Integrated responses of the SIP syncytium generate a major motility pattern in the colon

Abstract

The peristaltic reflex has been a central concept in gastrointestinal motility; however, evidence was published recently suggesting that post-stimulus responses that follow inhibitory neural responses provide the main propulsive force in colonic motility. This new concept was based on experiments on proximal colon where enteric inhibitory neural inputs are mainly nitrergic. However, the nature of inhibitory neural inputs changes from proximal to distal colon where purinergic inhibitory regulation dominates. In spite of the transition from nitrergic to purinergic regulation, post-stimulus responses and propulsive contractions were both blocked by antagonists of a conductance (ANO1) exclusive to interstitial cells of Cajal (ICC). How purinergic neurotransmission, transduced by PDGFRα⁺ cells, can influence ANO1 in ICC is unknown. We compared neural responses in proximal and distal colon. Post-stimulus responses were blocked by inhibition of nitrergic neurotransmission in proximal colon, but P2Y1 receptor antagonists were more effective in distal colon. Ca²⁺ entry through voltage-dependent channels (Ca_V3) enhances Ca²⁺ release in ICC. Thus, we reasoned that hyperpolarization caused by purinergic responses in PDGFRα⁺ cells, which are electrically coupled to ICC, might decrease inactivation of Ca_V3 channels and activate Ca²⁺ entry into ICC via anode-break upon cessation of inhibitory responses. Post-stimulus responses in distal colon were blocked by MRS2500 (P2Y1 receptor antagonist), apamin (SK channel antagonist) and NNC55-0396 (Ca_V3 antagonist). These compounds also blocked propagating contractions in mid and distal colon. These data provide the first clear demonstration that integration of functions in the smooth muscle-ICC-PDGFRα⁺ cell (SIP) syncytium generates a major motility behaviour. KEY POINTS: Propagating propulsive contractions initiated by the enteric nervous system are a major motility behaviour in the colon. A major component of contractions, necessary for propulsive contractions, occurs at cessation of enteric inhibitory neurotransmission (post-stimulus response) and is generated by interstitial cells of Cajal (ICC), which are electrically coupled to smooth muscle cells. The nature of enteric inhibitory neurotransmission shifts from proximal colon, where it is predominantly due to nitric oxide, to distal colon, where it is predominantly due to purine neurotransmitters. Different cells transduce nitric oxide and purines in the colon. ICC transduce nitric oxide, but another type of interstitial cell, PDGFRα⁺ cells, transduces input from purinergic neurons. However, the post-stimulus responses in proximal and distal colon are still generated in ICC. This paper explores how integrated behaviours of ICC, PDGFRα⁺ cells and smooth muscle cells accomplish propulsive motility in the colon.

Keywords: PDGFRα+ cells; colonic motility; enteric nervous system; interstitial cells of Cajal; smooth muscle cells.

Figure 1.. Comparison of electrical responses evoked by EFS in proximal and distal colon

A and B, representative traces showing electrical responses evoked by EFS (black bars; train duration 5 s, frequency 1–10 Hz) in circular muscles of proximal (A) and distal (B) colon. Arrows denote EJPs evoked upon initiation of EFS (A). Arrowheads (>) show examples of fast IJPs in A and B. Asterisks denote period of slow IJPs in in A and B. Expanded trace (inset in A) shows EJP evoked at 10 Hz. Small amplitude notches are stimulus artifacts. C–F, summary of data showing amplitudes of EJP (C), fast IJP (D), slow IJP (E) and PSD (F) in proximal (open circles; n = 8) and distal (filled circles; n = 8) colons. Numbers comparing data sets are P-values by unpaired t test.

Figure 2.. Comparison of contractile responses evoked by EFS in proximal and distal colon

A and B, representative traces showing contractile responses evoked by EFS (train duration 5 s, frequency 1–20 Hz) in proximal (A) and distal (B) colon. C and D, summary of data showing peak amplitudes (C) and the area under the curve (AUC, D) during EFS. E and F, summary of data showing peak amplitudes (E) and AUC (F) of the post-stimulus contraction (PSC); n = 6 in proximal colon and distal colon. Numbers comparing data sets are P-values by unpaired t test. Arrows in A and B denote PSCs.

Figure 3.. Comparison of contractile responses evoked by various train durations of EFS in proximal and distal colon

A and B, representative traces in proximal (A) and distal (B) colon showing the contractile responses evoked by EFS (5 Hz at train durations 1–20 s. C and D, summary of amplitude (C) and AUC (D) during EFS. E and F, summary of amplitude (E) and AUC (F) of PSC. n = 8 and n = 6 in proximal colon and distal colon, respectively. Numbers comparing data sets are P-values by unpaired t test.

Figure 4.. Comparison of electrical responses evoked by EFS in proximal and distal colon in the presence of MRS2500 and L-NNA

A and B, representative traces showing electrical responses evoked by EFS (train duration 5 s, 1–20 Hz) in proximal (A) and distal (B) colon in the presence of l-NNA and MRS2500 (MRS). Arrows denote post-stimulus responses (PSR). Dotted lines indicate resting potentials prior to EFS. C–E, summary of data showing amplitudes of EJPs in proximal colon (C) and distal colon (D) and PSRs (E). Open circles represent control responses and filled circles denote responses in the same muscles after pretreatment with l-NNA and MRS. Key in C also applies to D. E shows summary of PSR amplitudes following cessation of EFS in proximal (open circles) and distal (filled circles) colon (n = 5 each for proximal and distal colon). Numbers comparing data sets before and after l-NNA + MRS treatment are P-values by paired t test.

Figure 5.. Comparison of contractile responses evoked by EFS in proximal and distal colon in the presence of L-NNA, MRS2500 and atropine

A and B, representative traces showing mechanical responses of proximal (A) and distal (B) colon evoked by EFS (1–20 Hz, 20 s) in the presence of l-NNA and MRS2500 (MRS) and l-NNA, MRS and after further addition of atropine. Responses are shown at two sweep speeds. Expanded traces show responses of proximal (Aa–Ah) and distal (Ba–Bh) colon during EFS (between red lines in the traces) in greater detail. These are taken from the responses denoted by a–h in the long duration records (top traces in A and B). C and D, summary of data showing amplitude and AUC during EFS in the proximal (open circles; n = 6) and the distal (open triangles; n = 6) colon and after addition of l-NNA and MRS in the proximal (filled circles) and distal (filled triangles) colon. Black and red values denote P-values by paired t test before and after l-NNA + MRS2500 in proximal and distal colons, respectively. Due to spontaneous phasic activity, it was not possible to evaluate effects of atropine (Ae–Ah) quantitatively. We also noted residual inhibitory effects (arrows in Bf–Bh) in the presence of l-NNA and MRS after addition of atropine with EFS at 5 Hz and above. The residual inhibitory responses may have been due to additional release of VIP. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6.. Comparison of electrical and contractile responses evoked by EFS in proximal and distal colon in the presence of atropine and L-NNA

A and B, representative traces of electrical responses to EFS in the presence of atropine (atrop) and l-NNA in the proximal (A) and distal (B) colon. C–F, summary of data showing amplitude of fast IJP (fIJP) in the proximal (C; n = 6) and distal (D; n = 6) colons and the PSD in proximal (E) and distal (F) colons. Key in C applies to all summary data. G and H, representative traces showing contractile responses evoked by EFS (1–20 Hz; 20 s) in distal colon in control (G) and after addition of atropine and l-NNA (H). I and J, summary of data showing amplitude (I) and AUC (J) of PSC in control (open circles) and after addition of atropine and l-NNA (filled circles; n = 8). Numbers comparing data sets are P-values by paired t test.

Figure 7.. Comparison of electrical and contractile responses to EFS in proximal and distal colon in the presence of atropine and MRS2500

A and B, representative traces showing electrical responses in the presence of atropine (atrop) and MRS2500 (MRS) in proximal (A) and distal colon (B). C–F, summary of data showing the amplitudes of sIJP and PSD in proximal (C and E; n = 7) and distal (D and F; n = 8) colon. Key in C applies to all summary data. G–J, contractile responses in control (G and I) and in the presence of atropine and MRS2500 (H and J) in proximal and distal colon. J and K, summary of data shows effects during EFS (EFS-AUC) and after cessation of EFS (PSC-AUC) in proximal (K and M; n = 6) and distal (L and O, n = 6) colon. Open circles denote control responses and filled circles denote responses after atropine and MRS2500. Dotted lines denote the baselines of membrane potential and contractile responses. Numbers comparing data sets are P-values by paired t test.

Figure 8.. Effects of ANO1 antagonists on responses evoked by EFS and on CMMC propagation in mid and distal colon

A, representative trace showing effect of Ani9 on spontaneous contractions and responses to EFS in distal colon. Aa and Ab are portions of the trace in A denoted by a and b at an expanded time scale. Ani9 blocked PSCs suggesting these responses originate from events in ICC. B, summary of data showing AUC of PSC in control (open circles) and after addition of Ani9 (filled circles; n = 6). Numbers comparing data sets are P-values by paired t test. C and D, EFS-evoked electrical response in the distal colon in control (C) and in the presence of T16Ainh-AO1 (D). E, summary of data showing the amplitude of PSD in control (open circles) and after addition of Ani9 (filled circles; n = 6). Numbers comparing data sets are P-values by paired t test. T16Ainh-AO1 inhibited PSD, suggesting the electrical response after cessation of EFS originates in ICC. F, representative traces showing that Ani9 (red line; added to the mid/distal portion of the colon) blocked propagation of CMMCs through mid and distal colon. G, summary of data showing AUC of CMMCs in control (open circles) and after addition of Ani9 (filled circles; n = 6). Numbers comparing data sets are P-values by paired t test. This suggests that propagation of CMMCs depends upon activation of ANO1 in ICC. H, partitioned chamber used to separate proximal colon (chamber A) from mid and distal colon (chamber B). Force transducers (FT) were attached to wall of the proximal, mid and distal colon to record propagation of CMMCs via measurement of contractions (Tp, Tm and Td), respectively. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 9.. Effects of T-type Ca²⁺ channel antagonist on electrical and contractile responses evoked by EFS and on CMMC propagation in mid and distal colon

A, representative trace showing effect of NNC-55–0396 (NNC), a specific T-type Ca²⁺ channel antagonist, on spontaneous contractions and contractile responses occurring after cessation of EFS in distal colon. Aa and Ab are responses to nerve stimulation in A at expanded sweep speed. Ten seconds of EFS initiated significant PSC upon cessation of the stimulus (Aa). Note reduction in PSC after addition of NNC (Ab). B, summary of data showing AUC of PSC in control (open circles) and after addition of NNC55–0396 (filled circles; n = 6). Numbers comparing data sets are P-values by paired t test. C and D, electrical responses evoked in the distal colon by EFS before (C) and after (D) addition of NNC. E, summary of data showing the amplitude of PSD in control (open circles) and after addition of NNC55–0396 (filled circles; n = 6). Numbers comparing data sets are P-values by paired t test. F, NNC (shown by the line) added to mid/distal chamber B, as depicted in Fig. 8H, reduced propagation of CMMCs in the proximal, mid and distal colon. G, summary of data showing AUC of CMMC in control (open circles) and after addition of NNC (filled circles; n = 6). Numbers comparing data sets are P-values by paired t test. Tp, Tm and Td are contractions measured from the proximal, mid and distal colon, respectively.

Figure 10.. Spatio-temporal maps showing effects of EFS on Ca²⁺ transients in ICC-MY of distal colon

A–D, spatio-temporal maps (STM) of Ca²⁺ transients were tabulated from dynamic imaging of ICC-MY in the presence of atropine (A) and in the presence of atropine and l-NNA (C). During imaging, EFS (EFS, 5 Hz, 10 s) was applied (period of EFS is denoted by the dashed white box superimposed on the STM). B and D, summary of Ca²⁺ transient firing frequency (s⁻¹) in ICC-MY in the presence of atropine (B) and atropine + l-NNA (D) (n = 6, c = 14). l-NNA reduced but did not block PSR. E, STM of Ca²⁺ transients in the presence of atropine, l-NNA and NNC 55–0396. EFS (5 Hz, 10 s) applied during period denoted by white box. Slight curvature in the STM indicates a small movement during imaging. F, summary of Ca²⁺ transient firing frequency (s⁻¹) before, during and after EFS in the presence of atropine + l-NNA + NNC 55–0396. PSR was blocked by the T-channel antagonist (n = 6, c = 15). Numbers comparing data sets are P-values by repeated measure one-way ANOVA. ns, not significant. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 11.. Blocking post-junctional purinergic pathway inhibits PSR in ICC-MY of distal colon.

A and C, STM of Ca²⁺ transients in the presence of atropine and l-NNA (A) and in the presence of atropine, l-NNA and apamin (C). B and D, summary of Ca²⁺ transient firing frequency (s⁻¹) in ICC-MY in the presence of atropine + l-NNA (B) and atropine + l-NNA + apamin (D) (n = 6, c = 14). Apamin blocked PSR. Curvature of STM indicates movements during imaging. E and G, STM of Ca²⁺ transients in the presence of atropine and l-NNA (E) and in the presence of atropine, l-NNA and MRS 2500 (G). F and H, summary of Ca²⁺ transient firing frequency (s⁻¹) in the presence of atropine + l-NNA (F) and atropine + l-NNA + MRS 2500 (H) (n = 6, c = 17). Numbers comparing data sets are P-values by repeated measure one-way ANOVA. ns, not significant. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 12.. The effect of apamin and MRS2500 on CMMCs

A, representative trace showing the effect of apamin on CMMCs. B and C, expanded time scale from blue and red box in A. D, summary of data showing AUC of CMMC in control (open circles) and after addition of apamin (filled circles; n = 6). Numbers comparing data sets are P-values by paired t test. E, the effect of MRS2500 on CMMCs. F and G, expanded time scale from blue and red box in E. H, summary of data showing AUC of CMMC in control (open circles) and after addition of MRS2500 (filled circles; n = 6). Numbers comparing data sets are P-values by paired t test. Tp, Tm and Td are contractions measured from the proximal, mid and distal colon, respectively. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 13.. The effect of atropine, l-NNA and MRS2500 on the CMMC in wild type and W/W^v mutant

A, representative trace showing the effect of atropine, l-NNA and MRS2500 on the CMMC in wild type. Aa–Ac show excerpts from the record in A (a–c) at expanded time scales. B, summary of data showing AUC of CMMC in control (open circles), in the presence of atropine + l-NNA (filled circles) and atropine + l-NNA + MRS 2500 (open triangles; n = 6). Key in B applies to all summary data. Numbers comparing data sets are P-values by one-way ANOVA. C, the effect of atropine, l-NNA and MRS2500 on the CMMC in W/W^v mutant colon. Ca–Cc show excerpts from the record in C (a–c) at expanded time scales. D, summary of data showing frequency of CMMCs in control (open circles), in the presence of atropine + l-NNA (filled circles) and atropine + l-NNA + MRS 2500 (open triangles; n = 4). ND, not detectable.

Figure 14.. Model for post-stimulus responses and CMMC in distal colon

Figure shows schematic model of the restricted volumes formed by plasma membrane (PM)–endoplasmic reticulum (ER) junctions and key receptors and ionic conductances in cells of the SIP syncytium, ICC, SMC and PDGFRα⁺ cells. Cells are electrically coupled through gap junctions (GJ). Individual images from each cell-type represent snapshots of membrane events and Ca²⁺ transients during 3 phases of activity described in this study: (1) pre-stimulus; (2) inhibitory nerve stimulation; and (3) post-stimulus response. During the pre-stimulus phase stochastic, localized Ca²⁺ release (dotted arrows) in ICC and PDGFRα⁺ cells causes transient activation of ANO1 channels in ICC and SK3 channels in PDGFRα⁺ cells. Low open probability of T-type Ca²⁺ channels in ICC is due to relatively depolarized membrane potentials in SIP syncytium, putting membrane potential within the window current range for T-type channels. Minor influx of Ca²⁺ into ICC (dotted arrow) may stimulate Ca²⁺ release events during this phase. These conditions also lead to low open probability for L-type Ca²⁺ channels in SMCs (arrow) and low levels of activation of contractile elements (CE). During the second phase enteric inhibitory neurons release neurotransmitters, and purinergic mechanisms dominate in distal colon. β-NAD binds to P2Y1 receptors expressed by PDGFRα⁺ cells and increases Ca²⁺ release. Enhanced Ca²⁺ activates SK3 channels and causes hyperpolarization which influences membrane potentials of the other SIP cells. Hyperpolarization of ICC decreases open probability of T-type Ca²⁺ channels in ICC and leads to recovery from inactivation of these channels. Hyperpolarization of SMCs decreases open probability of L-type Ca²⁺ channels and inhibits contraction. Upon cessation of neurotransmission, repolarization occurs leading to anode-break activation of T-type Ca²⁺ channels in ICC. Ca²⁺ entry greatly increases Ca²⁺ release, due to Ca²⁺-induced Ca²⁺ release, and activation of ANO1 channels. Resulting depolarization conducts to SMCs, causing an increase in the open probability of L-type Ca²⁺ channels, generation of Ca²⁺ action potentials, Ca²⁺ entry and excitation–contraction coupling. Weight of arrows indicates relative levels of ion transport in each panel. Post-stimulus depolarization and contraction appear to be the major contractile events in colonic propulsive contractions (in mouse termed CMMCs). This model describes how integration of responses in SIP cells underlies a major motility behaviour in the colon. [Colour figure can be viewed at wileyonlinelibrary.com]