Ca²⁺ Signaling Is the Basis for Pacemaker Activity and Neurotransduction in Interstitial Cells of the GI Tract

Abstract

Years ago gastrointestinal motility was thought to be due to interactions between enteric nerves and smooth muscle cells (SMCs) in the tunica muscularis. Thus, regulatory mechanisms controlling motility were either myogenic or neurogenic. Now we know that populations of interstitial cells, c-Kit⁺ (interstitial cells of Cajal or ICC), and PDGFRα⁺ cells (formerly “fibroblast-like” cells) are electrically coupled to SMCs, forming the SIP syncytium. Pacemaker and neurotransduction functions are provided by interstitial cells through Ca²⁺ release from the endoplasmic reticulum (ER) and activation of Ca²⁺-activated ion channels in the plasma membrane (PM). ICC express Ca²⁺-activated Cl⁻ channels encoded by Ano1. When activated, Ano1 channels produce inward current and, therefore, depolarizing or excitatory effects in the SIP syncytium. PDGFRα⁺ cells express Ca²⁺-activated K⁺ channels encoded by Kcnn3. These channels generate outward current when activated and hyperpolarizing or membrane-stabilizing effects in the SIP syncytium. Inputs from enteric and sympathetic neurons regulate Ca²⁺ transients in ICC and PDGFRα⁺ cells, and currents activated in these cells conduct to SMCs and regulate contractile behaviors. ICC also serve as pacemakers, generating slow waves that are the electrophysiological basis for gastric peristalsis and intestinal segmentation. Pacemaker types of ICC express voltage-dependent Ca²⁺ conductances that organize Ca²⁺ transients, and therefore Ano1 channel openings, into clusters that define the amplitude and duration of slow waves. Ca²⁺ handling mechanisms are at the heart of interstitial cell function, yet little is known about what happens to Ca²⁺ dynamics in these cells in GI motility disorders.

22.1 Introduction

Interstitial cells of Cajal (ICC or c-Kit⁺) and platelet-derived growth factor receptor alpha (PDGFRα⁺) cells provide important regulatory behaviors in the generation of motility patterns in the gastrointestinal (GI) tract. ICC and PDGFRα⁺ cells are electrically coupled to smooth muscle cells (SMCs) [32, 44, 66], so changes in membrane conductances in these cells influence the excitability and contractility of SMCs. Together these cells make up an electrical syncytium known as the SIP syncytium [64]. The functions of interstitial cells were suggested by their anatomical characteristics and associations, such as formations of networks, abundant close proximity to varicose processes of motor neurons, and gap junction connectivity with SMCs. Thus, it was proposed that ICC might be pacemaker cells and innervated by motor neurons [24, 69, 79]. The role of PDGFRα⁺ cells, referred to as “fibroblast-like” by anatomists, was assumed to be only structural in nature. Recent work has begun to dissect the mechanisms of action of interstitial cells and reveal the many important functions of these cells in GI motility.

Major breakthroughs for understanding the functions of interstitial cells came when (i) mutants in which ICC failed to develop were used to investigate the role of ICC [36, 73, 74]: (ii) specific immunolabels for ICC and PDGFRα⁺ cells were discovered [42, 51, 52, 56, 70]; (iii) reporter strains were developed [31, 63, 83], allowing observation of specific types of interstitial cells in intact muscles and unequivocal identification of cells after enzymatic dispersion of muscle tissues [47, 51, 52, 70, 83]; (iv) Cre-loxP technology was used to allow highly specific deletion or expression of genes in ICC and allowed cell specific expression of genetically encoded Ca²⁺ sensors to monitor Ca²⁺ dynamics [3, 7, 39, 48, 68]. Evaluations of genes expressed in cellular components of the SIP syncytium showed highly specific expression of genes important for the functions of these cells such as Ano1 (originally called Tmem16a) in ICC [12] and Kcnn3 in PDGFRα⁺ cells [52]. Electrophysiological experiments on isolated cells using the patch clamp technique confirmed the importance of the highly expressed membrane conductances in pacemaker activity and responses to neurotransmitters [52, 83]. Activation of Ano1 channels results in an inward current, and activation of SK3 channels activates outward current. Thus, ICC and PDGFRα⁺ cells provide opposing regulatory inputs in the SIP syncytium.

The two major ionic currents of ICC and PDGFRα⁺ cells are activated by intracellular Ca²⁺, suggesting that Ca²⁺ dynamics regulate the electrophysiological behaviors of these cells. This chapter summarizes some of the current understanding about Ca²⁺ handling mechanisms in ICC and PDGFRα⁺ cells. Realization of the importance of Ca²⁺ dynamics has coincided with the ability to create transgenic mice with expression of genetically encoded Ca²⁺ sensors in ICC. Ca²⁺ dynamics in ICC can be monitored in these mice in situ, making it possible to follow the activation of cells, whether spontaneous or in response to neurotransmitters and other bioagonists. Cell-specific expression of genetically encoded sensors has not been accomplished for PDGFRα⁺ cells due to the lack of highly selective Cre recombinase strains for these cells. However, PDGFRα⁺ cells can be distinguished in intact muscles due to nuclear expression of eGFP in a reporter strain [52] and the ability to monitor Ca²⁺ transients in cell cytoplasms after loading with membrane-permeable Ca²⁺ sensors [5, 6].

22.2 Basal Ca²⁺ Transients in Interstitial Cells

There are two major classes of ICC, intramuscular cells and networks of interconnected cells. Intramuscular ICC (ICC-IM and ICC in the region of the deep muscular plexus of the small intestine, ICC-DMP) lie in close association with enteric motor nerve processes in most smooth muscle regions of the GI tract. In mice expressing GCaMPs exclusively in ICC, we have found that all intramuscular ICC studied generate spontaneous Ca²⁺ transients that originate from multiple sites within individual cells [3, 7, 16, 17]. These Ca²⁺ transients occur in a stochastic and localized manner and show no coupling between firing sites only a few microns from each other in the same cell or in cells nearby. The concept of coupled oscillators, used to describe the behaviors of ICC in the past [35, 72], is not apparent at this level of organization.

Ca²⁺ transients in ICC result from brief release events from intracellular Ca²⁺ stores. Drugs that block the uptake of Ca²⁺ into stores via SERCA pumps (CPA or thapsigargin) inhibit the generation of spontaneous Ca²⁺ transients within a few minutes [3, 16, 17]. Drugs that inhibit Ca²⁺ release through IP₃ receptor (IP₃R) channels or ryanodine (RyR) receptor channels also inhibit Ca²⁺ transients; however, the dependence upon IP₃Rs and RyRs varies between different types of ICC [3, 16, 17]. Ca²⁺ release appears to occur in microdomains (now known as endoplasmic reticulum–plasma membrane junctions; ER/PM junctions) formed by close associations between the ER and the PM [65]. In the excluded volumes of ER/PM junctions, Ca²⁺ released from the ER reaches high concentrations and activates Ca²⁺-dependent conductances, such as Ano1, in the plasma membrane. Indeed, ICC-DMP generate spontaneous transient inward currents (STICs) due to transient activation of Ano1 channels by Ca²⁺ release [85]. In this manner, the stochastic release of Ca²⁺ from many sites within individual cells and from thousands of ICC within tissues can generate a net inward current that is conducted to SMCs via gap junctions. Thus, ICC exert a net excitatory influence in the SIP syncytium (Fig. 22.1a).

Fig. 22.1

Schematics showing fundamental Ca²⁺ dynamics in ICC (a) and PDGFRα⁺ cells (b). These cells display localized Ca²⁺ transients due to release of Ca²⁺ from the endoplasmic reticulum (ER). The ER forms close associations with the plasma membrane (PM). These junctions are referred to as ER/PM junctions. High concentrations of [Ca²⁺]_i are achieved during Ca²⁺ transients in the excluded volumes of the ER/PM junctions. (a) The rise in [Ca²⁺]_i activates Ca²⁺-activated Cl⁻ channels in the PM (encoded by Ano1) of ICC, producing spontaneous transient inward currents (STICs) and depolarization. STICs activated by Ca²⁺ transients in ICC conduct to adjoining SMCs electrically coupled via gap junctions (not shown). Generation of Ca²⁺ transients is highly localized within cells and independent of Ca²⁺ transients even at nearby ER/PM junctions or events occurring in nearby cells (i.e., purpose of showing two discrete ER/PM junctions in the schematic). Coupling between active Ca²⁺ release sites has not been observed, even after addition of excitatory neurotransmitters. (b) The rise in [Ca²⁺]_i activates SK3 in the PM (encoded by Kcnn3) of PDGFRα⁺ cells, producing spontaneous transient outward currents (STOCs) and hyperpolarization or stabilization of membrane potential. STOCs activated by Ca²⁺ transients in PDGFRα⁺ cells conduct to adjoining SMCs that are electrically coupled via gap junctions (not shown)

When Ca²⁺ is released from stores into an ER/PM junction, some of the Ca²⁺ can be recovered via the SERCA pump, but some is lost through general diffusion to the bulk cytoplasm or to the extracellular space via plasma membrane Ca²⁺ ATPase (PMCA) or Na⁺/Ca²⁺ exchange, both of which are expressed by ICC [3, 16, 81]. Therefore, mechanisms to recover Ca²⁺ must exist to maintain the large ER-to-cytoplasm gradient and sustain the ability of the ER to release Ca²⁺. A major contributor to Ca²⁺ recovery in ICC occurs by the process of store-operated Ca²⁺ entry (SOCE) [60,61,62, 67, 71]. The apparatus for SOCE consists of a protein, stromal interaction protein (STIM) that spans the ER membrane. The portion of this protein within the ER lumen contains a Ca²⁺ sensor. When ER Ca²⁺ is depleted by release events, STIM molecules oligomerize, and the cytoplasmic portion of STIM binds to and activates ORAI, a highly selective Ca²⁺ channel in the PM. Thus, STIM and ORAI form a complex that senses a reduction in ER Ca²⁺ and activates Ca²⁺ entry to facilitate store refilling. Drugs that block ORAI can reduce or stop spontaneous Ca²⁺ transients in ICC [80].

While much less is known about Ca²⁺ dynamics in PDGFRα⁺ cells, these cells also have the ability to generate spontaneous Ca²⁺ transients [5, 6]. These events, however, couple to SK3 channels, that are also Ca²⁺ activated but cause the development of outward currents [52]. As a result of Ca²⁺ transients and activation of SK3 channels, isolated PDGFRα⁺ cells can generate spontaneous transient outward currents (STOCs). Generation of STOCs by many PDGFRα⁺ cells within GI muscles exerts a net hyperpolarizing or membrane-stabilizing effect, thereby reducing the excitability and contractility of muscles (Fig. 22.1b).

22.3 Neurotransduction by Interstitial Cells

Both types of interstitial cells in GI muscles contribute to neural regulation of motility. Although this topic has been somewhat controversial, some investigators have clung to the notion that SMCs are the cells innervated and responsible for post-junctional responses. Monitoring of Ca²⁺ signaling in ICC and PDGFRα⁺ cells clearly shows that these cells are innervated and display appropriate responses and temporal characteristics, which suggests that they mediate significant components of post-junctional responses. Blocking the ionic conductances specific to these cells, Ano1 or SK3, can reduce or block post-junctional electrical and mechanical responses to motor nerve stimulation [2, 4, 6, 19].

ICC form very close associations with the varicosities of motor neurons. This is not to say that such junctions are never found between varicosities and SMCs [57], but a morphometric study of esophageal muscles revealed a high propensity of these synaptic-like junctions between motor neurons and ICC [15]. These junctions may facilitate the availability of high concentrations of neurotransmitters near receptors of ICC. Expression of the appropriate receptors by ICC is another indication that these cells are involved in transduction of neural inputs. In the case of excitatory neurotransmission, ICC express type 3 muscarinic (M3) receptors and neurokinin type 1 (NK1) receptors [4, 12, 19, 43]. These receptors dominate responses to neurotransmitters released from enteric excitatory motor neurons (Fig. 22.2). The metabolic enzyme for ACh (acetylcholine esterase (AChE)) is expressed by enteric motor neurons [78], so it is likely that ACh is broken down rapidly in junctional spaces and sufficiently high concentrations of the transmitter may not reach muscarinic receptors expressed by SMCs. Our experiments have shown this to be the case in the murine gastric fundus, but after inhibition of AChE or when gastric ICC-IM fail to develop in the fundus, as in W/W^V mutants, thus removing the synaptic-like junctions, new post-junctional mechanisms are recruited, suggesting that in these conditions ACh reaches SMC receptors [10]. Enteric inhibitory responses due to release of nitric oxide (NO) are compromised in parts of the GI tract where ICC-IM are depleted in W/W^V mutants. ICC express soluble guanylate cyclase (sGC), the receptor for NO, suggesting these cells mediate at least a portion of post-junctional nitrergic responses [40, 41]. However, diffusion of NO may not be so heavily confined, and it may spread to other components of the SIP syncytium. Cell-specific knockdown of sGC suggests that nitrergic, post-junctional responses are mediated by ICC and SMCs [9, 28, 29, 54, 55]. Pathways for nitrergic inhibitory regulation are shown in Fig. 22.2.

Fig. 22.2

Elements and mechanisms of the SIP syncytium and how enteric and sympathetic motor neurons regulate the output of the SIP syncytium. The SIP syncytium is composed of SMCs, ICC (intramuscular and pacemaker types of ICC), and PDGFRα⁺ cells. Ca²⁺ release regulates the open probabilities of Ca²⁺-activated conductances in ICC and PDGFRα⁺ cells (see Fig. 22.1). Ca²⁺ release in ICC is regulated by excitatory and inhibitory neurotransmission by release of ACh and neurokinins from enteric excitatory enteric motor neurons (EEN) and NO from enteric inhibitory motor neurons (EIN). Pathways activated either increase (excitatory neurotransmission) or inhibit (nitrergic neurotransmission) the release of Ca²⁺ and therefore the activation of Ano1 channels in the PM. Ca²⁺ release in PDGFRα⁺ cells is regulated by release of purines from enteric inhibitory motor neurons and norepinephrine (NE) from sympathetic neurons. These neurotransmitters increase release of Ca²⁺ from the ER and activate SK3 channels, producing an outward current. Currents generated by ICC and PDGFRα⁺ cells conduct to SMCs and regulate the excitability of the SMC component of the SIP syncytium. SMCs also receive input from pacemaker ICC, such as ICC-MY. The pacemaker cells generate slow waves that cause periodic depolarizations of SMCs, activation of L-type Ca²⁺ channels (VDCC in SMCs), and phasic contractions of these cells. Active propagation of slow waves in the network of ICC-MY coordinates contractions of the muscles, producing behaviors such as gastric peristalsis and intestinal segmentation. Active propagation of slow waves depends upon T-type Ca²⁺ channels in ICC-MY (shown as VDCC in ICC). SMCs also receive direct stimulation by neurotransmission from, at a minimum, NO and NE. These neurotransmitters regulate smooth muscle contractions through electrophysiological responses (not shown) and by altering the Ca²⁺ sensitivity of contractile elements (CE). Based on the expression of receptors in SIP cells, stimulation of PDGFRα⁺ cells by inhibitory neuropeptides (e.g., VIP) is likely to occur

Effector pathways in ICC, including Ca²⁺ handling mechanisms and dominant ion channels, transduce neural inputs. Stimulation of intrinsic motor neurons with electrical field stimulation (EFS) under conditions favoring excitatory neurotransmission (i.e., by blocking inhibitory pathways) causes significant enhancement in the frequency and amplitude of Ca²⁺ transients in ICC-IM in the colon and ICC-DMP in the small intestine. Stimulation of ICC-DMP appears to be dominated through tachykinin release and binding of NK1 receptors [4], while these receptors are hardly functional and responses are dominated by M3 receptors in the colon [19]. M3 and NK1 receptors couple through G proteins (G_q/11) and activation of phospholipase Cβ, causing generation of IP₃ [1, 76]. Enhanced production of IP₃ causes dramatic increases in Ca²⁺ release from ER via IP₃Rs. These events, like spontaneous Ca²⁺ transients, activate Ano1 channels in the PM and elicit a depolarizing trend in the SIP syncytium, enhancing SMC excitability and contraction (Fig. 22.2). Although Ca²⁺ transient frequency increases in ICC, entrainment of Ca²⁺ release or coupled oscillations between discrete Ca²⁺ release sites have not been observed. Thus, no evidence for coupled oscillators is substantiated at this level of organization.

Opposite effects on Ca²⁺ transients are observed in ICC-IM and ICC-DMP in response to enteric inhibitory neural stimulation (i.e., when excitatory pathways are blocked). The inhibitory effects are mediated by NO and transduced through activation of sGC and generation of cGMP. Downstream from production of cGMP, the mechanisms for the inhibitory effects of NO on Ca²⁺ release in ICC are less well understood, but may occur through cGMP-dependent protein kinase I and phosphorylation of IP₃R-associated cGMP kinase substrate (IRAG) [26, 75]. Initiation of enteric inhibitory responses blocks Ca²⁺ transients for the duration of stimulation (Fig. 22.2). Inhibition of Ca²⁺ release causes deactivation of Ano1 channels and cessation of the basal inward current in the SIP syncytium. In colonic muscles, this resulted in a 9 mV hyperpolarization of muscles [17]. Hyperpolarization reduces the likelihood that the threshold for Ca²⁺ action potentials is reached in SMCs, so this is a mechanism through which nitrergic neurotransmission reduces SMC excitability and inhibits contractions.

A long-acknowledged phenomenon in GI muscles is tonic inhibition [77]. In some muscles, excitability and contractions are suppressed by ongoing release of inhibitory neurotransmitter, and SMC excitability and contractions of the muscles are greatly enhanced by TTX or by inhibition of nNOS. We found that tonic inhibition is linked to ongoing partial suppression of Ca²⁺ transients in ICC. TTX and antagonists of nNOS or sGC greatly increased the occurrence and amplitude of Ca²⁺ transients in ICC concomitantly with the increase in contractions [18]. The increased contractions resulting from suppression of tonic inhibition were reversed by an antagonist of Ano1 channels, supporting the idea that tonic inhibition in the proximal colon is mediated by nitrergic effects on ICC.

Enteric inhibitory neurons exhibit co-transmission whereby single neurons release multiple neurotransmitters. The synthetic enzyme for NO (nNOS) and the peptide VIP co-localize in enteric motor neurons [14, 45]. It is likely that these neurons also release purine neurotransmitters; however, this has never been shown definitively. A somewhat controversial story exists around the identity of the purine neurotransmitter in the GI tract. Classically, the transmitter was believed to be ATP [11], but more recent studies demonstrate that β-NAD⁺ fulfills the criteria for a neurotransmitter much better than ATP in mouse, monkey, and human GI muscles [38, 58]. Actually, enteric inhibitory neurons may release a cocktail of purines that include β-NAD⁺, ADPR, and Up4A [21, 22]. This story is detailed and not among the main topics of this review.

Another gene highly expressed by mouse and human PDGFRα⁺ cells is P2ry1 [5, 51, 52]. Activation of P2Y₁ receptors by several purines, including ATP, ADP, β-NAD⁺, and the highly selective agonist MRS2365, greatly increases the frequency and amplitude of Ca²⁺ transients in PDGFRα⁺ cells in gastric fundus and colon [5, 6]. P2Y₁ receptors are coupled through G proteins (G_q/11) [23], so like M3 and NK1 receptors in ICC, P2Y₁ agonists increase the activity of PLCβ, production of IP₃, and release of Ca²⁺ through IP₃Rs (Fig. 22.2). Interesting to note is that MRS2500, a selective antagonist of P2Y₁ receptors, blocked all of these responses except the responses of some PDGFRα⁺ cells to ATP. Genetic deactivation of P2ry1 ablates purinergic enteric inhibitory responses in mice [25, 37] and the increase in Ca²⁺ transients in PDGFRα⁺ cells caused by ADP, β-NAD⁺, and MRS2365. Responses to ATP, however, persist in some PDGFRα⁺ cells in GI muscles of P2ry1^−/− animals [5, 6]. Similar to the Ca²⁺ transients enhanced by P2Y₁ agonists, these compounds elicited STOCs in PDGFRα⁺ cells [52]. Confidence that purinergic inhibitory responses are mediated by PDGFRα⁺ cells increased dramatically when it was shown that large amplitude hyperpolarization responses are elicited in PDGFRα⁺ cells that are equivalent to purinergic inhibitory junction potentials (IJPs) elicited in whole muscles [50]. These responses, like IJPs, are blocked by apamin or MRS2365. Of importance, P2Y₁ agonists failed to cause hyperpolarization of SMCs isolated from the same muscles.

Evaluation of the transcriptomes of SIP cells has revealed other receptors in these cells that might mediate regulatory effects (Fig. 22.2). An example is the expression of α adrenergic receptors (Adra1a and Adra1b) in PDGFRα⁺ cells [30]. Expression of these receptors was verified by real-time PCR [48]. Processes of sympathetic neurons, as labeled with antibodies to tyrosine hydroxylase, are distributed in the plane of the myenteric plexus and within circular muscles in proximity to PDGFRα⁺ cells. As with P2Y₁ agonists, NE elicited STOCs in voltage-clamped PDGFRα⁺ cells that were blocked by both RS100329, a specific adrenergic α1a receptor antagonist, and apamin. In current-clamped PDGFRα⁺ cells, significant hyperpolarization responses were caused by NE. NE also initiated or increased Ca²⁺ transients in PDGFRα⁺ cells in situ, and these responses were also blocked by RS100329. Contractions of colonic muscles were inhibited by phenylephrine, and these responses were blocked by RS100329 and apamin. Inhibitory effects of phenylephrine did not occur in Adra1a^−/− mice. A preparation with the inferior mesenteric ganglion attached to the colon via the lumbar colonic nerve was used to isolate electrical stimulation of sympathetic neurons. Sympathetic nerve stimulation (SNS) caused hyperpolarization of colonic muscles that were partially blocked by prazosin and apamin [48]. SNS also inhibited colonic migrating motor complexes (CMMCs) through the mid and distal colon. This dramatic sympathetic effect on CMMCs did not occur in colons of Adra1a^−/− mice. Similar sympathetic neurotransduction in PDGFRα⁺ cells and regulation of contractions also occur in human colons [49].

22.4 Pacemaker Activity in Interstitial Cells

ICC generate pacemaker activity in the GI tract that is responsible for electrical slow waves [53, 83]. Networks of pacemaker cells occur along the boundaries of the muscle layers, between the circular and longitudinal muscle layers in the stomach, small bowel, and colon (ICC-MY) and at the submucosal surface of the circular muscle in the colon (ICC-SM). Recent studies have also shown that a type of pacemaker activity is also generated by the ICC along the serosal surface of the proximal colon (ICC-SS), and a type of ICC within muscle bundles (ICC-IM type II) appears to generate the high-frequency pacemaker activity responsible for tone in the internal anal sphincter [33]. A major difference between pacemaker types of ICC (i.e., ICC-MY and ICC-SM) and intramuscular types of ICC (i.e., ICC-IM and ICC-DMP) is the expression of voltage-dependent Ca²⁺ conductances in pacemaker cells [7, 12, 16, 27, 33, 82].

ICC-MY and ICC-SM generate spontaneous Ca²⁺ transients that activate Ano1 channels in the PMs of these cells (Fig. 22.2). In the small intestine, ICC-MY express T-type Ca²⁺ channels (Ca_V3.2) [12, 16, 27, 82]. These channels are activated by the small depolarizations caused by the activation of Ano1 channels (STICs). Increasing the open probability of T-type Ca²⁺ channels can result in development of a Ca²⁺ action potential that constitutes the upstroke phase of slow waves. The dV/dt of the slow wave upstroke, when recorded directly from ICC-MY, is 2 V/s in the mouse intestine and 11 V/s in the rabbit intestine [46]. As in other excitable cells connected by gap junctions, the upstroke phase of slow waves depolarizes neighboring ICC-MY, activates the T-type conductance in these cells, and regenerates the upstroke potential, facilitating active propagation of slow waves in the ICC-MY network. Propagation is seen optically as a coherent intracellular Ca²⁺ wave front that proceeds at about 2 mm/sec in ICC-MY networks in mouse small intestine [16, 59]. SMCs do not express the same ion channels as ICC-MY, and in spite of electrical coupling between SMCs and ICC-MY, SMCs cannot regenerate slow waves and sustain active propagation. Therefore, slow waves conduct passively and decay with distance in the SMC compartment of the SIP syncytium.

Active propagation of slow waves is an important factor in the generation of normal motility behaviors. For example, in the stomach of larger mammals, slow waves propagate without decrement for many centimeter from the dominant pacemaker in the orad corpus to the pylorus [8]. Slow wave propagation was studied in canine gastric muscles using a dual chamber apparatus where slow waves could be initiated by pacing in one chamber, and recordings could be made in the second, electrically isolated chamber at various distances from the site of initiation or after addition of antagonists of specific conductances [8]. Nicardipine had no effect on the dV/dt of the upstroke depolarization nor on the rate of propagation. Antagonists of T-type channels, however, dramatically reduced both the upstroke velocity and the propagation rate. At higher concentrations, these antagonists blocked active slow wave propagation, as did reducing extracellular Ca²⁺ to 0.5 mM. These experiments also showed that reducing the availability of T-type Ca²⁺ channels by depolarization, which causes voltage-dependent inactivation, also greatly reduced upstroke and propagation velocity. These experiments clearly showed that propagation of slow waves is dependent upon voltage-dependent activation of a Ca²⁺ conductance with characteristics of T-type channels.

The Ca²⁺ waves that sweep across a network of ICC-MY are also dependent upon T-type Ca²⁺ channels in the murine small intestine (Fig. 22.2) [16]. Clusters of Ca²⁺ transients occur regularly in jejunal ICC-MY networks at a frequency of about 30 cycles per min in the mouse. The coherent spread of waves and the clustering of Ca²⁺ transients are blocked by NNC 55-0396 and TTA-A2, two specific T-type Ca²⁺ channel antagonists. However, these compounds do not block all Ca²⁺ transients, and in fact the occurrence of Ca²⁺ transients in the presence of T-type Ca²⁺ channel antagonists reverts to a stochastic pattern, as seen in ICC-IM. These experiments suggest that stochastic Ca²⁺ release is a basic behavior of ICC, and addition of a voltage-dependent Ca²⁺ conductance organizes Ca²⁺ transients into periodic clusters of events. While T-type channels are of primary importance in the stomach and small intestine, L-type channels are also important in some ICC, such as those along the submucosal surface of the circular muscle (ICC-SM) in the murine colon [7]. Muscles of the murine proximal colon are relatively more depolarized than cells in the small bowel and stomach. It would be impractical for T-type channels to be the dominant conductance providing active propagation in these muscles because T-type channels are inactivated at the depolarized potentials of colonic muscles. Instead, ICC-SM in murine colon utilize L-type Ca²⁺ as the dominant conductance. However, when ICC-SM are hyperpolarized, T-type channels become available and contribute to slow wave propagation. Presence of both T- and L-type channels provides a safety factor such that slow waves can persist over a broad range of membrane potentials.

Ca²⁺ entry due to the activation of the voltage-dependent Ca²⁺ conductance elicits not only depolarization but also Ca²⁺-induced Ca²⁺ release (CICR) from the ER. Multiple firing sites generate these events both in the soma of ICC and in their processes. As above, Ca²⁺ entry organizes the otherwise stochastic Ca²⁺ transients into clusters of events. Ca²⁺ transients and corresponding activation of Ano1 channels through the network of ICC cause sustained net activation of the Cl⁻ conductance and clamp membrane potentials of ICC close to the equilibrium potential for the Cl⁻ gradient (about −10 mV; [84]). Ca²⁺ release events persist for more than a second or until Ca²⁺ stores are depleted to an extent where they cannot continue to release Ca²⁺. The durations of the Ca²⁺ transient clusters define the durations of the plateau phase of slow waves. Here again, the concept of coupled oscillators is fallacious. Ca²⁺ release events are independent of each other and are organized into clusters by propagation of Ca²⁺ action potentials (i.e., the upstroke of the slow wave event), Ca²⁺ entry, and CICR.

A question to be considered is why the plateau phase is sustained for more than a second. Ca²⁺ entry through T-type Ca²⁺ channels is brief because the ensuing depolarization rapidly inactivates these channels. Therefore, it is unlikely that entry of Ca²⁺ through T-type channels is capable of sustaining CICR. The increased open probability of Ano1 channels is maintained by elevated intracellular Ca²⁺. Thus, a source of Ca²⁺ is needed to maintain openings of Ano1 channels. Several potential sources are possible. The plateau phase of slow waves recorded from ICC is in the range of potentials that generate “window currents” from L-type Ca²⁺ channels [13]. Therefore, openings of these Ca²⁺ channels could provide persistent Ca²⁺ entry that could sustain CICR. This is likely a mechanism for sustaining the plateau in canine gastric antrum because nicardipine dramatically decreases and shortens the plateau phase of slow waves [8]. In contrast, dihydropyridines have little to no effect on slow waves in the small intestine. In murine, small intestine reverse mode Na⁺/Ca²⁺ exchange appears to be responsible for sustained Ca²⁺ entry during the plateau phase [81]. During the period of Ca²⁺ release from stores, Ca²⁺ entry can also occur through ORAI [80]. When ER Ca²⁺ stores are depleted, Ca²⁺ release terminates, the open probability for Ano1 channels drops to low levels, and membrane potential repolarizes. After repolarization, membrane potential rests at negative levels, Ca²⁺ entry is minimal, and the stores refill via SOCE [80].

There is another ICC behavior in which membrane potential of the SIP syncytium is conditioned or tuned into a range where SMCs become rhythmic. In colonic longitudinal muscle, rhythmic intercellular Ca²⁺ waves occur that are due to the periodic firing of Ca²⁺ action potentials in longitudinal muscle cells [34]. These events are inhibited by an Ano1 antagonist [20]. Ano1 is expressed in ICC but not in SMCs, so it is likely that the inward current due to Ano1 develops in the ICC along the serosal surface, ICC-SS. This hypothesis was investigated in muscles of murine proximal colon [20]. ICC-SS form network-like structures and fire stochastic, localized Ca²⁺ transients. These events did not spread cell to cell as observed in ICC-MY or ICC-SM. Thus, they appear to be a hybrid type of ICC with stellate morphologies reminiscent of pacemaker ICC, but with Ca²⁺ dynamics similar to ICC-IM and ICC-DMP. As in all other ICC, however, Ca²⁺ transients in ICC-SS activate Ano1 channels in the PM. The inward currents produced summate to provide a depolarizing influence on longitudinal muscle cells, bringing membrane potential into a range where the SMCs fire Ca²⁺ action potentials. Thus, this example of electrical and mechanical rhythmicity represents an emergent property due to the electrophysiological characteristics of ICC-SS and SMCs and electrical coupling between these cells.

22.5 Conclusions

Ca²⁺ imaging has provided revelations about the mechanisms and functions of interstitial cells in GI muscles. These cells have dynamic Ca²⁺ handling mechanisms that utilize activation of Ano1 or SK3 channels in the PM and conduction of electrical responses to SMCs to distribute slow waves and responses to neural inputs from enteric and sympathetic motor neurons. Much has been learned about the organization and functions of ICC and PDGFRα⁺ cells in normal GI muscles. It is now clear that disease or genetic mutations causing loss of function in interstitial cells can result in GI motor disorders. It is also possible that responses to immune mediators and phenotypic changes in interstitial cells could be a cause of fibrosis. However, we still lack knowledge about what causes defects or alters the phenotypes of interstitial cells and have no therapeutic means of restoring their functions if damaged in disease or aging. It is possible that defects in the SIP syncytium are the primary cause of motor disorders, and additional studies are needed to learn how to manipulate and potentially repair interstitial cell networks and connectivity with motor neurons. In-depth study of the pathophysiology of interstitial cells may provide new opportunities for therapeutics.