Sean Ward | University of Nevada, Reno (original) (raw)

Papers by Sean Ward

British Journal of Pharmacology, Apr 1, 1992

Gastroenterology, Oct 1, 1993

Proceedings of the National Academy of Sciences of the United States of America, Oct 15, 1996

bioRxiv (Cold Spring Harbor Laboratory), Oct 26, 2020

How Ca 2+ signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is u... more How Ca 2+ signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is unknown. This study shows how ICC modulate colonic motility via complex Ca 2+ signaling and defines Ca 2+ transients' sources using optogenetic techniques. .

American Journal of Physiology-gastrointestinal and Liver Physiology, Nov 1, 2010

American Journal of Physiology-gastrointestinal and Liver Physiology, May 1, 2003

The present study investigated the spatial organization of electrical activity in the canine rect... more The present study investigated the spatial organization of electrical activity in the canine rectoanal region and its relationship to motility patterns. Contraction and resting membrane potential (E m) were measured from strips of circular muscle isolated 0.5-8 cm from the anal verge. Rapid frequency [25 cycles/min (cpm)] E m oscillations (MPOs, 12 mV amplitude) were present across the thickness of the internal anal sphincter (IAS; 0.5 cm) and Em was constant (Ϫ52 mV). Between the IAS and the proximal rectum an 18 mV gradient in Em developed across the muscle thickness with the submucosal edge at Ϫ70 mV and MPOs were replaced with slow waves (20 mV amplitude, 6 cpm). Slow waves were of greatest amplitude at the submucosal edge. Nifedipine (1 M) abolished MPOs but not slow waves. Contractile frequency changes were commensurate with the changes in pacemaker frequency. Our results suggest that changing motility patterns in the rectoanal region are associated with differences in the characteristics of pacemaker potentials as well as differences in the sites from which these potentials emanate. interstitial cells of Cajal; smooth muscle; membrane potential; internal anal sphincter; rectum THE RECTOANAL REGION REPRESENTS the final site for controlling the storage, transport, and evacuation of gastrointestinal contents. The internal anal sphincter (IAS) is a thickening of the circular muscle layer at the distal end of the rectum. Under most circumstances, the IAS is closed and contributes to maintaining continence of fecal matter, liquids, and gases, whereas during the defecation reflex, it briefly relaxes to allow the passage of fecal matter. In contrast, although the rectum is generally empty, it can serve as a final site of storage before defecation as well as participating in the defecation reflex. The IAS and rectum are, therefore, anatomically linked but subserve different physiological roles (7, 13, 16, 18). These functional differences are likely to be associated with significant differences in the motility patterns in the two regions as well as the mechanisms controlling this activity.

The Journal of Physiology, Dec 1, 2004

Electrical slow waves in gastrointestinal (GI) muscles are generated by interstitial cells of Caj... more Electrical slow waves in gastrointestinal (GI) muscles are generated by interstitial cells of Cajal (ICC), and these events actively propagate through networks of ICC within the walls of GI organs. The mechanism by which spontaneously active pacemaker sites throughout ICC networks are entrained to produce orderly propagation of slow waves is unresolved. A three-chambered partition bath was used to test the effects of agents that affect metabolism, membrane potential and voltage-dependent Ca2+ entry on slow wave propagation in canine antral smooth muscle strips. Slow waves evoked by electrical field stimulation actively propagated from end to end of antral muscle strips with a constant latency between two points of recording. When the central chamber of the bath was perfused with low-temperature solutions, mitochondrial inhibitors, reduced extracellular Ca2+ or blockers of voltage-dependent Ca2+ channels, active propagation failed. Depolarization or hyperpolarization of the tissue within the central chamber also blocked propagation. Blockade of propagation by reduced extracellular Ca2+ and inhibitors of dihydropyridine-resistant Ca2+ channels suggests that voltage-dependent Ca2+ entry may be the ‘entrainment factor’ that facilitates active propagation of slow waves in the gastric antrum.

Proceedings of the National Academy of Sciences of the United States of America, Apr 21, 2022

Significance The peristaltic reflex elicits colonic migrating motor complexes (CMMCs) that are th... more Significance The peristaltic reflex elicits colonic migrating motor complexes (CMMCs) that are thought to be generated by enteric excitatory neurons stimulating smooth muscle cells (SMCs). We found that atropine did not block CMMCs and present a concept showing that poststimulus excitatory responses following nitrergic responses in interstitial cells of Cajal (ICC) are responsible for initiation of CMMCs. Ca 2+ transients in ICC are inhibited by nitrergic stimulation. After the inhibitory period, Ca 2+ transients are increased, activating currents that are conducted to SMCs and initiate propagated contractions. Poststimulus excitation generated by ICC explains the properties of CMMCs. This is important because current therapeutic manipulation of colonic transit is typically directed at strengthening cholinergic responses, whereas targeting nitrergic responses may be more suitable.

American Journal of Physiology-cell Physiology, Sep 1, 2001

Regulation of ATP-sensitive K ϩ channels by protein kinase C in murine colonic myocytes. Am J Phy... more Regulation of ATP-sensitive K ϩ channels by protein kinase C in murine colonic myocytes. Am J Physiol Cell Physiol 281: C857-C864, 2001.-We investigated the regulation of ATP-sensitive K ϩ (KATP) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10 Ϫ5 M) activated inward currents in the presence of high external K ϩ (90 mM) at a holding potential of Ϫ80 mV in dialyzed cells. Glibenclamide (10 Ϫ5 M) suppressed pinacidilactivated current. Phorbol 12,13-dibutyrate (PDBu; 2 ϫ 10 Ϫ7 M) inhibited pinacidil-activated current. 4-␣-Phorbol ester (5 ϫ 10 Ϫ7 M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of KATP channels was increased by pinacidil, and PDBu suppressed openings of K ATP channels. When cells were pretreated with chelerythrine (10 Ϫ6 M) or calphostin C (10 Ϫ7 M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10 Ϫ6 M). Acetylcholine (ACh; 10 Ϫ5 M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10 Ϫ7 M) decreased the effect of ACh. Cells dialyzed with protein kinase C ε-isoform (PKCε) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on KATP were blocked in these cells. Immunofluorescence and Western blots showed expression of PKCε in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates KATP in colonic muscle cells and that the effects of ACh on KATP are largely mediated by PKC. PKCε appears to be the major isozyme that regulates KATP in murine colonic myocytes.

Biology of Reproduction, Nov 8, 2021

Translational Andrology and Urology, Dec 1, 2016

ObjectiveThere is a growing body of evidence that gastrointestinal smooth muscle excitability is ... more ObjectiveThere is a growing body of evidence that gastrointestinal smooth muscle excitability is regulated by several different classes of interstitial cells [interstitial cells of Cajal (ICC) and PDGFRα(+) cells] that are electrically coupled to SMC. Thus, ‘myogenic’ activity results from the integrated behavior of the SMC/ICC/PDGFRα(+) cell (SIP) syncytium. Inputs from excitatory and inhibitory motor neurons are required to produce the complex motor patterns of the gut and coordinate GI motility. Motor neurons innervate these three cell types in the SIP syncytium, and receptors, second messenger pathways, and ion channels in these cells mediate postjunctional responses. Cholinergic neurotransmission in GI muscles from several species has long been thought to be dependent upon activation of a non-selective cation conductance in smooth muscle cells and the molecular candidates for mediating cholinergic excitation have been reported to be the transient receptor protein channels Trpc4 and Trpc6. However, we have shown that cholinergic responses in the GI tract involve ICC and in their absence these motor responses are greatly diminished or absent. We sought to determine the conductance(s) responsible for cholinergic motor responses in the colon.MethodsCre-LoxP recombinase technology was utilized to determine the role of the calcium-activated chloride conductance, Ano1 in post-junctional motor responses in the mouse colon in a cell-specific manner (Kit+ ICC). c-KitCreERT2/+ (Kit-Cre) mice and Ano f/f mice were crossed to generate c-KitCreERT2/+; Ano1f/f (mutants) and c-KitCreERT2/+; Ano1f/+ (controls) animals that were subsequently treated with tamoxifen to induce Cre recombinase expression in ICC. Confocal microscopy was used to determine the cell type Cre expression was switched on. Intracellular microelectrode recordings were performed to determine changes in post-junctional neural responses to nerve stimulation in c-KitCreERT2/+ (Kit-Cre) mice and Anof/f mice to generate c-KitCreERT2/+; Ano1f/f and c-KitCreERT2/+; Ano1f/+ animals mice treated with tamoxifen using electrical field stimulation (EFS).ResultsKnock down of Ano1 in Kit+ ICC using the Cre/Lox P technology caused a marked reduction or loss of excitatory junction potentials (EJPs) in colonic muscles of c-KitCreERT2/+; Ano1f/f in response to EFS compared to c-KitCreERT2/+; Ano1f/+ controls. These EJPs were atropine sensitive providing evidence that cholinergic muscarinic receptors were responsible for these excitatory responses.ConclusionsThese data provide evidence that Ano1 expressed in ICC and not smooth muscle cells is critically important for cholinergic excitatory post-junctional neural responses in colonic muscles.Funding SourcesNIH PO1 41315 and RO1 DK57236

British Journal of Pharmacology, May 1, 1996

oxide synthase by N0-nitro-L-arginine (L-NNA) reduced the neurogenic relaxation of precontracted ... more oxide synthase by N0-nitro-L-arginine (L-NNA) reduced the neurogenic relaxation of precontracted taenia coli only in the absence of atropine. The membrane hyperpolarization associated with the neurogenic relaxation was also reduced by inhibition of NOS only when atropine was absent. 2 The membrane hyperpolarization associated with the neurogenic relaxation of the taenia coli was inhibited by oxyhaemoglobin only in the absence of atropine. In the presence of atropine, oxyhaemoglobin did not reduce the ij.p. or nerve-evoked relaxation. 3 Inhibition of NOS by L-NNA did not affect the overflow of [3H]-ACh in response to electrical field stimulation (EFS), suggesting that, under the conditions of our experiments, endogenous NO did not modulate release of ACh. Sodium nitroprusside also had no effect on the neurogenic overflow of [3fH-ACh; however, noradrenaline significantly reduced [3H]-ACh overflow. 4 In summary, the postjunctional effects of neurally-released NO are not apparent in guinea-pig taenia coli when atropine is present. This implies muscarinic regulation of NO release or muscarinic regulation of another excitatory substance, such as tachykinin(s), that, when blocked, masks the postjunctional effects of NO. These data, together with previous studies, suggest a possible regulatory role for NO in enteric neurotransmission that may be more prominent in some species or tissues than others.

American Journal of Physiology-cell Physiology, Mar 1, 1993

We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical act... more We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical activity of intact circular smooth muscle from canine colon. TPeA (10 and 20 microM) increased slow-wave duration and "locked" the membrane potential around -30 mV plateau potential after several minutes of application, suggesting that K+ channels are essential for termination of colonic slow waves. Repolarization and normal slow-wave activity resumed after 20-30 min of washout. The patch-clamp technique was used to study the block of large-conductance Ca(2+)-activated K+ channels (BK channels) by TPeA and tetraethylammonium (TEA) in excised and cell-attached patches from isolated colonic smooth muscle cells. Channel block was characterized by a voltage-dependent dissociation constant [Kd(V)] for the binding of TEA and TPeA to a blocking site located a fraction of the distance across the membrane field (delta). The extracellular TEA binding site had a Kd(0) of 0.33 mM and a delta of 0.23. The extracellular TPeA binding site had a Kd(0) of 2.2 mM but showed significantly less voltage dependence (delta = 0.02). The intracellular binding site for TEA was of low affinity [Kd(0) = 76 mM]. Intracellular TPeA was the most potent blocker of BK channel current [Kd(0) = 11.7 microM]. The voltage dependence of block by intracellular TPeA (delta = -0.21) was not significantly different from that of intracellular TEA (delta = -0.3). Internal TPeA (10 microM) also blocked a 70-pS K+ channel and a 23-pS K+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)

American Journal of Physiology-cell Physiology, Nov 1, 2007

Bayguinov O, Ward SM, Kenyon JL, Sanders KM. Voltage-gated Ca 2ϩ currents are necessary for slow-... more Bayguinov O, Ward SM, Kenyon JL, Sanders KM. Voltage-gated Ca 2ϩ currents are necessary for slow-wave propagation in the canine gastric antrum.

Journal of Cellular and Molecular Medicine, Jun 28, 2012

An obstacle to understanding motor pathologies of the gastrointestinal (GI) tract is that the phy... more An obstacle to understanding motor pathologies of the gastrointestinal (GI) tract is that the physiology of some of the cellular components of the gut wall is not understood. Morphologists identified fibroblast-like cells in the tunica muscularis many years ago, but little is known about these interstitial cells because of inadequate techniques to identify these cells. Recent findings have shown that fibroblast-like cells express platelet-derived growth factor receptor α (PDGFRα) in mice and that antibodies for these receptors can be used to label the cells. We used immunohistochemical techniques to study the phenotype and intercellular relationships of fibroblast-like cells in the human colon. Fibroblast-like cells are labelled specifically with antibodies to PDGFRα and widely distributed through the tunica muscularis of human colon. These cells form discrete networks in the region of the myenteric plexus and within the circular and longitudinal muscle layers. Platelet-derived growth factor receptor α+ cells are distinct from c-Kit+ interstitial cells of Cajal and closely associated with varicose processes of neurons expressing substance P (excitatory motor neurons) or neuronal nitric oxide synthase (nNOS) (inhibitory motor neurons). Platelet-derived growth factor receptor α+ cells express small conductance Ca2+-activated K+ channels (SK3), which are likely to mediate purinergic neural regulation of colonic muscles. Our data suggest that PDGFRα+ cells may have an important role in transducing inputs from enteric motor neurons. This study identifies reagents and techniques that will allow investigation of this class of interstitial cells and help develop an understanding of the role of PDGFRα+ cells in the human GI tract in health and disease.

Neurogastroenterology and Motility, Apr 1, 2004

Specialized cells known as interstitial cells of Cajal (ICC) are distributed in specific location... more Specialized cells known as interstitial cells of Cajal (ICC) are distributed in specific locations within the tunica muscularis of the gastrointestinal tract and serve as electrical pacemakers, active propagation pathways for slow waves, and mediators of enteric motor neurotransmission. Recent morphological studies have provided evidence that motor neurotransmission in the gut does not occur through loosely defined synaptic structures between nerves and smooth muscle, but rather via synaptic‐like contacts that exist between varicose nerve terminals and intramuscular ICC (ICC‐IM). ICC‐IM are coupled to smooth muscle cells via gap junctions and electrical responses elicited in ICC are conducted to muscle cells. Electrophysiological studies of the stomach of wild‐type and mutant animals that lack ICC‐IM have provided functional evidence for the importance of ICC in cholinergic and nitrergic motor neurotransmission. The synaptic‐like contacts between nerve terminals and ICC‐IM facilitate rapid diffusion of transmitters to specific receptors on ICC. ICC‐IM also play a role in generating unitary potentials in the stomach that contribute to the excitability of the gastric fundus and antrum.

The FASEB Journal, Apr 22, 2023

Introduction: Motor functions of the stomach have traditionally been regarded to differ by stomac... more Introduction: Motor functions of the stomach have traditionally been regarded to differ by stomach region. However, to date there have been few studies on the gastric contractility of the human stomach. Objectives: The aim of the present study was therefore to examine the response to acetylcholine and electrical field stimulation (EFS) of the human fundus, corpus and antrum. Methods: Gastric muscles were obtained from 71 patients (44 males, 27 females with an average age of 60.7 yrs; 34 85 yrs) undergoing gastric cancer surgery. Muscle strips (n = 71) were isolated and attached to a fixed mount and to an isometric strain gauge. The muscle strips were studied In Vitro for their contractile responses to acetylcholine (n=25) and electrical field stimulation (EFS, n=46). N-nitro-L-arginine (L-NA), atropine and tetrodotoxin (TTX) were added to assess the nitro oxide-mediated changes and cholinergic neural pathway involved in EFS-induced contractions. Results: Spontaneous contractile activity was observed in muscle strips of stomach regions. In the antrum, acetylcholine induced increase in contraction frequency and peak contraction in dose dependent pattern, but no significant change was noticed in muscle tone. In corpus and fundus, acetylcholine induced dose-dependent increase in peak contraction and muscle tone, but there was no significant change in frequency. The response to EFS also differed by stomach region. EFS produced no significant change in contractile activity in 22 of 46 muscle strips. When EFS evoked frequency-dependent contraction or relaxation in muscle strips, contraction was primarily observed in the muscle strips from the antrum, and relaxation was primarily observed in the muscle strips of the corpus and fundus. The addition of LNA (100 uM) to the muscle bath converted the EFS-induced relaxation to contractions and increased the EFS-induced contractions. EFS-induced contractions were abolished by atropine (1uM) or TTX (1uM). Conclusions: Our study demonstrated that the response to acetylcholine and electrical field stimulation on human gastric fundus and corpus is different with that of human gastric antrum.

Biophysical Journal, Feb 1, 2018

In VSMCs, Ca 2þ-activated Clchannels (CaCCs) are encoded by the gene TMEM16A/Anoctamin 1 (ANO1). ... more In VSMCs, Ca 2þ-activated Clchannels (CaCCs) are encoded by the gene TMEM16A/Anoctamin 1 (ANO1). The mechanism by which ANO1 influence the excitability of VSMCs remains to be elucidated due to questionable pharmacology and lack of a reliable genetic knockdown mouse model of ANO1. The aim of this study was to re-evaluate the role of ANO1 in electromechanical coupling of pulmonary artery (PA) smooth muscle using newer generation ANO1 blockers and a novel smooth muscle-specific inducible ANO1 knockout mouse model (SMC-iANO1-KO). Wire myography was used to determine the vascular reactivity to 5-HT of PA from wild-type and SMC-iANO1-KO mice. Calcium imaging experiments were also carried out using SMC-iGCaMP3 mice, which genetically express the Ca 2þ biosensor GCaMP3 in smooth muscle cells. 5-HT elicited a dose-dependent contraction (0.01-30 mM) that was similarly inhibited ($50-70%) by the ANO1 blocker CaCC Inh-A01 (10 mM), the Ca V 1.2 blocker nifedipine (1 mM) or the SERCA2 pump inhibitor cyclopiazonic acid (CPA; 10 mM). Genetic ablation of ANO1 produced a reduction in 5-HT-induced tone ($ 60% at 1 mM 5-HT) that was similar to that produced by CaCC inh A01, nifedipine or CPA. Ca 2þ imaging experiments in the intact PA of SMC-iGCaMP3 mice revealed that 5-HT evoked spatially and temporally localized Ca 2þ transients. These Ca 2þ oscillations were potently inhibited by CaCC Inh-A01 or nifedipine, and were abolished by CPA. In conclusion, 5-HT elicited highly localized Ca 2þ oscillations that were promoted by Ca 2þ entry through Ca V 1.2, most likely involving transient depolarizations evoked by ANO1 activated by a balance between oscillatory SR Ca 2þ release through IP 3 receptors and Ca 2þ entry through Ca V 1.2. We propose that the stable agonistinduced PA contraction results from the integration of stochastic and localized Ca 2þ events supported by a microenvironment comprising ANO1, Ca V 1.2 and IP 3 receptors.

Scientific Reports, Jun 25, 2020

the internal anal sphincter (iAS) generates phasic contractions and tone. Slow waves (SWs) produc... more the internal anal sphincter (iAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of cajal (icc) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the iAS where only intramuscular icc (icc-iM) are found, however the evidence linking icc-iM to SWs is limited. this study examined the possible relationship between icc-iM and SWs by recording ca 2+ transients in mice expressing a genetically-encoded ca 2+-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type ca 2+ channels (cav L) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous ca 2+ transients not dependent on cav L or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., type ii cells (64% of total) generated rhythmic, global Ca 2+ transients at the SW frequency that were synchronised with neighbouring type ii cells and were abolished following blockade of either cav L or ANO1. Thus, the spatiotemporal characteristics of type ii cells and their dependence upon cav L and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the iAS. The internal anal sphincter (IAS) is responsible for approximately 70% of resting anal pressure; an important property for maintaining faecal continence 1,2. Reports suggest that approximately 43% of faecal incontinence cases may be related to disturbances in IAS motility 3. Unlike the majority of the gastrointestinal (GI) tract, the IAS spontaneously develops tone; a critical feature for raising pressure in the anal canal. While others have suggested that the IAS is a "purely tonic muscle" that develops tone as a result of enhanced myofilament sensitivity to Ca 2+ 4,5 , we have found that the IAS is fundamentally a phasic smooth muscle that generates tone as a result of the summation of phasic contractions 6-10. The IAS exhibits slow wave (SW) activity, the electrophysiological events that give rise to phasic contractions in many regions of the GI tract 11. SWs, phasic contractions and tone rely upon Ca 2+ influx via voltage-gated L-type Ca 2+ channels (Cav L) 8,12-14. Thus, mechanisms regulating Ca 2+ entry are clearly fundamental for tone development in the IAS. Interstitial cells of Cajal (ICC) are present throughout the GI tract and studies of ICC in non-sphincter muscles indicate that SWs are generated by specialised ICC located predominantly at the myenteric (ICC-MY) and/ or submucosal (ICC-SM) edges of the circular muscle layer 11. Pacemaker ICC in these regions are coupled electrically to one another and to adjacent smooth muscle cells (SMCs) via gap junctions allowing conduction of SWs from ICC to SMCs where excitation-contraction coupling occurs 15. These cells are typically highly-branched stellate-shaped cells 16,17. In contrast, another population of spindle-shaped intramuscular ICC (ICC-IM) are involved in neuromuscular transmission 16,18. ICC are also present in the IAS of various species but their distribution and morphology differs significantly from that of non-sphincteric muscles 19-23. Importantly, the density of ICC-MY and ICC-SM declines from rectum to IAS with only ICC-IM present in the distal IAS 21,23. SW amplitude and frequency are greatest in the distal IAS 8,23,24 and thus we hypothesise that ICC-IM could be the pacemaker cells that generate SWs in the IAS. IAS-SWs differ significantly from intestinal SWs. Intestinal SWs persist in the presence of antagonists of Cav L 25 , but IAS-SWs are inhibited by these antagonists, suggesting an essential role for Cav L in these events in

British Journal of Pharmacology, Apr 1, 1992

Gastroenterology, Oct 1, 1993

Proceedings of the National Academy of Sciences of the United States of America, Oct 15, 1996

bioRxiv (Cold Spring Harbor Laboratory), Oct 26, 2020

How Ca 2+ signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is u... more How Ca 2+ signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is unknown. This study shows how ICC modulate colonic motility via complex Ca 2+ signaling and defines Ca 2+ transients' sources using optogenetic techniques. .

American Journal of Physiology-gastrointestinal and Liver Physiology, Nov 1, 2010

American Journal of Physiology-gastrointestinal and Liver Physiology, May 1, 2003

The present study investigated the spatial organization of electrical activity in the canine rect... more The present study investigated the spatial organization of electrical activity in the canine rectoanal region and its relationship to motility patterns. Contraction and resting membrane potential (E m) were measured from strips of circular muscle isolated 0.5-8 cm from the anal verge. Rapid frequency [25 cycles/min (cpm)] E m oscillations (MPOs, 12 mV amplitude) were present across the thickness of the internal anal sphincter (IAS; 0.5 cm) and Em was constant (Ϫ52 mV). Between the IAS and the proximal rectum an 18 mV gradient in Em developed across the muscle thickness with the submucosal edge at Ϫ70 mV and MPOs were replaced with slow waves (20 mV amplitude, 6 cpm). Slow waves were of greatest amplitude at the submucosal edge. Nifedipine (1 M) abolished MPOs but not slow waves. Contractile frequency changes were commensurate with the changes in pacemaker frequency. Our results suggest that changing motility patterns in the rectoanal region are associated with differences in the characteristics of pacemaker potentials as well as differences in the sites from which these potentials emanate. interstitial cells of Cajal; smooth muscle; membrane potential; internal anal sphincter; rectum THE RECTOANAL REGION REPRESENTS the final site for controlling the storage, transport, and evacuation of gastrointestinal contents. The internal anal sphincter (IAS) is a thickening of the circular muscle layer at the distal end of the rectum. Under most circumstances, the IAS is closed and contributes to maintaining continence of fecal matter, liquids, and gases, whereas during the defecation reflex, it briefly relaxes to allow the passage of fecal matter. In contrast, although the rectum is generally empty, it can serve as a final site of storage before defecation as well as participating in the defecation reflex. The IAS and rectum are, therefore, anatomically linked but subserve different physiological roles (7, 13, 16, 18). These functional differences are likely to be associated with significant differences in the motility patterns in the two regions as well as the mechanisms controlling this activity.

The Journal of Physiology, Dec 1, 2004

Electrical slow waves in gastrointestinal (GI) muscles are generated by interstitial cells of Caj... more Electrical slow waves in gastrointestinal (GI) muscles are generated by interstitial cells of Cajal (ICC), and these events actively propagate through networks of ICC within the walls of GI organs. The mechanism by which spontaneously active pacemaker sites throughout ICC networks are entrained to produce orderly propagation of slow waves is unresolved. A three-chambered partition bath was used to test the effects of agents that affect metabolism, membrane potential and voltage-dependent Ca2+ entry on slow wave propagation in canine antral smooth muscle strips. Slow waves evoked by electrical field stimulation actively propagated from end to end of antral muscle strips with a constant latency between two points of recording. When the central chamber of the bath was perfused with low-temperature solutions, mitochondrial inhibitors, reduced extracellular Ca2+ or blockers of voltage-dependent Ca2+ channels, active propagation failed. Depolarization or hyperpolarization of the tissue within the central chamber also blocked propagation. Blockade of propagation by reduced extracellular Ca2+ and inhibitors of dihydropyridine-resistant Ca2+ channels suggests that voltage-dependent Ca2+ entry may be the ‘entrainment factor’ that facilitates active propagation of slow waves in the gastric antrum.

Proceedings of the National Academy of Sciences of the United States of America, Apr 21, 2022

Significance The peristaltic reflex elicits colonic migrating motor complexes (CMMCs) that are th... more Significance The peristaltic reflex elicits colonic migrating motor complexes (CMMCs) that are thought to be generated by enteric excitatory neurons stimulating smooth muscle cells (SMCs). We found that atropine did not block CMMCs and present a concept showing that poststimulus excitatory responses following nitrergic responses in interstitial cells of Cajal (ICC) are responsible for initiation of CMMCs. Ca 2+ transients in ICC are inhibited by nitrergic stimulation. After the inhibitory period, Ca 2+ transients are increased, activating currents that are conducted to SMCs and initiate propagated contractions. Poststimulus excitation generated by ICC explains the properties of CMMCs. This is important because current therapeutic manipulation of colonic transit is typically directed at strengthening cholinergic responses, whereas targeting nitrergic responses may be more suitable.

American Journal of Physiology-cell Physiology, Sep 1, 2001

Regulation of ATP-sensitive K ϩ channels by protein kinase C in murine colonic myocytes. Am J Phy... more Regulation of ATP-sensitive K ϩ channels by protein kinase C in murine colonic myocytes. Am J Physiol Cell Physiol 281: C857-C864, 2001.-We investigated the regulation of ATP-sensitive K ϩ (KATP) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10 Ϫ5 M) activated inward currents in the presence of high external K ϩ (90 mM) at a holding potential of Ϫ80 mV in dialyzed cells. Glibenclamide (10 Ϫ5 M) suppressed pinacidilactivated current. Phorbol 12,13-dibutyrate (PDBu; 2 ϫ 10 Ϫ7 M) inhibited pinacidil-activated current. 4-␣-Phorbol ester (5 ϫ 10 Ϫ7 M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of KATP channels was increased by pinacidil, and PDBu suppressed openings of K ATP channels. When cells were pretreated with chelerythrine (10 Ϫ6 M) or calphostin C (10 Ϫ7 M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10 Ϫ6 M). Acetylcholine (ACh; 10 Ϫ5 M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10 Ϫ7 M) decreased the effect of ACh. Cells dialyzed with protein kinase C ε-isoform (PKCε) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on KATP were blocked in these cells. Immunofluorescence and Western blots showed expression of PKCε in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates KATP in colonic muscle cells and that the effects of ACh on KATP are largely mediated by PKC. PKCε appears to be the major isozyme that regulates KATP in murine colonic myocytes.

Biology of Reproduction, Nov 8, 2021

Translational Andrology and Urology, Dec 1, 2016

ObjectiveThere is a growing body of evidence that gastrointestinal smooth muscle excitability is ... more ObjectiveThere is a growing body of evidence that gastrointestinal smooth muscle excitability is regulated by several different classes of interstitial cells [interstitial cells of Cajal (ICC) and PDGFRα(+) cells] that are electrically coupled to SMC. Thus, ‘myogenic’ activity results from the integrated behavior of the SMC/ICC/PDGFRα(+) cell (SIP) syncytium. Inputs from excitatory and inhibitory motor neurons are required to produce the complex motor patterns of the gut and coordinate GI motility. Motor neurons innervate these three cell types in the SIP syncytium, and receptors, second messenger pathways, and ion channels in these cells mediate postjunctional responses. Cholinergic neurotransmission in GI muscles from several species has long been thought to be dependent upon activation of a non-selective cation conductance in smooth muscle cells and the molecular candidates for mediating cholinergic excitation have been reported to be the transient receptor protein channels Trpc4 and Trpc6. However, we have shown that cholinergic responses in the GI tract involve ICC and in their absence these motor responses are greatly diminished or absent. We sought to determine the conductance(s) responsible for cholinergic motor responses in the colon.MethodsCre-LoxP recombinase technology was utilized to determine the role of the calcium-activated chloride conductance, Ano1 in post-junctional motor responses in the mouse colon in a cell-specific manner (Kit+ ICC). c-KitCreERT2/+ (Kit-Cre) mice and Ano f/f mice were crossed to generate c-KitCreERT2/+; Ano1f/f (mutants) and c-KitCreERT2/+; Ano1f/+ (controls) animals that were subsequently treated with tamoxifen to induce Cre recombinase expression in ICC. Confocal microscopy was used to determine the cell type Cre expression was switched on. Intracellular microelectrode recordings were performed to determine changes in post-junctional neural responses to nerve stimulation in c-KitCreERT2/+ (Kit-Cre) mice and Anof/f mice to generate c-KitCreERT2/+; Ano1f/f and c-KitCreERT2/+; Ano1f/+ animals mice treated with tamoxifen using electrical field stimulation (EFS).ResultsKnock down of Ano1 in Kit+ ICC using the Cre/Lox P technology caused a marked reduction or loss of excitatory junction potentials (EJPs) in colonic muscles of c-KitCreERT2/+; Ano1f/f in response to EFS compared to c-KitCreERT2/+; Ano1f/+ controls. These EJPs were atropine sensitive providing evidence that cholinergic muscarinic receptors were responsible for these excitatory responses.ConclusionsThese data provide evidence that Ano1 expressed in ICC and not smooth muscle cells is critically important for cholinergic excitatory post-junctional neural responses in colonic muscles.Funding SourcesNIH PO1 41315 and RO1 DK57236

British Journal of Pharmacology, May 1, 1996

oxide synthase by N0-nitro-L-arginine (L-NNA) reduced the neurogenic relaxation of precontracted ... more oxide synthase by N0-nitro-L-arginine (L-NNA) reduced the neurogenic relaxation of precontracted taenia coli only in the absence of atropine. The membrane hyperpolarization associated with the neurogenic relaxation was also reduced by inhibition of NOS only when atropine was absent. 2 The membrane hyperpolarization associated with the neurogenic relaxation of the taenia coli was inhibited by oxyhaemoglobin only in the absence of atropine. In the presence of atropine, oxyhaemoglobin did not reduce the ij.p. or nerve-evoked relaxation. 3 Inhibition of NOS by L-NNA did not affect the overflow of [3H]-ACh in response to electrical field stimulation (EFS), suggesting that, under the conditions of our experiments, endogenous NO did not modulate release of ACh. Sodium nitroprusside also had no effect on the neurogenic overflow of [3fH-ACh; however, noradrenaline significantly reduced [3H]-ACh overflow. 4 In summary, the postjunctional effects of neurally-released NO are not apparent in guinea-pig taenia coli when atropine is present. This implies muscarinic regulation of NO release or muscarinic regulation of another excitatory substance, such as tachykinin(s), that, when blocked, masks the postjunctional effects of NO. These data, together with previous studies, suggest a possible regulatory role for NO in enteric neurotransmission that may be more prominent in some species or tissues than others.

American Journal of Physiology-cell Physiology, Mar 1, 1993

We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical act... more We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical activity of intact circular smooth muscle from canine colon. TPeA (10 and 20 microM) increased slow-wave duration and "locked" the membrane potential around -30 mV plateau potential after several minutes of application, suggesting that K+ channels are essential for termination of colonic slow waves. Repolarization and normal slow-wave activity resumed after 20-30 min of washout. The patch-clamp technique was used to study the block of large-conductance Ca(2+)-activated K+ channels (BK channels) by TPeA and tetraethylammonium (TEA) in excised and cell-attached patches from isolated colonic smooth muscle cells. Channel block was characterized by a voltage-dependent dissociation constant [Kd(V)] for the binding of TEA and TPeA to a blocking site located a fraction of the distance across the membrane field (delta). The extracellular TEA binding site had a Kd(0) of 0.33 mM and a delta of 0.23. The extracellular TPeA binding site had a Kd(0) of 2.2 mM but showed significantly less voltage dependence (delta = 0.02). The intracellular binding site for TEA was of low affinity [Kd(0) = 76 mM]. Intracellular TPeA was the most potent blocker of BK channel current [Kd(0) = 11.7 microM]. The voltage dependence of block by intracellular TPeA (delta = -0.21) was not significantly different from that of intracellular TEA (delta = -0.3). Internal TPeA (10 microM) also blocked a 70-pS K+ channel and a 23-pS K+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)

American Journal of Physiology-cell Physiology, Nov 1, 2007

Bayguinov O, Ward SM, Kenyon JL, Sanders KM. Voltage-gated Ca 2ϩ currents are necessary for slow-... more Bayguinov O, Ward SM, Kenyon JL, Sanders KM. Voltage-gated Ca 2ϩ currents are necessary for slow-wave propagation in the canine gastric antrum.

Journal of Cellular and Molecular Medicine, Jun 28, 2012

An obstacle to understanding motor pathologies of the gastrointestinal (GI) tract is that the phy... more An obstacle to understanding motor pathologies of the gastrointestinal (GI) tract is that the physiology of some of the cellular components of the gut wall is not understood. Morphologists identified fibroblast-like cells in the tunica muscularis many years ago, but little is known about these interstitial cells because of inadequate techniques to identify these cells. Recent findings have shown that fibroblast-like cells express platelet-derived growth factor receptor α (PDGFRα) in mice and that antibodies for these receptors can be used to label the cells. We used immunohistochemical techniques to study the phenotype and intercellular relationships of fibroblast-like cells in the human colon. Fibroblast-like cells are labelled specifically with antibodies to PDGFRα and widely distributed through the tunica muscularis of human colon. These cells form discrete networks in the region of the myenteric plexus and within the circular and longitudinal muscle layers. Platelet-derived growth factor receptor α+ cells are distinct from c-Kit+ interstitial cells of Cajal and closely associated with varicose processes of neurons expressing substance P (excitatory motor neurons) or neuronal nitric oxide synthase (nNOS) (inhibitory motor neurons). Platelet-derived growth factor receptor α+ cells express small conductance Ca2+-activated K+ channels (SK3), which are likely to mediate purinergic neural regulation of colonic muscles. Our data suggest that PDGFRα+ cells may have an important role in transducing inputs from enteric motor neurons. This study identifies reagents and techniques that will allow investigation of this class of interstitial cells and help develop an understanding of the role of PDGFRα+ cells in the human GI tract in health and disease.

Neurogastroenterology and Motility, Apr 1, 2004

Specialized cells known as interstitial cells of Cajal (ICC) are distributed in specific location... more Specialized cells known as interstitial cells of Cajal (ICC) are distributed in specific locations within the tunica muscularis of the gastrointestinal tract and serve as electrical pacemakers, active propagation pathways for slow waves, and mediators of enteric motor neurotransmission. Recent morphological studies have provided evidence that motor neurotransmission in the gut does not occur through loosely defined synaptic structures between nerves and smooth muscle, but rather via synaptic‐like contacts that exist between varicose nerve terminals and intramuscular ICC (ICC‐IM). ICC‐IM are coupled to smooth muscle cells via gap junctions and electrical responses elicited in ICC are conducted to muscle cells. Electrophysiological studies of the stomach of wild‐type and mutant animals that lack ICC‐IM have provided functional evidence for the importance of ICC in cholinergic and nitrergic motor neurotransmission. The synaptic‐like contacts between nerve terminals and ICC‐IM facilitate rapid diffusion of transmitters to specific receptors on ICC. ICC‐IM also play a role in generating unitary potentials in the stomach that contribute to the excitability of the gastric fundus and antrum.

The FASEB Journal, Apr 22, 2023

Introduction: Motor functions of the stomach have traditionally been regarded to differ by stomac... more Introduction: Motor functions of the stomach have traditionally been regarded to differ by stomach region. However, to date there have been few studies on the gastric contractility of the human stomach. Objectives: The aim of the present study was therefore to examine the response to acetylcholine and electrical field stimulation (EFS) of the human fundus, corpus and antrum. Methods: Gastric muscles were obtained from 71 patients (44 males, 27 females with an average age of 60.7 yrs; 34 85 yrs) undergoing gastric cancer surgery. Muscle strips (n = 71) were isolated and attached to a fixed mount and to an isometric strain gauge. The muscle strips were studied In Vitro for their contractile responses to acetylcholine (n=25) and electrical field stimulation (EFS, n=46). N-nitro-L-arginine (L-NA), atropine and tetrodotoxin (TTX) were added to assess the nitro oxide-mediated changes and cholinergic neural pathway involved in EFS-induced contractions. Results: Spontaneous contractile activity was observed in muscle strips of stomach regions. In the antrum, acetylcholine induced increase in contraction frequency and peak contraction in dose dependent pattern, but no significant change was noticed in muscle tone. In corpus and fundus, acetylcholine induced dose-dependent increase in peak contraction and muscle tone, but there was no significant change in frequency. The response to EFS also differed by stomach region. EFS produced no significant change in contractile activity in 22 of 46 muscle strips. When EFS evoked frequency-dependent contraction or relaxation in muscle strips, contraction was primarily observed in the muscle strips from the antrum, and relaxation was primarily observed in the muscle strips of the corpus and fundus. The addition of LNA (100 uM) to the muscle bath converted the EFS-induced relaxation to contractions and increased the EFS-induced contractions. EFS-induced contractions were abolished by atropine (1uM) or TTX (1uM). Conclusions: Our study demonstrated that the response to acetylcholine and electrical field stimulation on human gastric fundus and corpus is different with that of human gastric antrum.

Biophysical Journal, Feb 1, 2018

In VSMCs, Ca 2þ-activated Clchannels (CaCCs) are encoded by the gene TMEM16A/Anoctamin 1 (ANO1). ... more In VSMCs, Ca 2þ-activated Clchannels (CaCCs) are encoded by the gene TMEM16A/Anoctamin 1 (ANO1). The mechanism by which ANO1 influence the excitability of VSMCs remains to be elucidated due to questionable pharmacology and lack of a reliable genetic knockdown mouse model of ANO1. The aim of this study was to re-evaluate the role of ANO1 in electromechanical coupling of pulmonary artery (PA) smooth muscle using newer generation ANO1 blockers and a novel smooth muscle-specific inducible ANO1 knockout mouse model (SMC-iANO1-KO). Wire myography was used to determine the vascular reactivity to 5-HT of PA from wild-type and SMC-iANO1-KO mice. Calcium imaging experiments were also carried out using SMC-iGCaMP3 mice, which genetically express the Ca 2þ biosensor GCaMP3 in smooth muscle cells. 5-HT elicited a dose-dependent contraction (0.01-30 mM) that was similarly inhibited ($50-70%) by the ANO1 blocker CaCC Inh-A01 (10 mM), the Ca V 1.2 blocker nifedipine (1 mM) or the SERCA2 pump inhibitor cyclopiazonic acid (CPA; 10 mM). Genetic ablation of ANO1 produced a reduction in 5-HT-induced tone ($ 60% at 1 mM 5-HT) that was similar to that produced by CaCC inh A01, nifedipine or CPA. Ca 2þ imaging experiments in the intact PA of SMC-iGCaMP3 mice revealed that 5-HT evoked spatially and temporally localized Ca 2þ transients. These Ca 2þ oscillations were potently inhibited by CaCC Inh-A01 or nifedipine, and were abolished by CPA. In conclusion, 5-HT elicited highly localized Ca 2þ oscillations that were promoted by Ca 2þ entry through Ca V 1.2, most likely involving transient depolarizations evoked by ANO1 activated by a balance between oscillatory SR Ca 2þ release through IP 3 receptors and Ca 2þ entry through Ca V 1.2. We propose that the stable agonistinduced PA contraction results from the integration of stochastic and localized Ca 2þ events supported by a microenvironment comprising ANO1, Ca V 1.2 and IP 3 receptors.

Scientific Reports, Jun 25, 2020

the internal anal sphincter (iAS) generates phasic contractions and tone. Slow waves (SWs) produc... more the internal anal sphincter (iAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of cajal (icc) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the iAS where only intramuscular icc (icc-iM) are found, however the evidence linking icc-iM to SWs is limited. this study examined the possible relationship between icc-iM and SWs by recording ca 2+ transients in mice expressing a genetically-encoded ca 2+-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type ca 2+ channels (cav L) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous ca 2+ transients not dependent on cav L or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., type ii cells (64% of total) generated rhythmic, global Ca 2+ transients at the SW frequency that were synchronised with neighbouring type ii cells and were abolished following blockade of either cav L or ANO1. Thus, the spatiotemporal characteristics of type ii cells and their dependence upon cav L and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the iAS. The internal anal sphincter (IAS) is responsible for approximately 70% of resting anal pressure; an important property for maintaining faecal continence 1,2. Reports suggest that approximately 43% of faecal incontinence cases may be related to disturbances in IAS motility 3. Unlike the majority of the gastrointestinal (GI) tract, the IAS spontaneously develops tone; a critical feature for raising pressure in the anal canal. While others have suggested that the IAS is a "purely tonic muscle" that develops tone as a result of enhanced myofilament sensitivity to Ca 2+ 4,5 , we have found that the IAS is fundamentally a phasic smooth muscle that generates tone as a result of the summation of phasic contractions 6-10. The IAS exhibits slow wave (SW) activity, the electrophysiological events that give rise to phasic contractions in many regions of the GI tract 11. SWs, phasic contractions and tone rely upon Ca 2+ influx via voltage-gated L-type Ca 2+ channels (Cav L) 8,12-14. Thus, mechanisms regulating Ca 2+ entry are clearly fundamental for tone development in the IAS. Interstitial cells of Cajal (ICC) are present throughout the GI tract and studies of ICC in non-sphincter muscles indicate that SWs are generated by specialised ICC located predominantly at the myenteric (ICC-MY) and/ or submucosal (ICC-SM) edges of the circular muscle layer 11. Pacemaker ICC in these regions are coupled electrically to one another and to adjacent smooth muscle cells (SMCs) via gap junctions allowing conduction of SWs from ICC to SMCs where excitation-contraction coupling occurs 15. These cells are typically highly-branched stellate-shaped cells 16,17. In contrast, another population of spindle-shaped intramuscular ICC (ICC-IM) are involved in neuromuscular transmission 16,18. ICC are also present in the IAS of various species but their distribution and morphology differs significantly from that of non-sphincteric muscles 19-23. Importantly, the density of ICC-MY and ICC-SM declines from rectum to IAS with only ICC-IM present in the distal IAS 21,23. SW amplitude and frequency are greatest in the distal IAS 8,23,24 and thus we hypothesise that ICC-IM could be the pacemaker cells that generate SWs in the IAS. IAS-SWs differ significantly from intestinal SWs. Intestinal SWs persist in the presence of antagonists of Cav L 25 , but IAS-SWs are inhibited by these antagonists, suggesting an essential role for Cav L in these events in