Interstitial cells of Cajal (ICC) regulate smooth muscle excitability and motility in the gastrointestinal (GI) tract. ICC in the deep muscular plexus (ICC-DMP) of the small intestine are aligned closely with varicosities of enteric motor neurons and thought to transduce neural responses. ICC-DMP generate Ca2+ transients that activate Ca2+ activated Cl- channels and generate electrophysiological responses. We tested the hypothesis that excitatory neurotransmitters regulate Ca2+ transients in ICC-DMP as a means of regulating intestinal muscles. High-resolution confocal microscopy was used to image Ca2+ transients in ICC-DMP within murine small intestinal muscles with cell-specific expression of GCaMP3. Intrinsic nerves were stimulated by electrical field stimulation (EFS). ICC-DMP exhibited ongoing Ca2+ transients before stimuli were applied. EFS caused initial suppression of Ca2+ transients, followed by escape during sustained stimulation, and large increases in Ca2+ transients after cessation of stimulation. Basal Ca2+ activity and the excitatory phases of Ca2+ responses to EFS were inhibited by atropine and neurokinin 1 receptor (NK1) antagonists, but not by NK2 receptor antagonists. Exogenous ACh and substance P (SP) increased Ca2+ transients, atropine and NK1 antagonists decreased Ca2+ transients. Neurokinins appear to be released spontaneously (tonic excitation) in small intestinal muscles and are the dominant excitatory neurotransmitters. Subcellular regulation of Ca2+ release events in ICC-DMP may be a means by which excitatory neurotransmission organizes intestinal motility patterns.

Spontaneous excitability and contractions of colonic smooth muscle cells (SMCs) are normally suppressed by inputs from inhibitory motor neurons, a behavior known as tonic inhibition. The post-junctional cell(s) mediating tonic inhibition have not been elucidated. We investigated the post-junctional cells mediating tonic inhibition in the proximal colon and whether tonic inhibition results from suppression of the activity of Ano1 channels, which are expressed exclusively in interstitial cells of Cajal (ICC). We found that tetrodotoxin (TTX), an inhibitor of nitric oxide (NO) synthesis, L-NNA, and an inhibitor of soluble guanylyl cyclase, ODQ, greatly enhanced colonic contractions. Ano1 antagonists, benzbromarone and Ani9 inhibited the effects of TTX, L-NNA and ODQ. Ano1 channels are activated by Ca2+ release from the endoplasmic reticulum (ER) in ICC, and blocking Ca2+ release with a SERCA inhibitor (thapsigargin) or a store-operated Ca2+ entry blocker (GSK 7975 A) reversed the effects of TTX, L-NNA and ODQ. Ca2+ imaging revealed that TTX, L-NNA and ODQ increased Ca2+ transient firing in colonic ICC. Our results suggest that tonic inhibition in the proximal colon occurs through suppression of Ca2+ release events in ICC. Suppression of Ca2+ release in ICC limits the open probability of Ano1 channels, reducing the excitability of electrically-coupled SMCs.

Key pointsr Rhythmic action potentials and intercellular Ca 2+ waves are generated in smooth muscle cells of colonic longitudinal muscles (LSMC).r Longitudinal muscle excitability is tuned by input from subserosal ICC (ICC-SS), a population of ICC with previously unknown function.r ICC-SS express Ano1 channels and generate spontaneous Ca 2+ transients in a stochastic manner.r Release of Ca 2+ and activation of Ano1 channels causes depolarization of ICC-SS and LSMC, leading to activation of L-type Ca 2+ channels, action potentials, intercellular Ca 2+ waves and contractions in LSMC.r Nitrergic neural inputs regulate the Ca 2+ events in ICC-SS. r Pacemaker activity in longitudinal muscle is an emergent property as a result of integrated processes in ICC-SS and LSMC.Abstract Much is known about myogenic mechanisms in circular muscle (CM) in the gastrointestinal tract, although less is known about longitudinal muscle (LM). Two Ca 2+ signalling behaviours occur in LM: localized intracellular waves not causing contractions and intercellular waves leading to excitation-contraction coupling. An Ano1 channel antagonist inhibited intercellular Ca 2+ waves and LM contractions. Ano1 channels are expressed by interstitial cells of Cajal (ICC) but not by smooth muscle cells (SMCs). We investigated Ca 2+ signalling in a novel population of ICC that lies along the subserosal surface of LM (ICC-SS) in mice expressing GCaMP6f in ICC. ICC-SS fired stochastic localized Ca 2+ transients. Such events have been linked to activation of Ano1 channels in ICC. Ca 2+ transients in ICC-SS occurred by release from stores most probably via inositol trisphosphate receptors. This activity relied on influx via store-operated Bernard T. Drumm received his PhD in Physiology in 2013. His research investigates how Ca 2+ signalling impacts the functions of visceral smooth muscle organs in the gastrointestinal and urinary tracts. His doctoral work, studying Ca 2+ wave propagation in urethral cells, was followed by a postdoctoral position and later research track faculty position at the University of Nevada, Reno (UNR), where he studied the regulation of pacemaker and neuroeffector mechanisms in gastrointestinal interstitial cells. Dr Drumm took up a position as Lecturer in the

Urethral smooth muscle cells (USMCs) generate myogenic tone and contribute to urinary continence. Currently, little is known about Ca signalling in USMCs in situ, and therefore little is known about the source(s) of Ca required for excitation-contraction coupling. We characterized Ca signalling in USMCs within intact urethral muscles using a genetically encoded Ca sensor, GCaMP3, expressed selectively in USMCs. USMCs fired spontaneous intracellular Ca waves that did not propagate cell-to-cell across muscle bundles. Ca waves increased dramatically in response to the α1 adrenoceptor agonist phenylephrine (10 μm) and to ATP (10 μm). Ca waves were inhibited by the nitric oxide donor DEA NONOate (10 μm). Ca influx and release from sarcoplasmic reticulum stores contributed to Ca waves, as Ca free bathing solution and blocking the sarcoplasmic Ca -ATPase abolished activity. Intracellular Ca release involved cooperation between ryanadine receptors and inositol trisphosphate receptors, as tetracaine and ryanodine (100 μm) and xestospongin C (1 μm) reduced Ca waves. Ca waves were insensitive to L-type Ca channel modulators nifedipine (1 μm), nicardipine (1 μm), isradipine (1 μm) and FPL 64176 (1 μm), and were unaffected by the T-type Ca channel antagonists NNC-550396 (1 μm) and TTA-A2 (1 μm). Ca waves were reduced by the store operated Ca entry blocker SKF 96365 (10 μm) and by an Orai antagonist, GSK-7975A (1 μm). The latter also reduced urethral contractions induced by phenylephrine, suggesting that Orai can function effectively as a receptor-operated channel. In conclusion, Ca waves in mouse USMCs are a source of Ca for excitation-contraction coupling in urethral muscles.

Cell, platelet derived growth factor receptor alpha positive interstitial cell; ROI, region of interest; SERCA, sarcoplasmic/endoplasmic reticulum Ca 2+-ATPase pump; SIP, cellular syncytium of smooth muscle cells, interstitial cells of Cajal and platelet derived growth factor receptor alpha positive interstitial cells; SMC, smooth muscle cell; SOCE, store operated Ca2 + entry.