E; green) under control conditions and in the presence of nicardipine; PTCL area (F; blue) and PTCL count (F; green) show the inhibitory effects of nicardipine on Ca²⁺ transients in ICC-SM. Summary graphs of Ca²⁺ PTCL activity in ICC-SM before and in the presence of nicardipine are shown in G (PTCL area/frame), (H) (PTCL count/frame), (I) the number of PTCL active sites and J (PTCL duration). Data were normalized to controls and expressed as percentages (%). Significance determined using unpaired t-test, ** = p<0.01, **** = p<0.0001, n = 8. All data graphed as mean ± SEM.

T-type Ca²⁺ channels contribute to Ca²⁺ transients in ICC-SM

T-type Ca²⁺ channels have been identified as the dominant voltage-dependent Ca²⁺ conductance responsible for Ca²⁺ entry and propagation of slow waves in ICC-MY of the small intestine (Kito et al., 2005; Zheng et al., 2014; ). T-type channel antagonists (Ni²⁺ and mibefradil) reduced the rate-of-rise of the upstroke component of the slow waves and higher concentrations attenuated slow wave activity (Kito et al., 2005; Hotta et al., 2007; Yoneda et al., 2003). As reported above, ICC-SM express T-type channels Cacna1h and Cacna1g. Therefore, the role of T-type Ca²⁺ channels in modulating Ca²⁺signaling in ICC-SM was evaluated with specific T-type channel antagonists, NNC 55–0396 (10 μM), TTA-A2 (10 μM) and Z-944 (1 μM). NNC 55–0396 reduced Ca²⁺ transient firing (Figure 10A&B), Ca²⁺ transients firing sites occurrence maps (Figure 10C&D) and firing sites. PTCL area and PTCL count traces show a reduction in Ca²⁺ transient firing (Figure 10E&F). PTCL area was reduced to 31.7 ± 3.3% (Figure 10E–G; n = 9) and PTCL count was reduced to 35.6 ± 4.5% (Figure 10H; n = 9). The number of firing sites was reduced by NNC 55–0396 to 37.6 ± 4.3% (Figure 10I; n = 9). The duration of PTCLs was also reduced by NNC 55–0396 to 73 ± 5.2% (Figure 10J; n = 9). TTA-A2 showed similar inhibitory effects on ICC-SM Ca²⁺ transients (Figure 10—figure supplement 1). PTCL area was reduced to 42 ± 5.8% (Figure 10—figure supplement 1E; n = 7) and PTCL count was reduced to 44.4 ± 6.2% (Figure 10—figure supplement 1F; n = 7) The number of firing sites was also reduced by TTA-A2 to 43.4 ± 6% (Figure 10—figure supplement 1G; n = 7). Z-944 significantly reduced ICC-SM Ca²⁺ transients but was somewhat less effective than NNC 55–0396 or TTA-A2. PTCL area was reduced to 56 ± 10% (Figure 10—figure supplement 1E; n = 5), and PTCL count was reduced to 53 ± 12% (Figure 10—figure supplement 1F; n = 5) The number of firing sites was also reduced by Z-944 to 53.4 ± 11% (Figure 10—figure supplement 1G; n = 5). The data suggest that T-type Ca²⁺ channels also contribute to the initiation and organization of Ca²⁺ transients in ICC-SM.

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T-type Ca²⁺ channel antagonist, NNC 55–0396 effects on ICC-SM Ca²⁺ transients.

**A and B** Representative heat-map images of Ca²⁺ transient particles in ICC-SM under control conditions A and in the presence of NNC 55–0396 (10 μM) (B). Active firing sites were color coded and plotted as an occurrence maps in the ICC-SM network under control C and NNC 55–0396 D conditions. Plots of Ca²⁺ transient particle activity of ICC-SM in control conditions and in the presence of NNC 55–0396 showing PTCL area (blue) and PTCL count (green) under control conditions E and in the presence of NNC 55–0396 F. Summary graphs of average percentage changes in PTCL area G, PTCL count H, the number of PTCL active sites I and PTCL duration J. Data were normalized to controls and expressed as percentages (%). Significance determined using unpaired t-test, **** = p<0.0001, n = 8. All data graphed as mean ± SEM.

Effects of membrane hyperpolarization on Ca²⁺ transients in ICC-SM

Experiments described above showed that Ca²⁺ transients in ICC-SM depend on voltage-dependent Ca²⁺ influx mechanisms (Figures 7–10). The role and contributions of the voltage-dependent channels expressed in ICC-SM (Ca_V 1.3 and Ca_V 1.2 and Ca_V 3.2) was further examined under conditions of membrane hyperpolarization induced by activation of K_ATP channels. K_ATP is functional in colonic SMCs but not in ICC (Huang et al., 2018). Therefore, these experiments were performed on preparations in which ICC-SM remained attached to the muscularis. Pinacidil produces hyperpolarization of SMCs via activation of K_ATP channels (Koh et al., 1998) and the hyperpolarization is expected to conduct to ICC, as they are electrically coupled. Pinacidil (10 μM), a selective K_ATP channel agonist, hyperpolarizes murine colonic muscles by ~10 mV (Kito et al., 2005; Koh et al., 1998; Kito and Suzuki, 2003). Pinacidil had no significant change to Ca²⁺ transient firing, except to reduce the duration of CTCs (Figure 11—figure supplement 1A–L). Ca²⁺ PTCL area was increased to 130.3 ± 24.3% (Figure 11—figure supplement 1G; p value = 0.24; n = 6) and PTCL count was increased to 121.8 ± 17.8% (Figure 11—figure supplement 1H; p value = 0.25; n = 6). The number of firing sites was not affected by pinacidil (Figure 11—figure supplement 1I; p value = 0.22; n = 6). The effects of pinacidil on global Ca²⁺ transients were evaluated. Pinacidil significantly reduced the duration of global Ca²⁺ transients from 1.78 ± 0.2 s to 0.89 ± 0.1 s (Figure 11—figure supplement 1J&K; n = 6). Reduction in the duration of Ca²⁺ transients was associated with a tendency for an increase in frequency, but this parameter did not reach significance. Ca²⁺ oscillation frequency under control conditions was 15.1 ± 1.1 cpm and in the presence of pinacidil was 16.5 ± 1.2 cpm (Figure 11—figure supplement 1J&L; p value = 0.45; n = 6).

The effects of nicardipine were tested in the presence of pinacidil. Under these conditions, nicardipine significantly reduced Ca²⁺ transients in ICC-SM (Figure 11G&H; n = 5). PTCL area was reduced to 38.5 ± 7.6% (Figure 11G; n = 5) and PTCL count was reduced to 42.3 ± 9.1% (Figure 11H; n = 5). In some regards, these results were surprising as membrane potential hyperpolarization might reduce contributions from L-type Ca²⁺ channels (Ca_V 1.2). One explanation is that Ca_V 1.3, which are abundant in ICC-SM and activate at more negative potentials than Ca_V 1.2 (Xu and Lipscombe, 2001) may contribute to Ca²⁺ entry at more hyperpolarized potentials. Further addition of NNC 55–0396 (10 μM) inhibited Ca²⁺ transients in ICC-SM to a greater extent. PTCL area was reduced to 9.0 ± 2% (Figure 11G; n = 5), and PTCL count was reduced to 11.2 ± 1.9% (Figure 11H; n = 5). The utilization of two Ca²⁺ conductances with different ranges of voltage-dependent activation for the initiation of CTCs provides a safety factor that insures persistence of pacemaker activity over a broad range of membrane potentials.

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The effects of membrane hyperpolarization and voltage-dependent Ca²⁺ entry block on submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transients.

(A) Ca²⁺-firing sites in ICC-SM are color coded and plotted in an occurrence map in the presence of pinacidil (10 μM). (B) Shows the effects of nicardipine (1 μM) in the continued presence of pinacidil. (C) shows effects of combining nicardipine and NNC 55–0396 (10 μM) in the continued presence of pinacidil. Traces of firing sites PTCL area (blue) and PTCL count (green) under each condition are shown in D pinacidil E pinacidil and nicardipine and F combination of pinacidil, nicardipine, and NNC 55–0396. Summary graphs of Ca²⁺ PTCL activity in ICC-SM in the presence of voltage-dependent Ca²⁺ channel antagonists (nicardipine and NNC 55–0396) are shown in G (PTCL area) and H (PTCL count). (I) The number of Ca^2+-firing events were tabulated during 2 s intervals before the initiation of Ca²⁺ transient clusters in ICC-SM (period of tabulation indicated by the gray box) and summarized in J under control conditions, in pinacidil, in pinacidil and nicardipine and in a combination of pinacidil, nicardipine, and NNC 55–0396. Data were normalized to controls and expressed as percentages (%) in G and H. Significance determined using one-way-ANOVA, * = p<0.1, ** = p<0.01, *** = p<0.001 **** = p<0.0001, n = 5. All data graphed as mean ± SEM.

Inhibiting voltage-dependent Ca²⁺ channels in the presence of pinacidil unmasked underlying Ca²⁺ transients that occurred more randomly than the clustered transients occurring normally (Figure 11F&I). We tabulated the number of Ca²⁺ events in the intervals between CTCs (calculated from a period of 2 s before the onset of a CTC). Underlying Ca²⁺ events were more frequent in the presence of pinacidil and nicardipine and increased again upon addition of NNC 55–0396 (Figure 11J; n = 5).

Contributions of intracellular Ca²⁺ stores and release channels in ICC-SM Ca²⁺activity

Previous studies have demonstrated that Ca²⁺ signaling in ICC-MY in the small intestine depends not only on Ca²⁺ influx but also on Ca²⁺ release from intracellular stores (Drumm et al., 2017). Ca²⁺ release from stores is also critical for generation of pacemaker currents and slow waves (Ward et al., 2000b; Zhu et al., 2015; Bayguinov et al., 2007). The role of Ca²⁺ release mechanisms in Ca²⁺ signaling in ICC-SM was also evaluated. Thapsigargin (1 μM; A SERCA pump antagonist) reduced, but did not block, Ca²⁺ transient firing in ICC-SM (Figure 12A–D). PTCL area was reduced to 29 ± 12% (Figure 12E; n = 6) and PTCL count was reduced to 22 ± 10% (Figure 12F; n = 6). The number of firing sites was reduced by thapsigargin to 21 ± 11% (Figure 12G; n = 6). The duration of PTCLs was increased by thapsigargin to 126.7 ± 9% (Figure 12H; n = 6). Cyclopiazonic acid (CPA, 10 μM), another SERCA antagonist, also reduced Ca²⁺ transient firing (Figure 12I–L). PTCL area was reduced to 36.1 ± 8.3% (Figure 12M; n = 5) and PTCL count was reduced to 29.1 ± 6.3% (Figure 12N; n = 5). The number of firing sites was reduced by CPA to 56 ± 9.5% (Figure 12O; n = 5). There was no significant change in the duration of PTCLs in the presence of CPA (Figure 12P; n = 5).

Figure 12

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Intracellular Ca²⁺ stores contributions in submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transients firing.

(A) Ca²⁺ activity of firing sites in ICC-SM are color coded and plotted in occurrence maps under control conditions and in the presence of thapsigargin (1 μM) (B). Traces of firing sites PTCL area (C; blue) and PTCL count (C; green) under control conditions and in the presence of thapsigargin PTCL area (D; blue) and PTCL count (D; green). Scale bars in C applies to traces in D. Summary graphs of Ca²⁺ PTCL activity in ICC-SM in the presence of thapsigargin are shown in E (PTCL area), (F) (PTCL count), (G) the number of PTCL active sites and H (PTCL duration; n = 6). CPA (SERCA pump inhibitor) reduced transients compared to control as shown in occurrence maps of firing sites **I and J** and Ca²⁺ activity traces **K and L**. Summary graphs of Ca²⁺ PTCL activity in ICC-SM in the presence of CPA are shown in M (PTCL area), (N) (PTCL count), (O) the number of PTCL active sites and P (PTCL duration; n = 5). Data were normalized to controls and expressed as percentages (%). Significance determined using unpaired t-test, * = p<0.1, ** = p<0.01, *** = p<0.001 **** = p<0.0001. All data graphed as mean ± SEM.

ER release channels RyRs and IP₃Rs amplify and sustain Ca²⁺ signaling via direct localized release of Ca²⁺ or Ca²⁺-induced Ca²⁺ release (CICR) (Kaßmann et al., 2019; Vierra et al., 2019; Amberg et al., 2006; Takeda et al., 2011; Fill and Copello, 2002; Dupont and Goldbeter, 1994; Thomas et al., 1996; Berridge, 2009). Contributions from RyRs and IP₃Rs to Ca²⁺ transients in ICC-SM were therefore investigated. Ryanodine (100 μM) significantly reduced Ca²⁺ event firing in ICC-SM (Figure 13A–F). PTCL area was reduced to 76.1 ± 1.6% (Figure 13G; n = 4) and PTCL count was reduced to 80.9 ± 2.7% (Figure 13H; n = 4). The number of firing sites was also reduced by ryanodine to 75.7 ± 2.1% (Figure 13I; n = 4). There was no significant change in the duration of PTCLs in the presence of ryanodine (Figure 13J; n = 5). We also noted that the greatest effects of ryanodine on Ca²⁺ transients occurred after the first ~300 ms of CTCs (Figure 13K&L; n = 4). Thus, ryanodine shortens the durations of the CTCs. Ca²⁺ release mechanisms via RyRs contribute to the overall patterns of Ca²⁺ waves in ICC-SM, as shown by the distribution plots of average percentages of firing sites during a Ca²⁺ wave (Figure 13L; n = 4).

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Ryanodine receptors (RyRs) contributing to Ca²⁺ release in submucosal interstitial cells of Cajal (ICC-SM).

(A) Representative heat-map image of an ICC-SM network from proximal colon showing total active Ca²⁺ PTCLs under control conditions and in the presence of ryanodine (100 μM) (B). **C and D** Ca^2+-firing sites are color coded and plotted in occurrence maps showing the effect of the ryanodine (100 μM), on Ca²⁺ transient clusters (CTCs) in ICC-SM. Traces of firing sites PTCL area (E; blue) and PTCL count (E; green) under control conditions and in the presence of ryanodine, PTCL area (F; blue) and PTCL count (F; green). Summary graphs of Ca²⁺ PTCL activity in ICC-SM in the presence of ryanodine are shown in G (PTCL area), (H) (PTCL count), (I) the number of PTCL active sites and J (PTCL duration; n = 4). (K) Overlaid occurrence maps showing Ca²⁺ firing during control conditions (all firing sites are in green) and in the presence of ryanodine (all firing sites are in red). Note how ryanodine shortened the duration of the total Ca²⁺ transient cluster (CTC). (L) Distribution plot of average percentages of firing sites during a Ca²⁺ wave, calculated for 1 s duration and plotted in 85 ms bins showing that ryanodine mainly blocked Ca²⁺ transients occurring after the first 257 ms intervals (n = 4). Data were normalized to controls and expressed as percentages (%). Significance determined using unpaired t-test, *** = p<0.001. All data graphed as mean ± SEM.

Xestospongin C (10 μM; An IP₃R antagonist) also reduced Ca²⁺ events in ICC-SM (Figure 13—figure supplement 1A–I; n = 4). PTCL area was significantly reduced to 78 ± 5.8% (Figure 13—figure supplement 1G; n = 4) and although both PTCL count and number were reduced to 75.3 ± 10.5% and 74.8 ± 9.3%, respectively, these effects did not reach statistical significance (PTCL count: p value = 0.08 and PTCL number: p value = 0.06; Figure 13—figure supplement 1H&I; n = 4). Xestospongin C displayed inhibitory effects similar to ryanodine; most of the inhibition of Ca²⁺ events occurred after the first ~400 ms of CTCs (Figure 13—figure supplement 1J&K; n = 4). Thus, Ca²⁺ release via IP₃Rs also contributes to the overall pattern of Ca²⁺ waves in ICC-SM, as shown by the distribution plots of average percentages of firing sites during a Ca²⁺ wave (Figure 13—figure supplement 1K; n = 4).

2-APB (100 μM) and tetracaine were also tested (100 μM; Figure 13—figure supplement 2A&B; n = 5) as secondary tests of the contributions of IP₃Rs and RyRs in CTCs. 2-APB reduced Ca²⁺ PTCL area to 61.1 ± 14.5% (Figure 13—figure supplement 2 Ai; n = 5) and reduced PTCL count to 58 ± 13.2% (Figure 13—figure supplement 2 Aii; n = 5). The number of firing sites was also reduced by 2-APB to 63.7 ± 11.3% (Figure 13—figure supplement 2 Aiii; n = 5). Tetracaine reduced Ca²⁺ PTCL area to 85 ± 5.1% (Figure 13—figure supplement 2 Bi; n = 5) and reduced PTCL count to 69.5 ± 7.8% (Figure 13—figure supplement 2 Bii; n = 5). The number of firing sites was also reduced by tetracaine to 71.3 ± 5.9% (Figure 13—figure supplement 2 Biii; n = 5).

The effects of 2-APB could be non-specific and may include effects on store-operated Ca²⁺ entry channels (SOCE; e.g. by blocking Orai channels). Previous studies have shown SOCE to be important for maintenance of Ca²⁺ stores and sustaining Ca²⁺ release from the ER (Zheng et al., 2018; Lyfenko and Dirksen, 2008; Trebak et al., 2013; Chen and Sanderson, 2017; Putney, 2011; Prakriya and Lewis, 2015). ICC-SM express Orai channels (Orai1 and Orai2; Figure 8B), so the role of SOCE in maintenance of Ca²⁺ transients was examined using an Orai antagonist. GSK 7975A (10 μM; An Orai antagonist) reduced the firing frequency of CTCs (Figure 14A&B). Firing site occurrence (Figure 14C&D) and PTCL counts and areas were reduced (Figure 14E&F). Ca²⁺PTCL area was reduced to 42.4 ± 9.4% (Figure 14E–G; n = 7), and PTCL count was reduced to 48 ± 7% (Figure 14H; n = 7). The number of firing sites was also inhibited by GSK 7975A to 47.5 ± 4.1% (Figure 14I; n = 7). There was no significant change in the duration of PTCLs in the presence of GSK 7975A (Figure 14J; n = 7).

Figure 14

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SOCE role in maintaining submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transients.

(A) Representative Heat-map image of an ICC-SM network showing total active Ca²⁺ PTCLs under control conditions and in the presence of GSK-7975A (10 μM, for 20 min) B. **C and D** Ca²⁺-firing sites are color coded and plotted in occurrence maps showing the effect of the SOCE channel antagonist, GSK-7975A (100 μM), on ICC-SM Ca²⁺ transients. Traces of PTCL area (E; blue) and PTCL count (E; green) under control conditions and in the presence of GSK-7975A, PTCL area (F; blue) and PTCL count (F; green). Summary graphs of Ca²⁺ PTCL activity in ICC-SM in the presence of GSK-7975A are shown in G (PTCL area), (H) (PTCL count), (I) the number of PTCL active sites, and J (PTCL duration; n = 7). Significance determined using unpaired t-test, **** = p<0.0001. All data graphed as mean ± SEM.

Discussion

This study characterized Ca²⁺ transients responsible for the pacemaker function of ICC-SM that contributes to contractile patterns in colonic motility. The sequence of activation from ICC-SM to colonic SMCs was quantified using two optogenetic sensors, expressed specifically in ICC or SMCs. Correlation analysis demonstrated a 1:1 relationship between CTCs in ICC-SM and Ca²⁺ signaling and contractile responses in SMCs. The CTCs in ICC-SM consisted of ~2 s bursts of activity from multiple sites within cells. Organization of the Ca²⁺ transients into clusters was due to voltage-dependent Ca²⁺ entry that appeared to be due to activation of both high-voltage activated Ca²⁺ channels (L-type encoded by Cacna1c and Cacna1d in ICC-SM) and low-voltage activated Ca²⁺ channels (T-type encoded by Cacna1h and possibly Cacna1g in ICC-SM). A portion of the Ca²⁺ transients making up CTCs were due to Ca²⁺ entry, as the earliest Ca²⁺ transients resolved in a cluster were not blocked by thapsigargin, CPA or antagonists of ryanodine and IP₃ receptors. The earliest events were blocked by nicardipine, suggesting that L-type Ca²⁺ channels are the dominant Ca²⁺ entry pathway at basal resting potentials. Ca²⁺ transients in ICC-SM were not as sensitive to block by STIM and Orai, as in other ICC (Zheng et al., 2018; Drumm et al., 2019a), However, the Orai antagonist reduced the frequency of CTCs and may have blocked these events completely with longer treatment periods. Our data show that Ca²⁺ entry is fundamental in ICC-SM Ca²⁺ transients. In fact, localized Ca²⁺ influx via clusters of L-type Ca²⁺ channels may cause localized elevation in [Ca²⁺]_i and activation of ANO1 channels directly. Localized Ca²⁺ entry events through L-type Ca²⁺ channels have been termed Ca²⁺sparklets, and these events could be involved in Ca²⁺ influx (Santana et al., 2008; Sonkusare et al., 2012; Navedo and Amberg, 2013a) and initiation of pacemaker activity in ICC-SM. However, the decrease in the frequency of CTCs by manipulations to reduce stored Ca²⁺ also suggest an important role for Ca²⁺ release in pacemaker activity, perhaps resulting from coupling between Ca²⁺ entry and CICR (Navedo and Santana, 2013b).

In this study, we developed a new preparation in which ICC-SM adherent to the submucosa was used to allow very high-resolution imaging without complications from muscular contractions. In situ preparations of this type in which the natural structure and connectivity between ICC are maintained may be valuable for future studies of cellular mechanisms responsible for pacemaker activity and factors that regulate or degrade pacemaker activity in pathophysiological conditions.

The pacemaker function of ICC-SM was demonstrated in a novel manner by simultaneous two-color optogenetic imaging with green (GCaMP6f) and red (RCaMP1.07) Ca²⁺ sensors expressed in ICC and SMCs, respectively. Imaging in this manner revealed the sequence of activation in ICC-SM and SMCs, showing clearly the frequency, onset and duration of Ca²⁺ transients in ICC-SM, the spatial spread of Ca²⁺ transients in ICC-SM networks, the development of Ca²⁺ transients in SMCs and tissue displacement (i.e. an optical indicator of muscle contraction). Correlation analysis demonstrated the coherence of these events. Ca²⁺ transients, lasting for about 2 s, propagated without decrement through networks of ICC-SM and preceded and likely initiated Ca²⁺ signaling and contractions in SMC, as was also suggested by intracellular microelectrode recordings from cells along the innermost surface of canine colonic muscles (Sanders et al., 1990). ICC-SM are electrically coupled to each other and the network of ICC-SM is coupled to SMCs via gap junctions, providing a means of electrical communication. Ca²⁺ transients initiate depolarization of ICC-SM via activation of ANO1 channels, depolarizing currents conduct to SMCs, and depolarization of SMCs activates Ca²⁺ entry, by increasing the open probability of L-type Ca²⁺ channels, and excitation-contraction coupling. Depolarizing signals from multiple ICC-SM influence the excitability of SMC, and the overall depolarization driving SMCs results from the summation of activity from the ICC-SM network.

Slow waves with characteristics similar to those found in the stomach and small intestine (i.e. relatively fast upstroke depolarization and a plateau phase) are generated along the submucosal surface of the CM layer in the colon (Smith et al., 1987a). It was discovered that peeling the submucosa from the innermost surface of CM blocked slow waves (Durdle et al., 1983). While the authors of that study thought this tissue was mostly connective tissue with possibly some adherent SMCs, it became clear that a population of pacemaker cells, ICC-SM, are present along the submucosal surface (Berezin et al., 1988; Ward et al., 1991). In the present study, we found that ICC-SM and the networks they form are preserved and remain functionally similar in isolated submucosal tissues to ICC-SM attached to the muscularis. It should be noted that ICC-SM were more adherent to CM in the distal colon, and it was more difficult to obtain ICC-SM/submucosal preparations from that region. Submucosal tissues with adherent ICC-SM were used in the current study to eliminate movement artifacts generated by muscle contractions that plague high-resolution Ca²⁺ imaging in most smooth muscle tissues.

While the frequency of pacemaker activity was relatively stable over time in a given preparation, the sequence of activation of individual ICC-SM within the network varied as a function of time, as previously observed in gastric (Hennig et al., 2004) and small intestinal (Park et al., 2006) ICC-MY networks. What appeared as global Ca²⁺ transients in low resolution imaging partitioned into clusters of localized Ca²⁺ transients when viewed with a ×60 objective at 30 frames/s or at higher acquisition speeds. Summation of the clustered events reproduced the frequency and duration of the Ca²⁺ waves observed at low resolution. Multiple firing sites, averaging ~8 per cell, were identified. This pattern of clustered Ca²⁺ transients (CTCs) was also observed in ICC-MY of the small intestine, the pacemaker cells in that region (Drumm et al., 2017). Organization of Ca²⁺ transients into clusters in ICC-SM was dependent upon voltage-dependent Ca²⁺ entry, and our data revealed that in contrast to ICC-MY of the small intestine, Ca²⁺ entry by both dihydropyridine-sensitive and insensitive mechanisms contributes to clustering and propagation of Ca²⁺ waves in intact networks. Nicardipine and isradipine reduced the occurrence of CTCs dramatically, and the T-channel antagonists, NNC 55–0396, TTA-A2, and Z-944 also reduced the occurrence and disordered the Ca²⁺ transients. Cacna1c, Cacna1d, and Cacna1h were expressed in purified ICC-SM, and the presence and function of these channels can explain the pharmacological observations. Channels resulting from Cacna1d (encoding Ca_Vα1D) activate at relatively hyperpolarized membrane potentials and their currents are partially inhibited by dihydropyridines (~50–70% of current density block) in comparison to Cacna1c gene products (Ca_Vα1C) (Xu and Lipscombe, 2001; Bell et al., 2001), but isradipine blocks Ca_Vα1C and Ca_Vα1D equally (Anekonda et al., 2011; Berjukow et al., 2000; Scholze et al., 2001). The fact that isradipine had no greater effect on the occurrence of clustered Ca²⁺ transients than nicardipine suggests that the L-type component of Ca²⁺ entry may be carried primarily by Ca_Vα1C channels. We have observed relatively robust expression of Cacna1d in a variety of ICC in mice (Drumm et al., 2017; Lee et al., 2017); however, the function of these channels has not been identified specifically.

Having three independent voltage-dependent Ca²⁺ conductances with different voltage-dependent properties coordinate clustering of Ca²⁺ transients provides a safety factor for preservation of pacemaker activity over a broad range of membrane potentials. In spite of overarching changes in membrane potential that might influence the availability of ion channels with narrow voltage-ranges, the broader range of activation potentials offered by expression and function of both L-type and T-type Ca²⁺ channels might protect against voltage-dependent inhibition of pacemaker activity. L-type channels are activated at less polarized potentials than T-type channels (Nowycky et al., 1985). Thus, a factor producing tonic hyperpolarization of the SIP syncytium (e.g. purinergic inhibitory neurotransmission; Gallego et al., 2008; Hwang et al., 2012) may tend to switch the dominant voltage-dependent Ca²⁺ entry mechanism from L-type to T-type Ca²⁺ channels. This concept was demonstrated by the decreased inhibitory effects of nicardipine and increased effects of NNC 55–0396 on Ca²⁺ transients after exposure of tissues to pinacidil. The opposite might be true if the SIP syncytium experiences a depolarizing influence (e.g. neurogenic or humerogenic).

Pinacidil hyperpolarizes colonic muscles through activation of K_ATP channels in SMCs (Koh et al., 1998). This compound increased the frequency and decreased the duration of CTCs. These results are consistent with the effects of pinacidil on electrical pacemaker activity in the small intestine where it increases the dV/dt_max of the upstroke depolarization and decreases the duration of slow waves (Kito et al., 2005). The increase in frequency may have been due to reduced inactivation and increased availability of Ca_Vα1D and T-type channels (Ca_Vα1H) at more hyperpolarized potentials. The decrease in the duration of CTCs may be due to a relative shift in the importance of T-type vs. L-type Ca²⁺ channels with hyperpolarization. In the presence of pinacidil, NNC 55–0396 had increased antagonistic effects on CTCs. Ca²⁺ currents via T-type channels inactivate rapidly, whereas L-type channel inactivation is slower and incomplete (Hirano et al., 1989; Tseng and Boyden, 1989; Xu and Best, 1992). Thus, the Ca²⁺ entry period for T-type currents is likely to be more transient than with L-type currents. Channel density in proximity to Ca²⁺ release channels may also affect the degree of coupling between Ca²⁺ entry and CICR, but, as yet, little is known about the structure and functional components of microdomains in ICC.

The importance of Ca²⁺ entry as the primary means of activation and organization of pacemaker activity in ICC-SM was shown by the discoordination of Ca²⁺ transients when extracellular Ca²⁺ was decreased and the incomplete effects of thapsigargin and CPA on Ca²⁺ transients. We noted tight clustering of Ca²⁺ transients at 2.5 and 2.0 mM [Ca²⁺]_o but the tightness of the CTCs disassociated when the driving force for Ca²⁺ entry (i.e. [Ca²⁺]_o was reduced to 1 mM), and frequency of CTCs was greatly reduced at concentrations lower than 1 mM. Our concept is that Ca²⁺ entry couples to CICR in ICC. Reducing the driving force for Ca²⁺ entry would be expected to reduce the probability for effective coupling to CICR. CICR would be negligible when Ca²⁺ entry falls below threshold levels. Concentrations of thapsigargin and CPA that blocked Ca²⁺ transients quantitatively in other ICC (Drumm et al., 2020a; Drumm et al., 2019a; Drumm et al., 2017) caused a partial block of Ca²⁺ transients in ICC-SM. In fact, these drugs caused a marked narrowing of the duration of the CTCs, and this led us to analyze the temporal characteristics of Ca²⁺ transients within clusters. Ca²⁺ transients at the beginning of the CTCs were unaffected by ryanodine and xestospongin C, but transients toward the end of the clusters were blocked. These data suggest that the initial Ca²⁺ transients may result primarily from Ca²⁺ entry, and CTCs are sustained by Ca²⁺ release. This concept is also supported by previous studies showing inhibition of slow waves by blocking Ca²⁺ entry (L-type and T-type Ca²⁺ channels) (Drumm et al., 2017).

We have searched for a preparation of pacemaker ICC that would allow us to investigate the underlying pacemaker activity. We have speculated that stochastic Ca²⁺ release events, occurring in all ICC (Sanders et al., 2014a), are responsible for the spontaneous transient depolarizations (STDs) observed in patch clamp recordings from isolated ICC (Zhu et al., 2009; Zhu et al., 2011). No simultaneous recordings of Ca²⁺ transients and membrane currents or potentials changes have been achieved yet, and the expected link between Ca²⁺ transients and STDs is based on the fact that these events have common pharmacology and sensitivity to drugs that interfere with Ca²⁺ release (Zhu et al., 2015). Ca²⁺ transients and the spontaneous transient inward currents (STICs) due to activation of ANO1 channels result in spontaneous transient membrane depolarizations (STDs). Temporal summation of STDs is likely to be the generator potentials that activate T-type or L-type Ca²⁺ currents and initiate propagating slow wave events. In this concept it is logical to suggest that inhibition of Ca²⁺ release should reduce the duration of the CTCs, and inhibition of Ca²⁺ entry should inhibit the organizing influence of Ca²⁺ entry and block CTCs. When CTCs are blocked, stochastic Ca²⁺ transients may be unleashed, as occur in ICC-IM (Drumm et al., 2019a) and ICC-DMP (Baker et al., 2016) that lack expression of voltage-dependent Ca²⁺ entry mechanisms. Block of CTCs and unmasking of stochastic Ca²⁺ transients was accomplished by hyperpolarization with pinacidil and reduction in the availability of L-type and T-type Ca²⁺ channels with nicardipine and NNC 55–0396. ICC-SM, as imaged in the current experiments provide a potent model for investigating basic pacemaker mechanisms and what happens to these events in response to neurotransmission, hormonal and paracrine inputs and pathological or inflammatory conditions.

Previous studies have supported a role for store-operated Ca²⁺ entry (SOCE) in maintaining Ca²⁺ release events in ICC (Zheng et al., 2018; Drumm et al., 2019a). This is logical because Ca²⁺ release is extremely dynamic in ICC, and it is likely that Ca²⁺ stores would be depleted without an effective recovery mechanism. SOCE depends upon the expression of Orai channels and the ER delimited activator of Orai, STIM, that senses ER Ca²⁺ and binds to and activates Orai channels when Ca²⁺ depletion of the ER occurs (Butorac et al., 2020). STIM and Orai are expressed in colonic ICC (Lee et al., 2017). However, an antagonist of Orai, GSK7975A, reduced the frequency of CTCs, but failed to block these events at a concentration effective in blocking Ca²⁺ transients in small intestinal ICC-MY (Zheng et al., 2018). STIM/Orai interactions appear to have a role in Ca²⁺ store maintenance in ICC-SM, but our data suggest that Ca²⁺ entry via L-type and T-type Ca²⁺ channels also provide Ca²⁺ entry mechanisms that may contribute to store refilling. It could also be suggested that GSK7975A, a well-known antagonist for Orai1 (IC₅₀ = 4 μM; Derler et al., 2013), is less effective on Orai2, the dominant isoform expressed in ICC-SM. However, studies on cortical neurons that express only Orai2 showed about 50% block of SOCE by GSK7975A (5 μM) (Chauvet et al., 2016).

In summary ICC-SM, as suggested from dissection and electrophysiological experiments (Smith et al., 1987a), are pacemaker cells distributed in an electrically coupled network along the submucosal surface of the CM layer. Our experiments demonstrate that Ca²⁺ transients in ICC-SM couple to activation of Ca²⁺ transients and contractions in neighboring SMCs. The contractile events, called ‘ripples’ by some authors in describing integrated colonic contractions (Corsetti et al., 2019; D'Antona et al., 2001), summate with the larger amplitude contractions emanating from the myenteric region of the tunica muscularis to produce mixing and propagated movements characteristic of colonic motility (Rosli et al., 2020). Data from this study suggest that voltage-dependent Ca²⁺ entry serves at least four important functions in the pacemaker activity of ICC-SM: (i) Propagation of activity within the ICC-SM network depends upon voltage-dependent Ca²⁺ entry, and the functions and voltage-dependent properties of three types of Ca²⁺ conductances appear to provide a safety factor that tends to preserve pacemaker activity over a broad range of membrane potentials (see Figure 15 and Figure 15—figure supplement 1). (ii) Ca²⁺ entry is the mechanism that organizes Ca²⁺ release events into CTCs. These events constitute the Ca²⁺ waves that propagate through ICC-SM networks, and cause slow wave depolarizations by activation of ANO1 channels. (iii) Ca²⁺ entry also appears to contribute to refilling of stores, as pacemaker activity was not as immediately dependent upon SOCE as in other ICC (Zheng et al., 2018; Drumm et al., 2019a). (iv) The observations that treatments expected to reduce Ca²⁺ release from stores and reduce coupling between Ca²⁺ entry and CICR reduced, but did not block CTCs, may indicate that transient Ca²⁺ entry (sparklets), possibly through activation of ANO1 channels and depolarization, may underlie the pacemaker functions of ICC-SM. Additional studies will be necessary to resolve these hypotheses in finer detail. The preparation of excised submucosal tissue with adherent ICC-SM removes movement artifacts from imaging and is likely to provide a powerful tool for improving resolution of pacemaker mechanisms and determining how regulatory and pathophysiological factors affect basic pacemaker mechanisms.

Figure 15 with 1 supplement see all

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Role of voltage-dependent Ca²⁺ entry in the pacemaker function of submucosal interstitial cells of Cajal (ICC-SM).

(A) Shows segments of plasma membrane (PM) and endoplasmic reticulum membrane (ER) that form PM-ER junctions and microdomains. At least three types of voltage-dependent Ca²⁺ channels (VDCC) are expressed in ICC-SM, Ca_V1.2, Ca_V1.3, and Ca_V3.2. These conductances, with voltage-dependent activation and inactivation properties spanning a broad range of negative potentials, insure maintenance of pacemaker activity under conditions of hyperpolarization or depolarization in ICC-SM. Pacemaker activity (1. Initial events) in ICC-SM could be due to spontaneous release of Ca²⁺ from stores in the ER and utilize either IP₃R or RYR receptors or both (Ca²⁺ sparks and puffs). However, our data cannot rule out the possibility that transient openings of voltage-dependent Ca²⁺ channels (sparklets) and amplification of Ca²⁺ in microdomains by CICR constitute the initial events of pacemaker activity. In this case, Ca²⁺ release from stores is not the primary pacemaker event but a secondary response to Ca²⁺ entry. Inhibition of Ca²⁺ release from stores would lead to reduced probability of CICR and decrease the frequency of CTCs. Our hypothesis is that Ca²⁺ entry and/or release from stores activates Ca²⁺-dependent Cl^- current due to ANO1 channels in the plasma membrane. Active propagation between cells in interstitial cells of Cajal (ICC) networks (Phase 2) was inhibited by blocking voltage-dependent Ca²⁺ channels. Active propagation may also require or depend upon amplification of Ca²⁺ in microdomains by CICR. The duration of Ca²⁺ entry is likely to be brief due to voltage-dependent inactivation of L- and T-type Ca²⁺ channels. The duration of CTCs appears to be enhanced by CICR (Phase 3). Our data show that the duration of CTCs is reduced by several manipulations known to inhibit Ca²⁺ release from stores. In Phase four store reloading may occur by multiple mechanisms and may include: (i) transient Ca²⁺ entry via sparklets, (ii) activation of SOCE via STIM/ORAI interactions, and (iii) the increase in Ca²⁺ entry that occurs via depolarization and activation of Ca²⁺ entry at the onset of each CTC. (B) A novel hypothesis emerges from this study suggesting that the pacemaker mechanism in non-voltage-clamped cells includes a cyclical, positive-feedback phenomenon that may be responsible for initiation of CTCs and relies on: (i) Ca²⁺ entry through voltage-dependent Ca²⁺ channels. Openings of clusters of these channels would generate sparklets; (ii) Ca²⁺ entry initiates CICR which amplifies [Ca²⁺]_i within microdomains; (iii) the rise in [Ca²⁺]_i activates ANO1 channels in the PM causing depolarization; (iv) depolarization enhances the open probability of voltage-dependent Ca²⁺ channels, increasing Ca²⁺ entry. This cycle creates positive feedback for Ca²⁺ entry, clustering of localized Ca²⁺ transients due to Ca²⁺ entry during the first 350–450 ms of CTCs and development of slow wave depolarizations in ICC-SM.

Materials and methods

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Antibody	Anti- c-Kit (Goat polyclonal)	R and D Systems	Cat# AF1356, RRID:AB_354750	IHC (1:500)
Antibody	Alexa-488 (donkey anti-goat IgG)	Invitrogen/ Thermo Fisher Scientific	(Cat# A32814, RRID:AB_2762838)	IHC (1:1000)
Sequence-based reagent	Kit, Ano1, Myh11,Uchl1, Cacna1c Cacna1d, Cacna1g Cacna1h Orai1,Orai Orai3	This paper	PCR primers	Suppl. Table 1
Chemical compound, drug	NNC 55–0396, TTA-A2	Alomone Labs	Cat# N-206 Cat# T-140
Chemical compound, drug	2-APB, tetracaine, nicardipine, pinacidil, EGTA	Millipore-Sigma	Cat# D9754; Cat# T7508; Cat# N7510; Cat# P154; Cat# E4378
Chemical compound, drug	Thapsigargin, isradipine, Z-944, CPA, ryanodine	Tocris Bioscience	Cat# 1138/1; Cat# 2004/10; Cat# 6367/10; Cat# 1235/10; Cat# 1329/1
Chemical compound, drug	GSK 7975A	Aobious	Cat# AOB4124
Chemical compound, drug	Xestospongin C (XeC)	Cayman Chemical	Cat# 64950
Software, algorithm	STMapAuto, Ca²⁺ Analysis Software	https://github.com/gdelvalle99/STMapAuto	https://doi.org/10.1016/j.ceca.2020.102260

Animals

Kit^+/copGFP mice (B6.129S7-^{Kittm1Rosay/J}; 5–8 wk old) were bred in house (Ro et al., 2010). GCaMP6f-floxed mice (Ai95 (RCL-GCaMP6f)-D) and C57BL/6 mice, their wild-type siblings, were purchased from Jackson Laboratories (Bar Harbor, MN, USA). Kit-iCre mice (c-Kit^+/Cre-ERT2) were gifted from Dr. Dieter Saur (Technical University Munich, Munich, Germany).

Generation of Kit-iCre-GCaMP6f/Acta2-RCaMP1.07 mice

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Acta2-RCaMP1.07 mice (tg(RP23-370F21-RCaMP1.07)B3-3Mik/J) express the fluorescent Ca²⁺ indicator RCaMP1.07 in SMCs under the control of the Acta2 locus promoter/enhancer regions were obtained from Jackson Laboratories (Bar Harbor, MN, USA). To generate cell-specific expression in two distinct cell types (ICC and SMCs) Acta2-RCaMP1.07 mice were bred with Kit^Cre‐ERT2/GCaMP6f^fl/fl mice. The offspring Kit-iCre-GCaMP6f/Acta2-RCaMP1.07 mice were identified by genotyping after receiving tamoxifen which served to delete the STOP cassette in the Cre-expressing cells; resulting in the expression of the fluorescent Ca²⁺ indicator protein, GCaMP6f. These mice allowed simultaneous, dual color imaging of ICC and SMCs. iCre mice were injected with tamoxifen (TAM; Intraperitoneal injection; IP) at 6–8 weeks of age (2 mg of TAM for three consecutive days), as described previously (Baker et al., 2016), to induce activation of the Cre recombinase and expression of optogenetic sensors. Mice were used for experiments 10–15 days after the tamoxifen injections. On days of experiments the mice were anaesthetized by inhalation of isoflurane (Baxter, Deerfield, IL, USA) and killed by cervical dislocation before excision of tissues.

The animals used, protocols performed and procedures in this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno (IACUC; Protocol: 00053).

Tissue preparation

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Colonic segments (2 cm in length, proximal region) were removed from mice after an abdominal incision and placed in Krebs-Ringer bicarbonate solution (KRB). The tissues were cut along the mesenteric border and intraluminal contents were washed away with KRB. Tissues were prepared by blunt dissection in two ways: (1) The submucosa layer was isolated after carefully removing the mucosal layer and the tunica muscularis. (2) The submucosa layer was left attached to the tunica muscularis after removal of the mucosa. The isolated submucosal layer preparation provided better imaging of ICC-SM by eliminating motion artifacts associated with muscle contractions. We used the isolated submucosal layer preparation in most cases in this study with the exception of two experiments where muscle attachments were necessary to test important questions (see Results).

Immunohistochemistry

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Colonic tissues from wild-type mice were processed to assess distribution of c-Kit immunoreactivity. Whole mounts of submucosal layer after removing the mucosa and tunica muscularis were fixed in 4% paraformaldehyde and visualized as described previously (Sanders et al., 2014b). Briefly, after block with 1% bovine serum albumin, colonic tissues were incubated with a polyclonal antibody raised against c-Kit (mSCFR, R and D Systems, MN, USA; 1:500 dilution in 0.5% Triton-X working solution) for 48 hr. Immunoreactivity was detected using Alexa-488 labeled donkey anti-goat IgG (1:1000 in PBS; Invitrogen, NY, USA). Colonic tissues were visualized using a Zeiss LSM 510 confocal microscope and images were constructed using Image J software (National Institutes of Health, MD, USA, http://rsbweb.nih.gov/ij). ICC (copKit) images were visualized using a spinning-disk confocal system (CSU-W1; spinning disk, Yokogawa Electric, Tokyo, Japan).

Cell sorting and quantitative PCR

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Kit^+/copGFP mice (B6.129S7-^{Kittm1Rosay/J}; 5–8 wks old) were used for evaluations of gene expression in ICC-SM. Cell-specific expression of the fluorescent reporter allows unequivocal identification of ICC (Ro et al., 2010). After cell dispersion, ICC-SM were sorted by fluorescence-activated cell sorting (FACS) and evaluated for purity as previously described (Baker et al., 2016). Total RNA was isolated using an Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare). qScript cDNA SuperMix (Quanta Biosciences), used according to the manufacturer’s instructions, was used to synthesize first-strand cDNA. Quantitative PCR (qPCR) was performed using Fast Sybr Green chemistry on the 7900HT Fast Real-Time PCR System (Applied Biosystems) and gene-specific primers (Supplementary file 1). Regression analysis was performed to generate standard curves from the mean values of technical triplicate qPCRs of log10 diluted cDNA samples. Evaluation of gene expression in ICC-SM was compared with expression in the unsorted cells from the submucosal tissue of Kit^+/copGFP mice.

Ca²⁺ imaging

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The isolated/intact submucosal layers were pinned to Sylgard coated dish and perfused with KRB solution at 37°C for a 60-min equilibration period. Ca²⁺ imaging was performed using a spinning-disk confocal system (CSU-W1; spinning disk, Yokogawa Electric, Tokyo, Japan) mounted on an upright Nikon Eclipse FN1 microscope equipped with several water immersion Nikon CFI Fluor lenses (10 × 0.3 NA, 20 × 0.5 NA, 40 × 0.8 NA, 60 × 0.8 NA and 100 × 1.1 NA) (Nikon Instruments, New York, USA). The system is equipped with two solid-state laser lines of 488 nm and 561 nm. The laser lines are combined with a borealis system (ANDOR Technology, Belfast, UK) to increase laser intensity and uniformity throughout the imaging FOV. The system also has two high-speed electron multiplying charged coupled devices (EMCCD) cameras (Andor iXon-Ultra 897 EMCCD Cameras; ANDOR Technology, Belfast, UK) to allow dual-color imaging simultaneously and maintain sensitive and fast speed acquisition at full frame of 512 × 512 active pixels as previously described (Baker et al., 2015). Briefly, images were captured, and image sequences were collected at 33 to 50 fps using MetaMorph software (MetaMorph Inc, TN, USA). In experiments with pharmacological agents, a control activity period of (30 s) was recorded prior of drug application into the chamber for 15 min.

Ca²⁺ imaging analysis

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Movies of Ca²⁺ transients in ICC-SM were imported, preprocessed and analyzed using a combination of three image analysis programs: (1) custom build software (Volumetry G8d, Dr. Grant Hennig); (2) Fiji/Image J (National Institutes of Health, MD, USA, http://rsbweb.nih.gov/ij); (3) Automated Spatio Temporal Map analysis plugin (STMapAuto), https://github.com/gdelvalle99/STMapAuto as described previously (Drumm et al., 2017; Drumm et al., 2019b; Leigh et al., 2020). Briefly, movies of Ca²⁺ transients (stacks of TIFF images) were imported into Volumetry G8d and motion stabilized, background subtracted and smoothed (Gaussian filter: 1.5 × 1.5 µm, StdDev 1.0). A particle analysis routine was employed using a flood-fill algorithm to enhance Ca²⁺ transient detection. particles (PTCLs) representing the areas of active Ca²⁺ signals in cells were saved as a coordinate-based particle movie and combined area and total number of PTCLs were calculated. To better isolate firing sites, only those particles that did not overlap with any particles in the previous frame but overlap with particles in the subsequent 70 ms were considered firing/initiation sites. To show the overall regions in cells where Ca²⁺ transients occurred, PTCLs were summed throughout the video to create an image of their occurrence. The spatial information in the Ca²⁺ occurrence maps data is an indication of each firing site that gives information on their temporal activation and provides no information on their spatial spread. This was implemented in our analysis to accommodate a large number of firing sites and effectively plot them in a 2D occurrence map.

Drugs and solutions

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All tissues were perfused continuously with KRB solution containing (mmol/L): NaCl, 5.9; NaHCO₃, 120.35; KCl, 1.2; MgCl₂, 15.5; NaH₂PO₄,1.2; CaCl₂, 2.5; and glucose, 11.5. The KRB solution was warmed to a physiological temperature of 37 ± 0.3°C and bubbled with a mixture of 97% O₂ – 3% CO₂. For experiments utilizing external solutions with 0 [Ca²⁺]_o, CaCl₂ was omitted and 0.5 mM ethylene glycol-bis (β-aminoethyl ether)-N, N, N’, N’–tetraacetic acid (EGTA) was added to the solution. NNC 55–0396 and TTA-A2 were purchased from Alomone Labs (Jerusalem, Israel). 2-aminoethyl-diphenylborinate (2-APB), tetracaine, nicardipine, pinacidil was purchased from Millipore-Sigma (St. Louis, Missouri, USA). Thapsigargin, isradipine, Z-944, CPA and ryanodine were purchased from Tocris Bioscience (Ellisville, Missouri, USA). GSK 7975A was purchased from Aobious (Aobious INC, MA, USA), and xestospongin C (XeC) was purchased from Cayman Chemical (Michigan, USA).

Statistical analysis

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Data is presented as the mean ± standard error unless otherwise stated. Statistical analysis was performed using either a Students t-test or one-way ANOVA with a Tukey post hoc test where appropriate. In all tests, p<0.05 was considered significant. When describing data, n refers to the number of animals used in a dataset. Probabilities < 0.05 are represented by a single asterisk (*), probabilities < 0.01 are represented by two asterisks (**), probabilities < 0.001 are represented by three asterisks (***) and probabilities < 0.0001 are represented by four asterisks (****). All statistical tests were performed using GraphPad Prism 8.0.1 (San Diego, CA).

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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Article and author information

Author details

Salah A Baker

Department of Physiology and Cell Biology, University of Nevada, Reno, School of Medicine, Reno, United States

Contribution
Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

For correspondence
sabubaker@med.unr.edu

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0002-1514-6876
Wesley A Leigh

Department of Physiology and Cell Biology, University of Nevada, Reno, School of Medicine, Reno, United States

Contribution
Data curation, Formal analysis, Investigation, Methodology

Competing interests
No competing interests declared
Guillermo Del Valle

Department of Physiology and Cell Biology, University of Nevada, Reno, School of Medicine, Reno, United States

Contribution
Formal analysis

Competing interests
No competing interests declared
Inigo F De Yturriaga

Department of Physiology and Cell Biology, University of Nevada, Reno, School of Medicine, Reno, United States

Contribution
Data curation, Formal analysis, Investigation

Competing interests
No competing interests declared
Sean M Ward

Department of Physiology and Cell Biology, University of Nevada, Reno, School of Medicine, Reno, United States

Contribution
Validation, Writing - review and editing

Competing interests
No competing interests declared
Caroline A Cobine

Department of Physiology and Cell Biology, University of Nevada, Reno, School of Medicine, Reno, United States

Contribution
Data curation, Formal analysis, Methodology, Writing - review and editing

Competing interests
No competing interests declared
Bernard T Drumm

Department of Physiology and Cell Biology, University of Nevada, Reno, School of Medicine, Reno, United States

Present address
Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Ireland

Contribution
Formal analysis, Investigation, Methodology, Writing - review and editing

Competing interests
No competing interests declared
Kenton M Sanders

Department of Physiology and Cell Biology, University of Nevada, Reno, School of Medicine, Reno, United States

Contribution
Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review and editing

Competing interests
No competing interests declared

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK-120759)

Salah A Baker
Kenton M Sanders

National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK-078736)

Caroline A Cobine

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors extend their sincere appreciation to Yulia Bayguinov for assistance with immunohistochemical experiments, Lauren O’Kane for assistance with qPCR experiments and David White and Emily Fox for assistance with FACS.

Ethics

Animal experimentation: The animals used, protocols performed and procedures in this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno (IACUC; Protocol# 00053).

Copyright

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Salah A Baker
Wesley A Leigh
Guillermo Del Valle
Inigo F De Yturriaga
Sean M Ward
Caroline A Cobine
Bernard T Drumm
Kenton M Sanders

(2021)

Ca²⁺ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the murine colon

eLife 10:e64099.

https://doi.org/10.7554/eLife.64099

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Mouse

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Cite this article

Distribution of Kit+ submucosal interstitial cells of Cajal (ICC-SM) in the murine colon.

Temporal sequence of Ca2+ transients firing in submucosal interstitial cells of Cajal (ICC-SM) and smooth muscle cells (SMCs).

Propagating Ca2+ waves in submucosal interstitial cells of Cajal (ICC-SM) network.

Simultaneous dual-color imaging of submucosal interstitial cells of Cajal (ICC-SM) and smooth muscle cells (SMCs) in the colon.

Submucosal interstitial cells of Cajal (ICC-SM) Ca2+ signaling activity is entrained.

Ca2+ transients in submucosal interstitial cells of Cajal (ICC-SM) arise from multiple firing sites.

Submucosal interstitial cells of Cajal (ICC-SM) Ca2+ transient initiation sites.

High spatial resolution ICC-SM Ca2+ signals composed of multiple Ca2+-firing sites.

The effect of lowering [Ca2+]o on Ca2+ transients in submucosal interstitial cells of Cajal (ICC-SM).

Molecular expression of genes related to Ca2+ entry channels.

L-type Ca2+ channel antagonist, nicardipine effects on submucosal interstitial cells of Cajal (ICC-SM) Ca2+ transients.

T-type Ca2+ channel antagonist, NNC 55–0396 effects on ICC-SM Ca2+ transients.

The effects of membrane hyperpolarization and voltage-dependent Ca2+ entry block on submucosal interstitial cells of Cajal (ICC-SM) Ca2+ transients.

Intracellular Ca2+ stores contributions in submucosal interstitial cells of Cajal (ICC-SM) Ca2+ transients firing.

Ryanodine receptors (RyRs) contributing to Ca2+ release in submucosal interstitial cells of Cajal (ICC-SM).

SOCE role in maintaining submucosal interstitial cells of Cajal (ICC-SM) Ca2+ transients.

Role of voltage-dependent Ca2+ entry in the pacemaker function of submucosal interstitial cells of Cajal (ICC-SM).

Author details

Salah A Baker

Contribution

For correspondence

Competing interests

Wesley A Leigh

Contribution

Competing interests

Guillermo Del Valle

Contribution

Competing interests

Inigo F De Yturriaga

Contribution

Competing interests

Sean M Ward

Contribution

Competing interests

Caroline A Cobine

Contribution

Competing interests

Bernard T Drumm

Present address

Contribution

Competing interests

Kenton M Sanders

Contribution

Competing interests

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Categories and tags

Research organism

Further reading

Distribution of Kit⁺ submucosal interstitial cells of Cajal (ICC-SM) in the murine colon.

Temporal sequence of Ca²⁺ transients firing in submucosal interstitial cells of Cajal (ICC-SM) and smooth muscle cells (SMCs).

Propagating Ca²⁺ waves in submucosal interstitial cells of Cajal (ICC-SM) network.

Submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ signaling activity is entrained.

Ca²⁺ transients in submucosal interstitial cells of Cajal (ICC-SM) arise from multiple firing sites.

Submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transient initiation sites.

High spatial resolution ICC-SM Ca²⁺ signals composed of multiple Ca^2+-firing sites.

The effect of lowering [Ca²⁺]_o on Ca²⁺ transients in submucosal interstitial cells of Cajal (ICC-SM).

Molecular expression of genes related to Ca²⁺ entry channels.

L-type Ca²⁺ channel antagonist, nicardipine effects on submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transients.

T-type Ca²⁺ channel antagonist, NNC 55–0396 effects on ICC-SM Ca²⁺ transients.

The effects of membrane hyperpolarization and voltage-dependent Ca²⁺ entry block on submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transients.

Intracellular Ca²⁺ stores contributions in submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transients firing.

Ryanodine receptors (RyRs) contributing to Ca²⁺ release in submucosal interstitial cells of Cajal (ICC-SM).

SOCE role in maintaining submucosal interstitial cells of Cajal (ICC-SM) Ca²⁺ transients.

Role of voltage-dependent Ca²⁺ entry in the pacemaker function of submucosal interstitial cells of Cajal (ICC-SM).