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Inhibitory motor neurons regulate several gastric motility patterns including receptive relaxation, gastric peristaltic motor patterns, and pyloric sphincter opening. Nitric oxide (NO) and purines have been identified as likely candidates that mediate inhibitory neural responses. However, the contribution from each neurotransmitter has received little attention in the distal stomach. The aims of this study were to identify the roles played by NO and purines in inhibitory motor responses in the antrums of mice and monkeys. By using wild-type mice and mutants with genetically deleted neural nitric oxide synthase ( Nos1 −/−) and P2Y1 receptors ( P2ry1 −/−) we examined the roles of NO and purines in postjunctional inhibitory responses in the distal stomach and compared these responses to those in primate stomach. Activation of inhibitory motor nerves using electrical field stimulation (EFS) produced frequency-dependent inhibitory junction potentials (IJPs) that produced muscle relaxations in both species. Stimulation of inhibitory nerves during slow waves terminated pacemaker events and associated contractions. In Nos1 −/− mice IJPs and relaxations persisted whereas in P2ry1 −/− mice IJPs were absent but relaxations persisted. In the gastric antrum of the non-human primate model Macaca fascicularis, similar NO and purine neural components contributed to inhibition of gastric motor activity. These data support a role of convergent inhibitory neural responses in the regulation of gastric motor activity across diverse species.

Slow waves (slow wavesICC) were recorded from myenteric interstitial cells of Cajal (ICC-MY) in situ in the rabbit small intestine, and their properties were compared with those of mouse small intestine. Rabbit slow wavesICCconsisted of an upstroke depolarization followed by a distinct plateau component. Ni2+and nominally Ca2+-free solutions reduced the rate-of-rise and amplitude of the upstroke depolarization. Replacement of Ca2+with Sr2+enhanced the upstroke component but decreased the plateau component of rabbit slow wavesICC. In contrast, replacing Ca2+with Sr2+decreased both components of mouse slow wavesICC. The plateau component of rabbit slow wavesICCwas inhibited in low-extracellular-Cl−-concentration (low-[Cl−]o) solutions and by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of Cl−channels, cyclopiazonic acid (CPA), an inhibitor of internal Ca2+pumps, or bumetanide, an inhibitor of Na+-K+-2Cl−cotransporter (NKCC1). Bumetanide also inhibited the plateau component of mouse slow wavesICC. NKCC1-like immunoreactivity was observed mainly in ICC-MY in the rabbit small intestine. Membrane depolarization with a high-K+solution reduced the upstroke component of rabbit slow wavesICC. In cells depolarized with elevated external K+, DIDS, CPA, and bumetanide blocked slow wavesICC. These results suggest that the upstroke component of rabbit slow wavesICCis partially mediated by voltage-dependent Ca2+influx, whereas the plateau component is dependent on Ca2+-activated Cl−efflux. NKCC1 is likely to be responsible for Cl−accumulation in ICC-MY. The results also suggest that the mechanism of the upstroke component differs in rabbit and mouse slow wavesICCin the small intestine.

The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα+ cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfrα egfp/+, Kit copGFP/+, and smMHC Cre-egfp mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were PDGFRα+ cells > ICC ≫ SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFRα+ cells whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα+ cells. The functional role of cGKI was investigated in cGKI −/− mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI −/− mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI +/+ and cGKI −/− mice although there was a small reduction in the cGKI −/− mouse. Nω-nitro-l-arginine (l-NNA) abolished responses during the first 20–30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI −/− mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.

Interstitial cells of mesenchymal origin form gap junctions with smooth muscle cells in visceral smooth muscles and provide important regulatory functions. In gastrointestinal (GI) muscles, there are two distinct classes of interstitial cells, c-Kit+interstitial cells of Cajal and PDGFRα+cells, that regulate motility patterns. Loss of these cells may contribute to symptoms in GI motility disorders.

Spontaneous contractions of the myosalpinx are critical for oocyte transport along the oviduct. Slow waves, the electrical events that underlie myosalpinx contractions, are generated by a specialized network of pacemaker cells called oviduct interstitial cells of Cajal (ICC-OVI). The ionic basis of oviduct pacemaker activity is unknown. Intracellular recordings and Ca2+imaging were performed to examine the role of extracellular and intracellular Ca2+sources in slow wave generation. RT-PCR was performed to determine the transcriptional expression of Ca2+channels. Molecular studies revealed most isoforms of L- and T-type calcium channels (Cav1.2,1.3,1.4,3.1,3.2,3.3) were expressed in myosalpinx. Reduction of extracellular Ca2+concentration ([Ca2+]o) resulted in the abolition of slow waves and myosalpinx contractions without significantly affecting resting membrane potential (RMP). Spontaneous Ca2+waves spread through ICC-OVI cells at a similar frequency to slow waves and were inhibited by reduced [Ca2+]o. Nifedipine depolarized RMP and inhibited slow waves; however, pacemaker activity returned when the membrane was repolarized with reduced extracellular K+concentration ([K+]o). Ni2+also depolarized RMP but failed to block slow waves. The importance of ryanodine and inositol 1,4,5 trisphosphate-sensitive stores were examined using ryanodine, tetracaine, caffeine, and 2-aminoethyl diphenylborinate. Results suggest that although both stores are involved in regulation of slow wave frequency, neither are exclusively essential. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor cyclopiazonic acid inhibited pacemaker activity and Ca2+waves suggesting that a functional SERCA pump is necessary for pacemaker activity. In conclusion, results from this study suggest that slow wave generation in the oviduct is voltage dependent, occurs in a membrane potential window, and is dependent on extracellular calcium and functional SERCA pumps.

The morphology of interstitial cells of Cajal (ICC) in the circular muscle layer of the cynomolgus monkey internal anal sphincter (IAS) and rectum and their relationship to sympathetic and nitrergic nerves were compared by dual-labeling immunohistochemistry. Contractile studies confirmed that nitrergic nerves participate in neural inhibition in both regions whereas sympathetic nerves serve as excitatory motor nerves only in the IAS. Muscle bundles extended from myenteric to submucosal edge in rectum but in the IAS bundles were further divided into “minibundles” each surrounded by connective tissue. Dual labeling of KIT and smooth muscle myosin revealed KIT-positive stellate-shaped ICC (ICC-IAS) within each minibundle. In the rectum intramuscular ICC (ICC-IM) were spindle shaped whereas stellate-shaped ICC were located at the myenteric surface (ICC-MY). ICC were absent from both the myenteric and submucosal surfaces of the IAS. Nitrergic nerves (identified with anti-neuronal nitric oxide synthase antibodies or NADPH diaphorase activity) and sympathetic nerves (identified with anti-tyrosine hydroxylase antibody) each formed a plexus at the myenteric surface of the rectum but not the IAS. Intramuscular neuronal nitric oxide synthase- and tyrosine hydroxylase-positive fibers were present in both regions but were only closely associated with ICC-IM in rectum. Minimal association was also noted between ICC-IAS and cells expressing the nonspecific neuronal marker PGP9.5. In conclusion, the morphology of rectal ICC-IM and ICC-MY is similar to that described elsewhere in the gastrointestinal tract whereas ICC-IAS are unique. The distribution of stellate-shaped ICC-IAS throughout the musculature and their absence from both the myenteric and submucosal surfaces suggest that ICC-IAS may serve as pacemaker cells in this muscle whereas their limited relationship to nerves suggests that they are not involved in neuromuscular transmission. Additionally, the presence of numerous minibundles, each containing both ICC-IAS and nerves, suggests that this muscle functions as a multiunit type muscle.

Interstitial cells of Cajal (ICC) have important functions in regulation of motor activity in the gastrointestinal tract. In murine small intestine, ICC are gathered in the regions of the myenteric plexus (ICC-MY) and the deep muscular plexus (ICC-DMP). These two classes of ICC have different physiological functions. ICC-MY are pacemaker cells and generate the slow-wave electrical rhythmicity of gastrointestinal organs. ICC-DMP form synaptic connections with the varicose nerve terminals of enteric motor neurons and are involved in reception and transduction of motor neurotransmission. Gene expression underlying specific functions of ICC classes is incompletely understood. In the present study, we used recently developed highly selective techniques to isolate the two functional ICC classes from enzymatically dispersed intestinal muscles by fluorescence-activated cell sorting. The transcriptomes of ICC-MY and ICC-DMP were investigated using oligonucleotide microarray analysis. Differential expression of functional groups of genes defined by standard gene ontology terms was also studied. There were substantial numbers of genes expressed more abundantly in ICC than in the tunica muscularis, and we also detected marked phenotypic differences between ICC-MY and ICC-DMP. Notably, genes related to cell junction, process guidance, and vesicle trafficking were upregulated in ICC. Consistent with their specific functions, metabolic and Ca2+transport genes were relatively upregulated in ICC-MY, whereas genes for signaling proteins involved in transduction of neurotransmitter functions were relatively upregulated in ICC-DMP. Our results may lead to the identification of novel biomarkers for ICC and provide directions for further studies designed to understand ICC function in health and disease.

Specific functions of interstitial cells of Cajal (ICC) have been linked to distinct classes that differ by morphology and distribution. In the small intestine, slow wave-generating ICC are located in the myenteric region (ICC-MY), whereas ICC that mediate neuromuscular neurotransmission occur either throughout the circular muscle layer (intramuscular ICC, ICC-IM) or in association with the deep muscular plexus (ICC-DMP). Selective isolation of ICC to characterize specific properties has been difficult. Recently, neurokinin-1 receptors have been detected in murine ICC-DMP and neurons but not in ICC-MY. Here we identified and isolated ICC-DMP/IM by receptor-mediated internalization of fluorescent substance P and Kit immunofluorescence. Specificity of labeling was verified by confocal microscopy. Mouse and human ICC-DMP/IM were detected in suspension by fluorescent microscopy and harvested for RT-PCR with micropipettes. The isolated cells expressed Kit but not markers for neurons, smooth muscle, or antigen-presenting cells. ICC-DMP expressed neurokinin-1 receptor, M2 and M3 muscarinic receptors, P2Y1 and P2Y4 purinergic receptors, VIP receptor 2, soluble guanylate cyclase-1 subunits, and protein kinase G. L- or T-type Ca2+ channels were not detected in these cells. ICC-MY and ICC-DMP were simultaneously detected and enumerated by flow cytometry and sorted to purity by fluorescence-activated cell sorting. In summary, functional classes of ICC have distinct molecular identities that can be used to selectively identify and harvest these cells with, for example, receptor-mediated uptake of substance P and Kit immunofluorescence. ICC-DMP express neurotransmitter receptors and signaling intermediate molecules that are consistent with their role in neuromuscular neurotransmission.

Spontaneous electrical pacemaker activity occurs in tunica muscularis of the gastrointestinal tract and drives phasic contractions. Interstitial cells of Cajal (ICC) are the pacemaker cells that generate and propagate electrical slow waves. We used Ca2+ imaging to visualize spontaneous rhythmicity in ICC in the myenteric region (ICC-MY) of the murine small intestine. ICC-MY, verified by colabeling with Kit antibody, displayed regular Ca2+ transients that occurred after electrical slow waves. ICC-MY formed networks, and Ca2+ transient wave fronts propagated through the ICC-MY networks at ∼2 mm/s and activated attached longitudinal muscle fibers. Nicardipine blocked Ca2+ transients in LM but had no visible effect on the transients in ICC-MY. β-Glycyrrhetinic acid reduced the coherence of propagation, causing single cells to pace independently. Thus, virtually all ICC-MYs are spontaneously active, but normal activity is organized into propagating wave fronts. Inhibitors of dihydropyridine-resistant Ca2+ entry (Ni2+ and mibefradil) and elevated external K+ reduced the coherence and velocity of propagation, eventually blocking all activity. The mitochondrial uncouplers, FCCP, and antimycin and the inositol 1,4,5-trisphosphate receptor-inhibitory drug, 2-aminoethoxydiphenyl borate, abolished rhythmic Ca2+ transients in ICC-MY. These data show that global Ca2+ transients in ICC-MYs are a reporter of electrical slow waves in gastrointestinal muscles. Imaging of ICC networks provides a unique multicellular view of pacemaker activity. The activity of ICC-MY is driven by intracellular Ca2+ handling mechanisms and entrained by voltage-dependent Ca2+ entry and coupling of cells via gap junctions.

Pacemaker potentials were recorded in situ from myenteric interstitial cells of Cajal (ICC-MY) in the murine small intestine. The nature of the two components of pacemaker potentials (upstroke and plateau) were investigated and compared with slow waves recorded from circular muscle cells. Pacemaker potentials and slow waves were not blocked by nifedipine (3 μM). In the presence of nifedipine, mibefradil, a voltage-dependent Ca2+channel blocker, reduced the amplitude, frequency, and rate of rise of upstroke depolarization (d V/d tmax) of pacemaker potentials and slow waves in a dose-dependent manner (1–30 μM). Mibefradil (30 μM) changed the pattern of pacemaker potentials from rapidly rising, high-frequency events to slowly depolarizing, low-frequency events with considerable membrane noise (unitary potentials) between pacemaker potentials. Caffeine (3 mM) abolished pacemaker potentials in the presence of mibefradil. Pinacidil (10 μM), an ATP-sensitive K+channel opener, hyperpolarized ICC-MY and increased the amplitude and d V/d tmaxwithout affecting frequency. Pinacidil hyperpolarized smooth muscle cells and attenuated the amplitude and d V/d tmaxof slow waves without affecting frequency. The effects of pinacidil were blocked by glibenclamide (10 μM). These data suggest that slow waves are electrotonic potentials driven by pacemaker potentials. The upstroke component of pacemaker potentials is due to activation of dihydropyridine-resistant Ca2+channels, and this depolarization entrains pacemaker activity to create the plateau potential. The plateau potential may be due to summation of unitary potentials generated by individual or small groups of pacemaker units in ICC-MY. Entrainment of unitary potentials appears to depend on Ca2+entry during upstroke depolarization.

Interstitial cells of Cajal in the deep muscular plexus (ICC-DMP) of the small intestine express excitatory neurotransmitter receptors. We tested whether ICC-DMP are functionally innervated by cholinergic neurons in the murine intestine. Muscles were stimulated by intrinsic nerves and ACh and processed for immunohistochemistry to determine these effects on PKC-epsilon activation. Under control conditions, PKC-epsilon-like immunoreactivy (PKC-epsilon-LI) was only observed in myenteric neurons within the tunica muscularis. Electrical field stimulation or ACh caused translocation of neural PKC-epsilon-LI from the cytosol to a peripheral compartment. After stimulation, PKC-epsilon-LI was found in spindle-shaped cells in the DMP. These cells were identified as ICC-DMP by Kit-LI and vimentin-LI. PKC-epsilon-LI in ICC-DMP and translocation of PKC epsilon-LI in neurons were blocked by tetrodotoxin or atropine, suggesting that these responses were due to activation of muscarinic receptors. Western blots also confirmed translocation of PKC-epsilon-LI. In conclusion, PKC-epsilon translocation is linked to muscarinic receptor activation in ICC-DMP and a subpopulation of myenteric neurons. These studies demonstrate that ICC-DMP are functionally innervated by excitatory motoneurons.

Electrical and mechanical activity of the circular muscle layer in the rectoanal region of the gastrointestinal tract undergoes considerable changes in the site of dominant pacemaking activity, frequency, and waveform shape. The present study was performed to determine whether changes in the structural organization of the circular layer or in the density, distribution, and ultrastructure of interstitial cells of Cajal (ICC) could account for this heterogeneity in electrical and mechanical activities. Light microscopy revealed that the structural organization of the circular muscle layer underwent dramatic morphological changes, from a tightly packed layer with poorly defined septa in the proximal rectum to one of discrete muscle bundles separated by large septae in the internal anal sphincter. Kit immunohistochemistry revealed a dense network of ICC along the submucosal and myenteric borders in the rectum, whereas in the internal anal sphincter, ICC were located along the periphery of muscle bundles within the circular layer. Changes in electrical activity within the circular muscle layer can be partially explained by changes in the structure of the muscle layer and changes in the distribution of ICC in the rectoanal region of the gastrointestinal tract.

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 moscillations (MPOs, 12 mV amplitude) were present across the thickness of the internal anal sphincter (IAS; 0.5 cm) and E m was constant (−52 mV). Between the IAS and the proximal rectum an 18 mV gradient in E mdeveloped across the muscle thickness with the submucosal edge at −70 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 μM) 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.

An antibody directed against Kit protein was used to investigate the distribution of interstitial cells of Cajal (ICC) within the murine colon. The ICC density was greatest in the proximal colon and decreased along its length. The distribution of the different classes of ICC in the aganglionic colons of lethal spotted ( ls/ls) mice was found to be similar in age-matched wild-type controls. There were marked differences in the electrical activities of the colons from ls/ls mutants compared with wild-type controls. In ls/ls aganglionic colons, the circular muscle was electrically quiescent compared with the spontaneous spiking electrical activity of wild-type tissues. In ls/ls aganglionic colons, postjunctional neural responses were greatly affected. Inhibitory junction potentials were absent or excitatory junction potentials inhibited by atropine were observed. In conclusion, the distribution of ICC in the ganglionic and aganglionic regions of the colons from ls/ls mutants appeared similar to that of wild-type controls. The electrical activity and neural responses of the circular layer are significantly different in aganglionic segments of ls/ls mutants.

Several human motility disorders have been shown to be associated with loss or defects in interstitial cells of Cajal (ICC) networks. Because tissue samples for these studies were taken from patients with well-advanced motility problems, it is difficult to determine whether the loss of ICC is a cause or a consequence of the disease process. To establish the cause-and-effect relationship of ICC loss in motility disorders, it may be feasible to use animal models in which ICC are lost as motility dysfunction develops. Several models with defects in ICC networks have been developed, and these include animals with defects in the Kit signaling pathway (e.g., white-spotting mutants that have defects in Kit receptors; steel mutants that have mutations in stem cell factor, the ligand for Kit; and animals that are chronically treated with reagents that block Kit or downstream signaling proteins). ICC do not die when Kit signaling is blocked, rather, they redifferentiate into a smooth musclelike phenotype. Diabetic animals (NOD/LtJ mice), animals with chronic bowel obstruction, and inflammatory bowel models also have defects in ICC networks that have been associated with motility disorders. By studying these models with molecular and genomic techniques it may be possible to determine the signals that cause loss of ICC and find ways of restoring ICC to dysfunctional tissues. This article discusses recent progress in the utilization of animal models to study the consequences of losing ICC on the development of motility disorders.

Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα+cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα+cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues.

Interstitial cells of Cajal (ICC) are the pacemaker cells in gastrointestinal (GI) muscles. They also mediate or transduce inputs from enteric motor nerves to the smooth muscle syncytium. What is known about functional roles of ICC comes from developmental studies based on the discovery that ICC express c- kit. Functional development of ICC networks depends on signaling via the Kit receptor pathway. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells within the tunica muscularis, and the loss of ICC in some motility disorders. Manipulating Kit signaling with reagents to block the receptor or downstream signaling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies of the development of these cells within the tunica muscularisand also allowed the study of specific functions of different classes of ICC in several regions of the GI tract. This article examines the role of ICC in GI motility, focusing on the functional development and maintenance of ICC networks in the GI tract and the phenotypic changes that can occur when the Kit signaling pathway is disrupted.

Located within the tunica muscularis of the gastrointestinal (GI) tract are networks of cells known as interstitial cells of Cajal (ICC). ICC are critical for important basic functions of GI motility such as generation and propagation of slow-wave pacemaker activity and reception of regulatory inputs from the enteric nervous system. We have developed a novel procedure to identify and isolate individual ICC from freshly dispersed cell preparations of the murine small intestine and gastric fundus and to determine differential transcriptional expression We have compared the expression profiles of pacemaker ICC isolated from the murine small intestine (IC-MY) and ICC involved in neurotransmission from the gastric fundus (IC-IM). We have also compared expression profiles between ICC and smooth muscle cells (SMC) and between freshly isolated ICC and cultured ICC. Cultured ICC express smooth muscle myosin, whereas freshly dispersed ICC do not. All cell types express muscarinic receptor types M2and M3, neurokinin receptors NK1and NK3, and inhibitory receptor VIP-1, whereas only cultured ICC and SMC express VIP-2. Both cultured and freshly dispersed IC-IM and IC-MY express the soluble form of stem cell factor, whereas SMC from the gastric fundus express only the membrane-bound form.

Voltage-activated K+(KV) channels play an important role in regulating the membrane potential in excitable cells. In gastrointestinal (GI) smooth muscles, these channels are particularly important in modulating spontaneous electrical activities. The purpose of this study was to identify the molecular components that may be responsible for the KV currents found in the canine GI tract. In this report, we have examined the qualitative expression of eighteen different KV channel genes in canine GI smooth muscle cells at the transcriptional level using RT-PCR analysis. Our results demonstrate the expression of KV1.4, KV1.5, KV1.6, KV2.2, and KV4.3 transcripts in all regions of the GI tract examined. Transcripts encoding KV1.2, KVβ1.1, and KVβ1.2 subunits were differentially expressed. KV1.1, KV1.3, KV2.1, KV3.1, KV3.2, KV3.4, KV4.1, KV4.2, and KVβ2.1 transcripts were not detected in any GI smooth muscle cells. We have also determined the protein expression for a subset of these KV channel subunits using specific antibodies by immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry demonstrated that KV1.2, KV1.4, KV1.5, and KV2.2 are expressed at the protein level in GI tissues and smooth muscle cells. KV2.1 was not detected in any regions of the GI tract examined. These results suggest that the wide array of electrical activity found in different regions of the canine GI tract may be due in part to the differential expression of KV channel subunits.

Recently platelet-derived growth factor-α-positive cells (PDGFRα+cells), previously called “fibroblast-like” cells, have been described in the muscle layers of the gastrointestinal tract. These cells form networks and are involved in purinergic motor neurotransduction. Examination of colon from mice with enhanced green fluorescent protein (eGFP) driven from the endogenous Pdgfra (PDGFRα-eGFP mice) revealed a unique population of PDGFRα+cells in the mucosal layer of colon. We investigated the phenotype and potential role of these cells, which have not been characterized previously. Expression of PDGFRα and several additional proteins was surveyed in human and murine colonic mucosae by immunolabeling; PDGFRα+cells in colonic mucosa were isolated from PDGFRα-eGFP mice, and the gene expression profile was analyzed by quantitative polymerase chain reaction. We found for the first time that PDGFRα was expressed in subepithelial cells (subepithelial PDGFRα+cells) forming a pericryptal sheath from the base to the tip of crypts. These cells were in close proximity to the basolateral surface of epithelial cells and distinct from subepithelial myofibroblasts, which were identified by expression of α-smooth muscle actin and smooth muscle myosin. PDGFRα+cells also lay in close proximity to varicose processes of nerve fibers. Mouse subepithelial PDGFRα+cells expressed Toll-like receptor genes, purinergic receptor genes, 5-hydroxytryptamine (5-HT) 4 receptor gene, and hedgehog signaling genes. Subepithelial PDGFRα+cells occupy an important niche in the lamina propria and may function in transduction of sensory and immune signals and in the maintenance of mucosal homeostasis.

We have developed and characterized an organ culture system that maintains the viability of colonic smooth muscles. Morphological, mechanical, electrical, and molecular properties of cultured canine colonic circular muscles were determined. Strips of circular muscle were cultured for up to 6 days. The smooth muscle phenotype was retained during culture; muscles contracted to agonists and responded to electrical field stimulation, suggesting that intrinsic nerves also survived in culture. Morphological analysis showed identifiable smooth muscle cells, enteric neurons, and interstitial cells, but some alterations in ultrastructure were also observed. Mechanical responses to acetylcholine suggested that the muscles developed supersensitivity during the culture period. The resting membrane potentials of cells near the submucosal surface of the circular muscle layer decreased from -82 mV on day 0 to -55 mV on day 3. Similar changes in the resting potential gradient occur when colonic muscles are treated with inhibitors of the Na(+)-K(+)-ATPase. Resting potentials of day 3 muscles remained constant in low external K+ (0.1 mM), suggesting little contribution of the pump to resting potential. Northern analysis of RNA from muscles cultured up to 6 days showed that the alpha 2-isoform of the pump decreased. The data suggest that organ-cultured strips of smooth muscle may provide a useful tool for evaluating electrical and mechanical events in conjunction with molecular analysis of functional components.

The effects of beta-adrenergic receptor stimulation on the electrical and mechanical activity of canine colonic circular muscles were compared with forskolin (Fsk), a known stimulator of adenylate cyclase. The actions of isoproterenol (Iso) were mediated by beta 2-receptors. Iso and Fsk increased intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in both the presence and absence of acetylcholine (ACh), whereas ACh (0.3 microM) alone reduced cAMP levels. These agents caused inhibition of spontaneous and ACh-induced contractions. Inhibition was associated with a reduction in the amplitude and duration of electrical slow waves recorded near the submucosal border. Near the myenteric border, Iso and Fsk hyperpolarized the membrane by up to 30 mV and changed the pattern of electrical rhythmicity. These effects were mimicked by 8-bromo-cAMP (1-3 mM). Contractile inhibition with Fsk and Iso was associated with a decrease in the amplitude and duration of Ca2+ transients measured with fura-2 fluorescence. cAMP (10-300 microM) reduced the Ca2+ sensitivity of the contractile apparatus in muscles permeabilized with staphylococcal alpha-toxin. The actions of Iso appear linked to cAMP. We hypothesize that cAMP produces relaxation both by modulation of membrane ionic channels with a consequent decline in the entry of Ca2+ as well as through a decrease in the sensitivity of the contractile apparatus to Ca2+.

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)

The plateau phase of electrical slow waves in phasic gastrointestinal muscles is critical for excitation-contraction coupling. The plateau appears to depend upon a balance between inward Ca2+ current and outward K+ currents that is sustained for several seconds. Voltage-dependent, non-Ca(2+)-dependent K currents were studied in canine colonic circular muscle cells using the whole cell patch-clamp technique. At room temperature, depolarization activated a slow outward current that showed little inactivation during 500 ms. Increasing the temperature to 37 degrees C significantly increased the rate of activation of voltage-dependent outward current. The onset of the outward current overlapped the transient inward Ca2+ current, suggesting that this K current may act as a brake on the upstroke depolarization of electrical slow waves in intact muscles. Voltage-dependent outward current was sustained for the duration of test pulses. This current balanced the sustained inward current that was also activated at physiological test potentials. The outward current evoked by test pulses positive to -20 mV inactivated by at least 50% within 500 ms. Half inactivation occurred at -36 mV. Voltage-dependent K current was reduced by 4-aminopyridine (4-AP; 1-5 mM), but difference currents obtained by subtracting currents elicited from holding potentials of -45 mV from currents obtained from holding potentials of -100 mV were not affected by 4-AP (1 mM). Studies were also performed on intact muscles to test the effects of 4-AP on electrical slow waves. 4-AP increased the amplitude and rate of rise of the upstroke potential and increased the amplitude and prolonged the plateau phase of slow waves. These data suggest that a rapidly activating, inactivating, voltage-dependent K current participates in electrical slow waves of colonic circular smooth muscles.

Electrical events in longitudinal and circular muscles of the colon are different. Longitudinal muscles generate action potentials superimposed upon small depolarizations termed myenteric potential oscillations and circular muscles generate slow wave events that persist for several seconds. Differences between circular and longitudinal muscles may be related to the potassium channels these cells express. We have studied Ca(2+)-dependent and voltage-dependent K currents of isolated longitudinal cells with the whole cell patch-clamp technique. Test depolarizations positive to -40 mV yielded a transient inward current followed by a large sustained outward current. Blockade of the inward Ca2+ current reduced the amplitude of the outward current. Outward current was also reduced by tetraethylammonium (TEA; 1 mM), suggesting that a component of the outward current is Ca2+ dependent. After blockade of the Ca(2+)-dependent outward current, a voltage- and time-dependent component of outward current remained. The activation and inactivation properties and sensitivity to TEA and 4-aminopyridine (4-AP) were characterized. The voltage-dependent outward current in longitudinal cells had different properties than the voltage-dependent K currents in circular muscle cells (i.e., more negative inactivation, less sensitivity to 4-AP). TEA (1-5 mM) increased the amplitude and frequency of action potentials in intact longitudinal muscles; 4-AP (1 mM) had little effect on electrical activity of longitudinal muscles. The data suggest that differences in electrical behavior of the 2 muscle layers may be related to the expression of different species of K channels.

Nonadrenergic, noncholinergic (NANC) nerves regulate slow waves along the submucosal border of the canine proximal colon. Experiments were performed to determine the role of nitric oxide (NO) in NANC responses. NANC responses are characterized by hyperpolarization and reduction in slow-wave amplitude and duration during the period of stimulation. This is followed by a "rebound" excitation (increase in amplitude and duration) of the slow wave immediately after the stimulus. These responses were blocked by L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase and by tetrodotoxin (TTX). Exogenous NO mimicked NANC responses, including the poststimulus rebound excitation. Responses to NO were unaffected by L-NAME or TTX. Responses to NANC nerve stimulation and NO were blocked by oxyhemoglobin but not by methemoglobin. Rebound excitation was reduced by pretreatment with indomethacin, suggesting that an eicosanoid may mediate this phase of NANC responses. Taken together, these data suggest that NO mediates NANC nerve responses in the proximal colon. NO appears to directly cause the inhibitory response, but the rebound response may depend on release of an eicosanoid.

Intracellular recordings were made from isolated circular muscle bundles of rat gastric fundus. The majority of cells generated an ongoing discharge of electrical activity that were ≤10 mV in amplitude (unitary potentials). A second pattern of electrical activity was recorded in less than 1% of all impalements. This electrical activity was characterized by high frequency, large amplitude spontaneous transient depolarizations (STDs) with a maximum rate of rise (d V/d tmax) of 0.5 V/s. Injection of the fluorescent dye propidium iodide into cells and double labeling with an antibody against the Kit receptor revealed that unitary potentials were recorded from circular smooth muscle cells (CSMC), whereas STDs were generated by intramuscular interstitial cells of Cajal (ICC-IM). Sustained injection periods (>15 min) resulted in the spread of dye between CSMC, between ICC-IM, and between CSMC and ICC-IM. Two types of STDs were observed, regularly occurring continuous STDs and irregular noisy bursting STDs. The amplitude of STDs varied between the two types of STDs. Single units summed to develop STDs with a maximum amplitude of 30 mV. Sodium nitroprusside (3 μM) induced membrane hyperpolarization and abolished unitary potentials generated by CSMC. In contrast, the amplitude of STDs generated by ICC-IM was increased with membrane hyperpolarization. Hyperpolarization induced by pinacidil (10 μM) also increased the amplitude of STDs and enhanced d V/d tmax. These observations indicate that STDs generated in ICC-IM spread passively to the adjacent CSMC to evoke the discharge of unitary potentials in the gastric fundus.

Previous evidence suggests that nonadrenergic, noncholinergic (NANC) inhibitory neurotransmission in visceral muscles may be mediated by nitric oxide (NO). We have demonstrated that NO and the NO carrier S-nitrosocysteine can mimic the hyperpolarization in colonic muscle caused by nerve stimulation. The finding that S-nitrosocysteine breaks down fast enough to cause inhibitory junction potential (IJP)-like hyperpolarizations suggests that NO could be stored as a nitrosothiol in secretory vesicles in nerve terminals. Oxyhemoglobin blocked hyperpolarization responses to NO and S-nitrosocysteine and NANC IJPs. These findings suggest that NO is a biologically active transmitter substance in NANC inhibitory neurotransmission. NO enhanced the open probability of Ca(2+)-activated K+ channels in isolated colonic muscle cells. These channels may mediate the hyperpolarization response to NANC neurotransmission in colonic muscles.

The effects of calyculin A, a phosphatase inhibitor isolated from the marine sponge Discodermia calyx, on the electrical activity of colonic and gastric muscles were studied. Calyculin A reduced the amplitude and duration of slow waves, primarily by inhibiting the plateau component. Okadaic acid, another phosphatase inhibitor, also reduced the amplitude and duration of gastric slow waves. The mechanism of action of calyculin A was investigated by studying its effects on inward currents of isolated gastric and colonic myocytes. Calyculin A reduced the amplitude of the peak and the sustained components of the inward current. Okadaic acid had similar effects. These data suggest that phosphorylation of Ca2+ channels of gastrointestinal smooth muscles may inhibit Ca2+ currents. This mechanism may provide an important means of regulating the currents responsible for excitation-contraction coupling in these muscles.

Experiments were performed to determine the involvement of nitric oxide synthase in the generation of nonadrenergic, noncholinergic (NANC) inhibitory junction potentials (IJPs) in the canine proximal colon. Smooth muscle cells were impaled near the myenteric border between the circular and longitudinal layers. Cells exhibited rhythmic myenteric potential oscillations. IJPs were evoked with electrical field stimulation in the presence of drugs to block adrenergic and cholinergic neurotransmission. N omega-nitro-L-arginine, methyl ester (L-NAME), a nitric oxide synthase inhibitor, progressively reduced the amplitude of IJPs. The effect of L-NAME was reversed by L-Arg but not by the stereoisomer D-Arg. IJPs disrupt the regular pattern of myenteric potential oscillations. This effect was also blocked by L-NAME and reversed by L-Arg. These experiments suggest that a product of the nitric oxide synthetic pathway is involved in NANC neurotransmission in the canine proximal colon.

Morphological and electrophysiological experiments were performed to characterize the pacemaker areas of the circular muscle in the canine proximal colon. Morphological studies showed interstitial cells of Cajal lining the submucosal surface of the circular layer and the septal structures that separate the circular layer into bundles. Electrical measurements suggested that slow waves may propagate into the thickness of the circular muscle in a regenerative manner along the surface of these septa. Removal of the submucosal pacemaker region blocked generation of slow waves in nonseptal regions of the circular muscle, but slow-wave activity continued in the circular muscle near septa. These data suggest that slow-wave pacemaker activity is not limited to a two-dimensional surface at the submucosal surface but extends into the interior of the circular layer along septal invaginations. Experiments were also performed to determine the dominance of pacemaker activity (i.e., septal vs. submucosal), and examples were found in which both areas appeared to initiate slow waves in intact muscles. Other studies showed that slow waves could propagate across septa, suggesting some form of electrical coupling between circular muscle bundles. This study provides a more complete view of the structure and function of pacemaker areas in the canine proximal colon.