Interstitial cells of Cajal (ICC) generate pacemaker activity (slow waves) in gastrointestinal (GI) smooth muscles, but the mechanism(s) of pacemaker activity are controversial. Several conductances, such as Ca 2+ -activated Cl − channels (CaCC) and non-selective cation channels (NSCC) have been suggested to be involved in slow wave depolarization. We investigated the expression and function of a new class of CaCC, anoctamin 1 (ANO1), encoded by Tmem16a, which was discovered to be highly expressed in ICC in a microarray screen. GI muscles express splice variants of the Tmem16a transcript in addition to other paralogues of the Tmem16a family. ANO1 protein is expressed abundantly and specifically in ICC in all regions of the murine, non-human primate (Macaca fascicularis) and human GI tracts. CaCC blocking drugs, niflumic acid and 4,4 -diisothiocyano-2,2 -stillbene-disulfonic acid (DIDS) reduced the frequency and blocked slow waves in murine, primate, human small intestine and stomach in a concentration-dependent manner. Unitary potentials, small stochastic membrane depolarizations thought to underlie slow waves, were insensitive to CaCC blockers. Slow waves failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16a tm1Bdh/tm1Bdh ) and did not develop subsequent to birth in organ culture, as in wildtype and heterozygous muscles. Loss of function of ANO1 did not inhibit the development of ICC networks that appeared structurally normal as indicated by Kit antibodies. These data demonstrate the fundamental role of ANO1 in the generation of slow waves in GI ICC.

Key points• Normal colonic motility is regulated by excitatory and inhibitory motor neurons, and previous studies have shown that both components of neural regulation are important for normal propulsion of colonic contents.• Inhibitory neural control consists of two main components, and the major neurotransmitters have been identified as nitric oxide and purines; we investigated the nature of the receptors responsible for purine inhibitory motor control of the colon using mice with P2Y1 receptors deactivated.• Inhibitory control of the colon by purine neurotransmitters was dramatically decreased in these animals and transit of fecal pellets was delayed.• Inhibitory responses to purine neurotransmission and exogenous β-NAD, a neurotransmitter candidate, were completely abolished in P2Y1 receptor knockouts.• These studies demonstrate the importance of purinergic neural regulation of colonic motility and suggest this form of neural regulation depends upon P2Y1 receptors to receive and transduce inhibitory neural signals.Abstract Activation of enteric inhibitory motor neurons causes inhibitory junctional potentials (IJPs) and muscle relaxation in mammalian gastrointestinal (GI) muscles, including humans. IJPs in many GI muscles are bi-phasic with a fast initial hyperpolarization (fIJP) due to release of a purine neurotransmitter and a slower hyperpolarization component (sIJP) due to release of nitric oxide. We sought to characterize the nature of the post-junctional receptor(s) involved in transducing purinergic neural inputs in the murine colon using mice with genetically deactivated P2ry1. Wild-type mice had characteristic biphasic IJPs and pharmacological dissection confirmed that the fIJP was purinergic and the sIJP was nitrergic. The fIJP was completely absent in P2ry1 −/− mice and the P2Y1 receptor antagonist MRS2500 had no effect on electrical activity or responses to electrical field stimulation of intrinsic nerves in these mice. Contractile experiments confirmed that purinergic responses were abolished in P2ry1 −/− mice. Picospritzing of neurotransmitter candidates (ATP and its primary metabolite, ADP) and β-NAD (and its primary metabolite, ADP-ribose, ADPR) caused transient hyperpolarization responses in wild-type colons, but responses to β-NAD and ADPR were completely abolished in P2ry1 −/− mice. Hyperpolarization and relaxation responses to ATP and ADP were retained in colons of P2ry1 −/− mice. Video imaging revealed that transit of fecal pellets was significantly delayed in colons from P2ry1 −/− mice. These data demonstrate the importance of purinergic neurotransmission in regulating colonic motility and confirm pharmacological experiments suggesting that purinergic neurotransmission is mediated via P2Y1 receptors.

Smooth muscle layers of the gastrointestinal tract consist of a heterogeneous population of cells that include enteric neurons, several classes of interstitial cells of mesenchymal origin, a variety of immune cells and smooth muscle cells (SMCs). Over the last number of years the complexity of the interactions between these cell types has begun to emerge. For example, interstitial cells, consisting of both interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha-positive (PDGFRα + ) cells generate pacemaker activity throughout the gastrointestinal (GI) tract and also transduce enteric motor nerve signals and mechanosensitivity to adjacent SMCs. ICC and PDGFRα + cells are electrically coupled to SMCs possibly via gap junctions forming a multicellular functional syncytium termed the SIP syncytium. Cells that make up the SIP syncytium are highly specialized containing unique receptors, ion channels and intracellular signaling pathways that regulate the excitability of GI muscles. The unique role of these cells in coordinating GI motility is evident by the altered motility patterns in animal models where interstitial cell networks are disrupted. Although considerable advances have been made in recent years on our understanding of the roles of these cells within the SIP syncytium, the full physiological functions of these cells and the consequences of their disruption in GI muscles have not been clearly defined. This review gives a synopsis of the history of interstitial cell discovery and highlights recent advances in structural, molecular expression and functional roles of these cells in the GI tract.

BACKGROUND & AIMS Interstitial cells of Cajal (ICC) express the receptor tyrosine kinase, KIT, the receptor for stem cell factor. In the gastrointestinal (GI) tract, ICC are pacemaker cells that generate spontaneous electrical slow waves, and mediate inputs from motor neurons. Absence or loss of ICC are associated with GI motility disorders, including those consequent of diabetes. Studies of ICC have been hampered by the low density of these cells and difficulties in recognizing these cells in cell dispersions. METHODS Kit+/copGFP mice harboring a copepod super green fluorescent protein (copGFP) complementary DNA, inserted at the Kit locus, were generated. copGFP+ ICC from GI muscles were analyzed using confocal microscopy and flow cytometry. copGFP+ ICC from the jejunum were purified by a fluorescence-activated cell sorter and validated by cell-specific markers. Kit+/copGFP mice were crossbred with diabetic Lep+/obmice to generate mice compound Kit+/copGFP;Lepob/ob mutant mice. copGFP+ ICC from compound transgenic mice were analyzed by confocal microscopy. RESULTS copGFP in Kit+/copGFP mice colocalized with KIT immunofluorescence and thus was predominantly found in ICC. In other smooth muscles, mast cells were also labeled, but these cells were relatively rare in the murine GI tract. copGFP+ cells from jejunal muscles were Kit+ and free of contaminating cell-specific markers. Kit+/copGFP; Lepob/ob mice displayed ICC networks that were dramatically disrupted during the development of diabetes. CONCLUSIONS Kit+/copGFP mice offer a powerful new model to study the function and genetic regulation of ICC phenotypes. Isolation of ICC from animal models will help determine the causes and responses of ICC to therapeutic agents.

Enteric motor neurotransmission is essential for normal gastrointestinal (GI) motility. Controversy exists regarding the cells and ionic conductance(s) that mediate post-junctional neuroeffector responses to motor neurotransmitters. Isolated intramuscular ICC (ICC-IM) and smooth muscle cells (SMCs) from murine fundus muscles were used to determine the conductances activated by carbachol (CCh) in each cell type. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1) is expressed by ICC-IM but not resolved in SMCs, and CCh activated a Cl conductance in ICC-IM and a non-selective cation conductance in SMCs. We also studied responses to nerve stimulation using electrical-field stimulation (EFS) of intact fundus muscles from wild-type and Ano1 knockout mice. EFS activated excitatory junction potentials (EJPs) in wild-type mice, although EJPs were absent in mice with congenital deactivation of Ano1 and greatly reduced in animals in which the CaCC-Ano1 was knocked down using Cre/loxP technology. Contractions to cholinergic nerve stimulation were also greatly reduced in Ano1 knockouts. SMCs cells also have receptors and ion channels activated by muscarinic agonists. Blocking acetylcholine esterase with neostigmine revealed a slow depolarization that developed after EJPs in wild-type mice. This depolarization was still apparent in mice with genetic deactivation of Ano1. Pharmacological blockers of Ano1 also inhibited EJPs and contractile responses to muscarinic stimulation in fundus muscles. The data of the present study are consistent with the hypothesis that ACh released from motor nerves binds muscarinic receptors on ICC-IM with preference and activates Ano1. If metabolism of acetylcholine is inhibited, ACh overflows and binds to extrajunctional receptors on SMCs, eliciting a slower depolarization response.