Septal Interstitial Cells of Cajal Conduct Pacemaker Activity to Excite Muscle Bundles in Human Jejunum
2007; Elsevier BV; Volume: 133; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2007.06.024
ISSN1528-0012
AutoresHyun–Tai Lee, Grant W. Hennig, Neal Fleming, Kathleen D. Keef, Nick J. Spencer, Sean M. Ward, Kenton M. Sanders, Terence K. Smith,
Tópico(s)Diet and metabolism studies
ResumoBackground & Aims: Like the heart, intestinal smooth muscles exhibit electrical rhythmicity, which originates in pacemaker cells surrounding the myenteric plexus, called interstitial cells of Cajal (ICC-MY). In large mammals, ICC also line septa (ICC-SEP) between circular muscle (CM) bundles, suggesting they might be necessary for activating muscle bundles. It is important to determine their functional significance, because a loss of ICC in humans is associated with disordered motility. Our aims were therefore to determine the role of ICC-SEP in activating the thick CM in the human jejunum. Methods: The mucosa and submucosa were removed and muscle strips were cut and pinned in cross-section so that the ICC-MY and ICC-SEP networks and the CM could be readily visualized. The ICC networks and CM were loaded with the Ca2+ indicator fluo-4, and pacemaker and muscle activity was recorded at 36.5 ± 0.5°C. Results: Ca2+ imaging revealed that pacemaker activity in human ICC-MY can entrain ICC-SEP to excite CM bundles. Unlike the heart, pacemaker activity in ICC-MY varied in amplitude, propagation distance, and direction, leading to a sporadic activation of ICC-SEP. Conclusions: ICC-SEP form a crucial conduction pathway for spreading excitation deep into muscle bundles of the human jejunum, necessary for motor patterns underlying mixing. A loss of these cells could severely affect motor activity. Background & Aims: Like the heart, intestinal smooth muscles exhibit electrical rhythmicity, which originates in pacemaker cells surrounding the myenteric plexus, called interstitial cells of Cajal (ICC-MY). In large mammals, ICC also line septa (ICC-SEP) between circular muscle (CM) bundles, suggesting they might be necessary for activating muscle bundles. It is important to determine their functional significance, because a loss of ICC in humans is associated with disordered motility. Our aims were therefore to determine the role of ICC-SEP in activating the thick CM in the human jejunum. Methods: The mucosa and submucosa were removed and muscle strips were cut and pinned in cross-section so that the ICC-MY and ICC-SEP networks and the CM could be readily visualized. The ICC networks and CM were loaded with the Ca2+ indicator fluo-4, and pacemaker and muscle activity was recorded at 36.5 ± 0.5°C. Results: Ca2+ imaging revealed that pacemaker activity in human ICC-MY can entrain ICC-SEP to excite CM bundles. Unlike the heart, pacemaker activity in ICC-MY varied in amplitude, propagation distance, and direction, leading to a sporadic activation of ICC-SEP. Conclusions: ICC-SEP form a crucial conduction pathway for spreading excitation deep into muscle bundles of the human jejunum, necessary for motor patterns underlying mixing. A loss of these cells could severely affect motor activity. Different strategies have evolved to conduct excitation deep into muscle in order to simultaneously release the Ca2+ necessary to excite many fibrils to maximize force. Microscopically, invaginations of the plasma membrane (T-tubules) conduct action potentials deep into cardiac and skeletal muscle myocytes.1Berne R.M. Levy M.N. The cardiovascular system.in: Berne R.M. Levy M.N. Physiology. 3rd ed. Mosby, St. Louis1993: 382-385Google Scholar, 2Murphy R.A. Muscle.in: Berne R.M. Levy M.N. Physiology. 3rd ed. Mosby, St. Louis1993: 281-291Google Scholar Macroscopically, in order to activate the large ventricles of the heart a specialized conduction system originates in the atrioventricular node and consists of the bundle of His, which divides to run down both sides of the septa, separating each ventricle, where it ramifies into Purkinje fibers supplying the ventricles.1Berne R.M. Levy M.N. The cardiovascular system.in: Berne R.M. Levy M.N. Physiology. 3rd ed. Mosby, St. Louis1993: 382-385Google Scholar This conduction system consists of specialized myocytes connected by gap junctions. The smooth muscle layers of the gastrointestinal tract of mammals also exhibit rhythmic depolarizations, referred to as slow waves, which initiate the rhythmic contractions of motility patterns.3Sanders K.M. Koh S.D. Ward S.M. Interstitial cells of Cajal as pacemakers in the gastrointestinal tract.Annu Rev Physiol. 2006; 68: 307-343Crossref PubMed Scopus (509) Google Scholar, 4Edwards F.R. Hirst G.D.S. An electrical description of the generation of slow waves in the antrum of the guinea-pig.J Physiol. 2005; 564: 213-232Crossref PubMed Scopus (33) Google Scholar, 5Farrelly A.M. Ro S. Callaghan B.P. et al.Expression and function of KCNH2 (HERG) in the human jejunum.Am J Physiol. 2003; 284: G883-G895Google Scholar, 6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Park K.J. Hennig G.W. Lee H.T. et al.Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ.Am J Physiol. 2006; 290: C1411-C1427Crossref PubMed Scopus (95) Google Scholar, 8Bauer A.J. Sarr M.G. Szurszewski J.H. Opioids inhibit neuromuscular transmission in circular muscle of human and baboon jejunum.Gastroenterology. 1991; 101: 970-976PubMed Google Scholar, 9Stark M.E. Bauer A.J. Sarr M.G. et al.Nitric oxide mediates inhibitory nerve input in human and canine jejunum.Gastroenterology. 1993; 104: 398-409PubMed Google Scholar, 10Smith T.K. Reed J.B. Sanders K.M. Interaction of two electrical pacemakers in muscularis of canine proximal colon.Am J Physiol. 1987; 252: C290-C299PubMed Google Scholar, 11Ward S.M. Sanders K.M. Pacemaker activity in septal structures of canine colonic circular muscle.Am J Physiol. 1990; 259: G264-G273PubMed Google Scholar, 12Horiguchi K. Semple G.S.A. Sanders K.M. et al.Distribution of pacemaker function through the tunica muscularis of the canine gastric antrum.J Physiol. 2001; 537: 237-250Crossref PubMed Scopus (88) Google Scholar In small mammals, the pacemaker apparatus consists of a 2-dimensional network of stellate cells, which lie between the longitudinal muscle (LM) and circular muscle (CM) layers, called the myenteric interstitial cells of Cajal (ICC-MY).3Sanders K.M. Koh S.D. Ward S.M. Interstitial cells of Cajal as pacemakers in the gastrointestinal tract.Annu Rev Physiol. 2006; 68: 307-343Crossref PubMed Scopus (509) Google Scholar, 7Park K.J. Hennig G.W. Lee H.T. et al.Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ.Am J Physiol. 2006; 290: C1411-C1427Crossref PubMed Scopus (95) Google Scholar The ICC-MY are connected to one another and to the neighboring smooth muscle layers by gap junctions.3Sanders K.M. Koh S.D. Ward S.M. Interstitial cells of Cajal as pacemakers in the gastrointestinal tract.Annu Rev Physiol. 2006; 68: 307-343Crossref PubMed Scopus (509) Google Scholar The ICC and muscle cells appear to develop from common mesenchymal precursor cells.13Torihashi S. Ward S.M. Sanders K.M. Development of c-Kit–positive cells and the onset of electrical rhythmicity in murine small intestine.Gastroenterology. 1997; 112: 144-155Abstract Full Text PDF PubMed Scopus (209) Google Scholar Pacemaker tissues in the gut are more extensive than in the heart. The network of ICC is also required for active propagation of slow waves, so it runs around the entire circumference and along the length of the organs that are active phasically. In small mammals, ICC-MY can readily activate both muscle layers of the gastrointestinal tract.7Park K.J. Hennig G.W. Lee H.T. et al.Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ.Am J Physiol. 2006; 290: C1411-C1427Crossref PubMed Scopus (95) Google Scholar, 14Hennig G.W. Hirst G.D.S. Park K.J. et al.Propagation of pacemaker activity in the guinea-pig antrum.J Physiol. 2004; 556: 585-599Crossref PubMed Scopus (64) Google Scholar The structure of gastrointestinal pacemaker networks is more complex in larger mammals. In addition to the 3-dimensional ICC-MY network, ICC also line septa (ICC-SEP) separating CM bundles, as in the canine stomach and colon.11Ward S.M. Sanders K.M. Pacemaker activity in septal structures of canine colonic circular muscle.Am J Physiol. 1990; 259: G264-G273PubMed Google Scholar, 12Horiguchi K. Semple G.S.A. Sanders K.M. et al.Distribution of pacemaker function through the tunica muscularis of the canine gastric antrum.J Physiol. 2001; 537: 237-250Crossref PubMed Scopus (88) Google Scholar Gap junctions have been observed between ICC-SEP and between ICC-SEP and adjacent smooth muscle cells.11Ward S.M. Sanders K.M. Pacemaker activity in septal structures of canine colonic circular muscle.Am J Physiol. 1990; 259: G264-G273PubMed Google Scholar Myocytes in CM bundles are arranged orthogonal to the long axis of the gut, so that they can contract as a thick ring. Muscle bundles are also electrically coupled to each other via anastomosing branches.15Gabella G. Arrangement of smooth muscle cells and intramuscular septa in the taenia coli.Cell Tissue Res. 1977; 184: 195-212Crossref PubMed Scopus (25) Google Scholar Although action potentials are likely to spread rapidly around the intestine along a CM bundle,16Tomita T. Spread of excitation in smooth muscle.Prog Clin Biol Res. 1990; 327: 361-373PubMed Google Scholar they can propagate only a short distance (approximately 100–200 μm) orthogonal to the direction of muscle fibers.17Spencer N.J. Hennig G.W. Smith T.K. Electrical rhythmicity and spread of action potentials in longitudinal muscle of guinea pig distal colon.Am J Physiol. 2002; 282: G904-G917PubMed Google Scholar, 18Stevens R.J. Publicover N.G. Smith T.K. Induction and organization of Ca2+ waves by enteric neural reflexes.Nature. 1999; 399: 62-66Crossref PubMed Scopus (65) Google Scholar, 19Hennig G.W. Smith C.B. O'Shea D.M. et al.Patterns of intracellular and intercellular Ca2+ waves in the longitudinal muscle layer of the murine large intestine in vitro.J Physiol. 2002; 543: 233-253Crossref PubMed Scopus (54) Google Scholar, 20Tack J. Smith T.K. Calcium imaging of gut activity.Neurogastroenterol Motil. 2004; 16: 86-95Crossref PubMed Scopus (21) Google Scholar Therefore, a specialized conduction system such as ICC-SEP may be required to organize the excitability of thick CM layers.11Ward S.M. Sanders K.M. Pacemaker activity in septal structures of canine colonic circular muscle.Am J Physiol. 1990; 259: G264-G273PubMed Google Scholar At present, little is known about the role of ICC-SEP in coordinating the spread on slow waves in large mammals. However, this has become an important question, because loss of ICC is associated with numerous human motility disorders.21Kubota M. Kanda E. Ida K. et al.Severe gastrointestinal dysmotility in a patient with congenital myopathy: causal relationship to decrease of interstitial cells of Cajal.Brain Dev. 2005; 27: 447-450Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 22Vanderwinden J.M. Rumessen J.J. Interstitial cells of Cajal in human gut and gastrointestinal disease.Microsc Res Tech. 1999; 47: 344-360Crossref PubMed Scopus (181) Google Scholar, 23Lee J.I. Park H. Kamm M.A. et al.Decreased density of interstitial cells of Cajal and neuronal cells in patients with slow-transit constipation and acquired megacolon.J Gastroenterol Hepatol. 2005; 20: 1292-1298Crossref PubMed Scopus (93) Google Scholar Our aims were, therefore, to investigate the structure of the pacemaker in human small intestinal muscles and to determine how pacemaker activity spreads from the ICC-MY network to muscle bundles within the interior of the CM layer. The segments of human small intestine used in this study were obtained from the jejunum of obese patients of either sex ranging in age from 28 to 56 years as surgical waste tissues during gastric bypass operations performed for morbid obesity.5Farrelly A.M. Ro S. Callaghan B.P. et al.Expression and function of KCNH2 (HERG) in the human jejunum.Am J Physiol. 2003; 284: G883-G895Google Scholar, 6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar The protocol was approved by the Human Subjects Research Committees at the University of Nevada and the University of California Davis. A segment of jejunum was opened along the mesenteric border, and the mucosa and submucosa were removed by sharp dissection. Thin strips (0.5–1.0 mm thick, 10 mm long) were cut parallel to the LM with a double-bladed scalpel.5Farrelly A.M. Ro S. Callaghan B.P. et al.Expression and function of KCNH2 (HERG) in the human jejunum.Am J Physiol. 2003; 284: G883-G895Google Scholar, 10Smith T.K. Reed J.B. Sanders K.M. Interaction of two electrical pacemakers in muscularis of canine proximal colon.Am J Physiol. 1987; 252: C290-C299PubMed Google Scholar These full-muscle-thickness (15 × 15 mm square; approximately 1 mm thick) preparations were pinned serosal side up, CM down, to the base of the organ bath. Strips of LM were then peeled away to reveal the underlying ICC-MY network.6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Both preparations were continuously perfused with oxygenated Krebs solution (see below) at 36.5 ± 0.5°C and equilibrated for 4 hours before the dye loading procedure. The methods for dye loading ICC have been published in detail elsewhere.6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Park K.J. Hennig G.W. Lee H.T. et al.Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ.Am J Physiol. 2006; 290: C1411-C1427Crossref PubMed Scopus (95) Google Scholar, 14Hennig G.W. Hirst G.D.S. Park K.J. et al.Propagation of pacemaker activity in the guinea-pig antrum.J Physiol. 2004; 556: 585-599Crossref PubMed Scopus (64) Google Scholar, 18Stevens R.J. Publicover N.G. Smith T.K. Induction and organization of Ca2+ waves by enteric neural reflexes.Nature. 1999; 399: 62-66Crossref PubMed Scopus (65) Google Scholar, 19Hennig G.W. Smith C.B. O'Shea D.M. et al.Patterns of intracellular and intercellular Ca2+ waves in the longitudinal muscle layer of the murine large intestine in vitro.J Physiol. 2002; 543: 233-253Crossref PubMed Scopus (54) Google Scholar After equilibration, tissues were incubated with fluo-4 (Molecular Probes, Eugene, OR) dissolved in a Krebs solution with 0.8% dimethyl sulfoxide and 0.2% Cremophor EL for 20 minutes at 25°C. The final loading concentration of fluo-4 in the tissue bath was 50 μg/5 mL (approximately 10 μmol/L). The dye primarily loaded into the exposed ICC network. Following incubation, tissues were re-perfused with Krebs solution to allow for de-esterification and trapping of the dye in cells.6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Park K.J. Hennig G.W. Lee H.T. et al.Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ.Am J Physiol. 2006; 290: C1411-C1427Crossref PubMed Scopus (95) Google Scholar, 14Hennig G.W. Hirst G.D.S. Park K.J. et al.Propagation of pacemaker activity in the guinea-pig antrum.J Physiol. 2004; 556: 585-599Crossref PubMed Scopus (64) Google Scholar Preparations were then illuminated by a 100-W high-pressure mercury burner and viewed with a BX50WI upright microscope fitted with epi-fluorescence using ×4, ×10, and ×20 lenses (Olympus UMPlanF; Olympus, Melville, NY). Appropriate filters produced excitation of fluo-4 between 460 and 490 nm, and passed emissions >515 nm (peaks: excitation, 490 nm; emission, 515 nm). Image sequences were captured at 15.6 frames per second using a Cascade 512B camera (Roper Scientific Inc., Trenton, NJ) and MetaMorph 6.26 software (Molecular Devices Corp, Downington, PA).6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Image sequences were analyzed using custom written software (Volumetry G6a, G.W.H.).6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Park K.J. Hennig G.W. Lee H.T. et al.Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ.Am J Physiol. 2006; 290: C1411-C1427Crossref PubMed Scopus (95) Google Scholar, 14Hennig G.W. Hirst G.D.S. Park K.J. et al.Propagation of pacemaker activity in the guinea-pig antrum.J Physiol. 2004; 556: 585-599Crossref PubMed Scopus (64) Google Scholar, 19Hennig G.W. Smith C.B. O'Shea D.M. et al.Patterns of intracellular and intercellular Ca2+ waves in the longitudinal muscle layer of the murine large intestine in vitro.J Physiol. 2002; 543: 233-253Crossref PubMed Scopus (54) Google Scholar Ca2+-induced fluorescence was measured in individual cells and characteristics of Ca2+ transients including frequency, time to peak, duration, and amplitude were calculated. In selected regions in the field of view (FOV), spatiotemporal (ST) maps were constructed. Fluorescence was averaged for each pixel row (horizontal) or column (vertical) within the region of interest (ROI), and the resulting lines of averaged pixels, representing fluorescence from many cells, were placed alongside each other from left to right, creating an ST map of Ca2+-induced fluorescence for the selected region (time progressing to the right). Velocity of propagation of Ca2+ waves was calculated from ST maps. For display purposes, all ST maps and individual frames presented are subtracted from the average background. The unit of Ca2+-induced fluorescence is an arbitrary unit, intensity unit, corresponding to 1 pixel intensity change captured on a 16-bit gray scale. The F/F0 or F/Favg ratio was not used because of bleaching over the long recording periods. To create images of the network of ICC-MY and ICC-SEP and muscle in cross-sectional preparations, the average Ca2+ fluorescence was summed over 1000 frames. Using live labeling with Kit antibodies6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Park K.J. Hennig G.W. Lee H.T. et al.Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ.Am J Physiol. 2006; 290: C1411-C1427Crossref PubMed Scopus (95) Google Scholar we verified the presence of rhythmic fluorescent Ca2+ transients in identified ICC-MY. After Ca2+-induced fluorescence was recorded, tissues were labeled with the AB-4 combination of murine anti-CD117/Kit/stem cell receptor monoclonal antibodies (clones K69+, K44.2 K45; 5 μg mL−1; Lab Vision Corp, Fremont, CA) for 1 hour at room temperature. Tissues were then washed with Krebs solution for 30 minutes before being incubated in Alexa Fluor 594 goat antimouse IgG (1:200; Molecular Probes) for 1 hour. Following secondary antibody labeling, tissues were again washed for 30 minutes before being examined. Labeled ICC were visualized using a Texas Red filter set (peaks: excitation, 560 nm; emission, 645 nm; Filter Set 41004, Chroma Technology, Rockingham, VT) and images overlaid with Ca2+-induced fluorescence signals. Results are expressed as mean ± standard error. Data were evaluated by the paired or unpaired Student's t test; P < .05 was accepted as representing a statistical difference. The letter "n" refers to the number of tissues (1 or 2 tissues were taken from each segment of jejunum). The Krebs solution used in this study contained (in mmol/L): 120.4 NaCl, 5.9 KCl, 15.5 NaHCO3, 11.5 glucose, 1.2 MgCl2, 1.2 NaH2PO4, and 2.5 CaCl2. The solution had a pH of 7.3–7.4 at 36.5°C when bubbled with a mixture of 97% O2 and 3% CO2. Cremophor EL, dimethyl sulfoxide, 18β-glycyrrhetinic acid, nicardipine, and tetrodotoxin were purchased from Sigma-Aldrich Co (St. Louis, MO). Labeling cross-sectional preparations with antibodies to the Kit receptor3Sanders K.M. Koh S.D. Ward S.M. Interstitial cells of Cajal as pacemakers in the gastrointestinal tract.Annu Rev Physiol. 2006; 68: 307-343Crossref PubMed Scopus (509) Google Scholar, 6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Park K.J. Hennig G.W. Lee H.T. et al.Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ.Am J Physiol. 2006; 290: C1411-C1427Crossref PubMed Scopus (95) Google Scholar, 14Hennig G.W. Hirst G.D.S. Park K.J. et al.Propagation of pacemaker activity in the guinea-pig antrum.J Physiol. 2004; 556: 585-599Crossref PubMed Scopus (64) Google Scholar enabled us to identify the complex architecture of the pacemaker network in the human small intestine. The ICC-MY network varied in thickness (100–250 μm) along the bowel, becoming thicker between muscle bundles to form dense, triangular areas of ICC-MY that were continuous with ICC-SEP that extended along the outside of individual CM bundles (Figure 1A–C and Movie 1; See supplemental material online at www.gastrojournal.org). ICC-SEP extended along the surface of CM bundles (Figure 1A–C), and we resolved penetration of the ICC-SEP to a depth of at least 607 ± 52 μm (52 ± 5% of the total CM thickness). The maximum height and width of a CM bundle averaged 1153 ± 48 μm and 546 ± 42 μm (n = 5), respectively. Cross-sectional preparations allowed dynamic resolution of the spread of pacemaker activity through the ICC-MY network and into ICC-SEP. This was not possible using flat-sheet preparations.6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Penetration of slow waves through ICC-SEP resulted in activation of muscle bundles within the CM layer. In preparations where only the ICC-MY and ICC-SEP were loaded with fluo-4, their activity could be studied without contamination of Ca2+ signals from either the adjacent LM or CM. Spontaneous rhythmic Ca2+ transients occurred in ICC-MY (frequency, 6.50 ± 0.34 c/min; range, 4 to 9 c/min; n = 16; duration, 7.44 ± 0.22 s; n = 26) (Figure 1D and E). The identity of these active cells was confirmed by spatially imaging Ca2+ transients and comparing their location with Kit antibody labeling of ICC (Figure 1B–E).6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 7Park K.J. Hennig G.W. Lee H.T. et al.Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ.Am J Physiol. 2006; 290: C1411-C1427Crossref PubMed Scopus (95) Google Scholar, 14Hennig G.W. Hirst G.D.S. Park K.J. et al.Propagation of pacemaker activity in the guinea-pig antrum.J Physiol. 2004; 556: 585-599Crossref PubMed Scopus (64) Google Scholar Each Ca2+ transient was biphasic, consisting of a rapid upstroke phase (time to peak, 0.43 ± 0.01 second; n = 25), followed by a prolonged plateau phase (time to peak, 2.51 ± 0.09 seconds; n = 25; duration, 6.18 ± 0.22 seconds; n = 25; Figure 2C), which is similar in waveform to electrical slow waves and Ca2+ transients in flat-sheet preparations (Movie 2; See supplemental material online at www.gastrojournal.org).6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar The generation of pacemaker activity in ICC-MY was not dependent on neural activity, as it was unaffected by tetrodotoxin (1 μmol/L; n = 5).6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 18Stevens R.J. Publicover N.G. Smith T.K. Induction and organization of Ca2+ waves by enteric neural reflexes.Nature. 1999; 399: 62-66Crossref PubMed Scopus (65) Google Scholar Pacemaker activity originated in ICC-MY near the surface of either the LM (n = 8) or the CM (n = 3) and propagated toward the opposite muscle, while at the same time propagating along the network at a velocity of 4.04 ± 0.49 mm/s (n = 7). As pacemaker activity propagated along the ICC-MY network, it also conducted into ICC-SEP lying along the edges of CM bundles (Figure 2A and D, Movie 3; See supplemental material online at www.gastrojournal.org). In many cases the spread of activity in ICC-MY failed to activate all ICC-SEP within the FOV. We also noted that activation of any given ICC-SEP was not obligatory during every pacemaker cycle. Both the upstroke and plateau phases of the pacemaker Ca2+ transient declined in amplitude in a linear manner down the ICC-SEP, reaching 37% of their initial amplitude at the myenteric border at a depth of around 240 μm (Figure 2B). The rate of rise of the plateau phase, which is linearly dependent upon the size of the upstroke phase,8Bauer A.J. Sarr M.G. Szurszewski J.H. Opioids inhibit neuromuscular transmission in circular muscle of human and baboon jejunum.Gastroenterology. 1991; 101: 970-976PubMed Google Scholar also decreased as Ca2+ transients propagated through ICC-SEP (Figure 2A and D). However, ICC-SEP, like the cells within the conduction system of the heart, were also capable of generating rhythmic pacemaker activity when they were observed to be disconnected from ICC-MY. Rhythmic Ca2+ transients in ICC-SEP that appeared to be disconnected from ICC-MY occurred at a lower frequency than those in ICC-MY (ICC-SEP 3.07 ± 0.64 c/min vs ICC-MY 6.36 ± 0.44 c/min; n = 7, P < .01). In cross-sectional preparations where the muscle was also loaded with fluo-4, pacemaker activity could be observed to spread along the ICC-MY network, leading to the activation of robust Ca2+ transients (amplitude, 1/2; duration, 0.94 ± 0.06 seconds; n = 13) in one or more CM bundles, an example of which is shown in Figure 3A and B. Unlike the heart, however, each pacemaker cycle in ICC-MY did not necessarily activate all CM bundles within a given FOV (Figure 3A and B). If pacemaker activity in ICC-SEP caused the muscle to reach threshold, a rapid, large-amplitude Ca2+ transient was seen to propagate from the surface of the CM bundle into its interior (Figure 3C). It was not surprising that all the muscle bundles within the FOV were not activated simultaneously, given that not all the ICC-SEP were activated at the same time (Figure 3A and C). In fact, activation of CM bundles in relation to a passing wave of pacemaker activity appeared to be irregular, because a particular bundle could be activated several times in succession, several bundles could be activated simultaneously, or activity could switch between adjacent muscle bundles (Figure 4A–C,Movie 4; See supplemental material online at www.gastrojournal.org). It could be argued that the random activation of CM bundles was somewhat artificial, given the narrow width of our cross-sectional preparations. This seems unlikely, however, because a similar random activation of CM bundles was observed in flat-sheet preparations (Figure 4D). These preparations often showed a stepwise propagation across muscle bundles that could reverse direction of propagation after one or more cycles (Figure 4D). The rapid Ca2+ transients in muscle bundles were abolished by nicardipine (1 μmol/L; n = 6), suggesting that they were generated by Ca2+-dependent action potentials through activation of L-type Ca2+ channels.3Sanders K.M. Koh S.D. Ward S.M. Interstitial cells of Cajal as pacemakers in the gastrointestinal tract.Annu Rev Physiol. 2006; 68: 307-343Crossref PubMed Scopus (509) Google Scholar, 5Farrelly A.M. Ro S. Callaghan B.P. et al.Expression and function of KCNH2 (HERG) in the human jejunum.Am J Physiol. 2003; 284: G883-G895Google Scholar, 6Lee H.T. Hennig G.W. Fleming N.W. et al.The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine.Gastroenterology. 2007; 132: 1852-1865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 17Spencer N.J. Hennig G.W. Smith T.K. Electrical rhythmicity and spread of acti
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