555 research outputs found
LabPatch, an acquisition and analysis program for patch-clamp electrophysiology
No full text An acquisition and analysis program, “LabPatch,” has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/ . </jats:p
Figure 1: Effect of TNF and IL-10 on the clearance of
No full textHospital Leiden, Netherlands; and Department of Psychonomics, Free University Amsterdam (D I Boomsma PhD) Correspondence to: Dr Rudi G J Westendorp, Department of Clinical Epidemiology, University Hospital Leiden, C0-P, 2300 RC Leiden, Netherlands Genetic influence on cytokine production and fatal meningococcal disease Rudi G J Westendorp, Jan A M Langermans, Tom W J Huizinga, Abdul H Elouali, Cornelis L Verweij, Dorret I Boomsma, Jan P Vandenbrouke Introduction There is a strong genetic component to fatal infectious disease. Adoptees have a fivefold increased risk of fatal infectious disease if a biological parent has died from infection. 1 By contrast, death from infection in an adoptive parent resulted in no excess relative risks of death. Our knowledge of the factors that contribute to this genetic susceptibility is limited. Tumour necrosis factor (TNF) is a decisive proinflammatory mediator in the host defence to infection. Treatment with recombinant human TNF can pr
Assessment of Healthy Colonic Motility Patterns, Colonic Dysmotility, and its Association with Autonomic Nervous System Dysfunction
No full textIntroduction: Functional motility disorders of the colon are poorly defined. Hence, patients with chronic colonic motor dysfunction are treated or undergo surgery without proper diagnosis. Most colonic motility assessment centers around the largest propagating motor pattern in the colon- the High Amplitude Propagating Pressure Wave (HAPW). However, there is no consensus regarding a definition of this important motor pattern. Additionally, no consideration is given to other aspects of colonic motility such as colo-ano-rectal coordination and control by the autonomic nervous system (ANS). The aim of this thesis was to improve understanding of HAPWs and other features of colonic motility in health and constipation, understand how autonomic dysfunction is related to observations in patients, and evaluate the effect of neuromodulation of the ANS. Methods: Motility was assessed in healthy volunteers and patients using water-perfused High Resolution Colonic Manometry (HRCM). To assess the association between ANS and colonic motor activity Heart Rate Variability (HRV) was measured in patients. Spatiotemporal maps were created using HRCM to analyse and quantify colonic motor activity following baseline, and interventions which included proximal balloon distention, meal, and rectal bisacodyl. Low-Level Light Therapy (LLLT) was also applied during HRCM as a method of neuromodulation, to observe its effect on colonic motility. Results: Normal HAPWs are those which have an amplitude of more than 50 mmHg and belong to one of 3 categories: proximally originating, proximal continuing, and transverse/descending. The best intervention sequence to generate these during HRCM assessment is baseline, proximal balloon distention, meal, rectal bisacodyl. Based on their responses to these interventions and the type of HAPWs present, patients could be classified into strong responder, weak responder and non-responder groups. Overall, patients in the strong responder group were most similar to healthy volunteers both with regard to motility and ANS control. Conversely, the weak and non-responders had showed decreased or no motility with decreased parasympathetic input and occasionally sympathetic inhibition. Additionally, other features of motility such as the sphincter of O'Beirne, and lack of colo-ano-rectal coordination were found to lead to constipation even in presence of normal HAPWs. LLLT shows promise in initiating colonic motor activity through neuromodulation of the sacral defecation center. Conclusions: HAPWs can be defined into one of three categories and used to categorize patients based on their HAPW response to different interventions. However, other aspects of colonic motility such as the colo-ano-rectal coordination and autonomic nervous system control of colonic motility should be taken into consideration in diagnosis of constipation, as they can point towards more non-invasive treatment methods such as neuromodulation using LLLT.ThesisMaster of Science in Medical Sciences (MSMS
Intrinsic electrophysiological properties of interstitial cells of Cajal and smooth muscle cells
No full textThe gastrointestinal (GI) tract is a hollow tubular organ that runs through the length of the central body. To move, mix, and compartmentalize ingesta through this tract, different patterns of motility are needed. This thesis is concerned with the myogenic control of motility through the pacemaker network of interstitial cells of Cajal (ICC) and the smooth muscle cells (SMC). Using patch clamp techniques, the electrophysiological properties of single ICC and SMC were examined. Previous research suggested the possibility of a specialized cell type generating the pacemaker slow wave potentials: the network of ICC that resides in the Auerbach's plexus region of the small intestine. An isolation procedure was developed and optimized to harvest single ICC that can survive short term culture and allow examination by patch clamp. Single cell patch clamp recordings demonstrated the presence of slow wave-like voltage oscillations driven by active current oscillations that match all properties seen in whole intestinal tissue slow waves. With different recording modes, whole cell currents, voltage and current oscillations were recorded from the same cell, showing that ICC are electrophysiologically unique and that the active inward current driving the slow wave-like oscillations are not voltage dependent. The isolated single ICC were demonstrated to have a specific tyrosine receptor marker protein for ICC, Kit , by selective RT-PCR amplification. The slow wave-like oscillations had a reversal potential consistent with a non-specific cation conductance. Although previous research had been done on single smooth muscle cells, there is currently no consensus on the cellular ionic currents present. In this thesis, analysis of different recordings demonstrated that there are at least four main groups of SMCs with different whole cell current profiles. Different cellular ionic currents were found specifically in different groups, and can be confirmed by reconstructing single channel recordings. One cellular outward current was chosen for further investigation-a fast activating and inactivating transient outward current. This current was characterized by common protocols and with a novel ramp analysis. Characterization revealed two distinct transient outward currents with different kinetic properties. Finally, spontaneous transient outward currents (STOCs) have been recorded in 25% of smooth muscle cells, reflecting quantal Ca2+ release from the intracellular stores to the plasmalemma calcium dependent potassium channels. Therefore, the study of STOCs gives direct information not only on the activities of intracellular Ca2+ stores, but also on the kinetics of Ca2+ release and reuptake in the microenvironment where STOCs originate. From these results, a simple model for GI motility was developed to account for the cellular interactions between nerve, ICC, and smooth muscle.Doctor of Philosophy (PhD
THE ROLE OF LUMINAL 5-HT4 RECEPTORS IN COLONIC MOTILITY
No full textFunctional gastrointestinal (GI) disorders including constipation and Irritable Bowel
Syndrome (IBS) constitute the most widespread digestive disorders that could involve GI
dysmotility and altered serotonin (5-HT) signaling. Current treatments include oral intake
of prokinetic drugs such as serotonin sub-type 4 receptor (5-HT4) agonists that activate 5-
HT4 receptors located on nerves in the gut wall. However, these receptors are also found
on the luminal side of enterochromaffin cells in the colonic epithelium where more than
90% of the body’s 5-HT is synthesized. Therefore, activation of luminal 5-HT4 receptors
by using a delivery system that releases the drug inside the colonic lumen without it being
first absorbed in the upper GI tract, can result in the release of 5-HT and increase in colonic
motility. This could significantly minimize the adverse side effects associated with
systemic absorption of such drugs. In this study, first the rabbit animal model was used to
test the effects of prucalopride after administration inside the colon (ex vivo). Results
showed significant increase in propulsive motor patterns and their properties such as
pressure and force. Such potent prokinetic effects occurred even in the presence of
simulated fecal impaction, an acute complication of chronic constipation. Using highresolution
colonic manometry (HRCM), all aspects of propulsive motility including the
colo-anal reflex and simultaneous pressure waves (SPW) were studied in vivo in healthy
volunteers; then, the effects of intraluminal prucalopride was evaluated with HRCM in a
human case study. Similar to the animal model, marked increase in propulsive motor
activity was observed. This project shows the SPW and the colo-anal reflex have potential
diagnostic values in patients with colonic dysmotility or abdominal bloating and
prucalopride incorporated in colon-specific drug delivery systems has the potential to
become the preferred prokinetic for the treatment of constipation. It also encourages further
research into the role of luminal 5-HT in generating normal colonic motor function.ThesisMaster of Science in Medical Sciences (MSMS
Electrical Communication Between Different Cell Types in the Colonic Musculature
No full textThe major cell types in the canine colon musculature are interstitial cells of Cajal (ICC), circular muscle (CM) cells and longitudinal muscle (LM) cells. In isolated muscle strip studies, spontaneous membrane potential oscillations (slow waves) are generated in the submucosal border of the circular muscle where a gap junctionally well-coupled network of ICC and CM is found. CM devoid of LM and submucosal pacemaker region (CM preparations) are spontaneously quiescent. The research undertaken was to understand the mechanism of slow wave propagation into the circular muscle and to investigate the consequences to the electrical activity in CM after coupling with different electrical activities from different cells types. Our results show that CM cells, although spontaneously quiescent because of high K+ conductance, are excitable and can actively participate in slow wave generation. The electrical oscillations induced in the CM preparations could easily be potentiated by an L-type Ca2+ channel activator, Bay K 8644, and abolished by a L-type Ca2+ antagonist, D600, suggesting involvement of the conductance in the induced activity. The induced oscillations are similar to the SLAPs in the longitudinal muscle which shows that it is not necessary to have a specialized pacemaker cells for generating SLAPs. Using a cross sectioned preparation with all intact muscle layers, we also showed that the heterogeneity in the electrical activity of CM, such as: the resting membrane potential gradient, depolarization of plateau potential in the myenteric border and "apparent" decay in slow wave amplitude, is due to electrical interactions between different intrinsic activities from different cell types. Morphological evidence was obtained for the possible communication pathways in the submucosal and the myenteric borders of the circular muscle. Different coupling mechanisms in different areas were hypothesized. In addition, the 3-dimensional aspects of the submucosal ICC network in the ca.nine colon were clarified.ThesisMaster of Engineering (ME
Cellular origin and regulation of the electrical slow wave in the murine small intestinal musculature
No full textThe electrical pacemaker slow wave is responsible for the generation of anally propagating phasic contractions underlying the peristaltic motor activity of the gastrointestinal musculature. Yet, the cellular origins of the slow wave and mechanisms of the slow wave regulation or generation still remain unresolved and constituted primary goals of the current thesis. As described in detail in Chapters Three-Six, spontaneously genetic knock out mice with genetic mutations affecting the structure (W / Wν mice), expression (Wbd / Wbd mice), or the ligand (Sl / Sld mice) of the kit tyrosine kinase receptor were shown to lack both the network of interstitial cells of Cajal associated with the myenteric plexus and the slow wave activity in the small intestine, hence, supporting the proposed role of the interstitial cells of Cajal as pacemaker cells responsible for the slow wave generation. In the absence of the slow wave, the mutant musculature was either electrically quiescent or showed action potentials in regular or irregular patterns as recorded with a standard microelectode technique. The observed action potentials were also clearly distinguished from the slow waves by their shape and pharmacological sensitivities to L-type Ca2+ channel and K+ channel blockade. The mechanisms of the slow wave generation and regulation are addressed in Chapters Seven-Nine. The data indicate that the slow wave generation involves primarily Na+ and Ca2+ conductances not mediated by TTX- or mexiletine-sensitive Na+ channels, gadolinium sensitive nonselective cation channels, or L-type Ca2+ channels. Cl- channels may be also involved in the regulation but not in the slow wave initiation. Pharmacological agents acting on cytosolic Ca2+ , SR Ca2+ ATPase, and intracellular Ca 2+ release mechanisms support the role of intracellular Ca 2+ release mechanisms, sensitive to IP3 , in the regulation of the slow wave frequency and amplitude. Furthermore, activation of the Ca 2+ induced Ca2+ release (CICR) mechanism leads to depolarization not mediated predominantly by chloride channels nor likely by KCa channels. The CICR may be also involved in the regulation of the slow wave. These experiments importantly identify intracellular metabolic pathways that may potentially lead to the development of therapeutic approaches aimed at treating certain gastrointestinal motor disorders by modifying the slow wave frequency or amplitude.Doctor of Philosophy (PhD
Pacemaker Activity and Intercellular Communication in the Intestinal Musculature
No full textKnowledge of the origin and characteristics of the intestinal pacemaker activity, and the characteristics of intercellular communication throughout the musculature is instrumental for the understanding of the mechanisms through which gastrointestinal (GI) motility is regulated. This thesis makes a significant contribution to provide electrophysiological and morphological evidence supporting the hypothesis that interstitial cells of Cajal (ICCs) are the Gl pacemaker cells. The pacemaker activity of the GI tract triggers the slow-wave.type action potentials (slow waves) which are coherent with the phasic contractions for facilitating peristaltic movement. Origins of the slow waves at different portions of the GJ tract always coincide with the locations of the ICCs. The objectives of this study were to identify the cellular origin of the pacemaker activity and to thoroughly investigate the mechanism of intercellular communication in the canine colon using electrophysiological and microscopic techniques. The cellular origin of the pacemaker activity was further examined by studying simultaneously the ontogenesis of the pacemaker activity and the ICCs in the neonatal mouse small intestine. The cellular origin of the pacemaker activity was studied by employing the photodynamic property of methylene blue (Chapter 3). We previously demonstrated that the submuscular ICCs of the canine colon selectively accumulate methylene blue. In this undertaken study, we further illustrated that incubation with 50 μM methylene blue and subsequent intense illumination resulted in abolition of the pacemaker activity. Following methylene blue incubation, intense illumination first changed the mitochondrial conformation in the ICCs from very condensed to orthodox, and progressively imposed more severe damages, such as swollen and ruptured mitochondria, loss of cytoplasmic contrast and detail, and rupture of the plasma membrane. No damage was seen in smooth muscle cells and nerves. The correlation between selective lesioning of ICCs and loss of the pacemaker activity strongly supports that ICCs play an essential role in the generation of the pacemaker activity. The regulatory mechanism of the pacemaker frequency was investigated with a focus on the effects of cyclopiazonic acid (CPA), a specific inhibitor of the endoplasmic reticulum (ER) Ca²⁺-pump (Chapter 4). CPA dose dependently decreased the pacemaker frequency. Similarly, chelating cytosolic Ca²⁺ with BAPTA also decreased the pacemaker frequency. The pacemaker frequency was also decreased by neomycin (inhibiting inositol 1,4,5-triphosphate (IP₃) synthesis) and caffeine (inhibiting the IP₃-sensitive Ca²⁺ channels in the ER membrane). Electron microscopy showed that the smooth ER forms an extensive network of subsurface cisternae which is closely associated with large areas of the cytoplasmic face of the plasma membrane. These structures were the most extensive in the ICCs, slightly less in branching smooth muscle cells and far less in circular muscle cells. Based on the electrophysiological and morphological observations, we hypothesize that the Ca²⁺ refilling cycle of the IP3-sensitive calcium stores associated with the plasma membrane, determines the frequency of the pacemaker activity generated by the submuscular ICC-smooth-muscle network of the canine colon. The ontogenesis of the pacemaker activity and the ICCs in the small intestine of neonatal mice was studied to further substantiate the pacemaker role of ICCs (Chapter 5). The pacemaker component of the slow waves was fingerprinted by its resistance to L-type Ca²⁺-channel blockers and sensitivity to cyclopiazonic acid, CPA, (Chapter 4). All isolated musculature of the neonatal mouse small intestine (newborn, unfed-7 days old) spontaneously generated action potentials. The presence of the pacemaker component in different age groups was examined by verapamil, a L-type Ca²⁺ channel blocker, and CPA. In conclusion, electrophysiological and morphological evidences were obtained to demonstrate that both the pacemaker activity and the, ICC network were immature at birth but fully developed in 2 days old neonatal mice. Communication between the longitudinal and the circular muscle layers are essential for producing co-ordinated motility in the musculature. Through electrophysiological measurements with microelectrodes, the study of neurobiotin spread using confocal microscopy and the investigation of the cellular structure at the electron-microscopic level, the cellular mechanisms of communication between the two muscle layers was studied. We positively demonstrated the existence of low-resistance pathways. We also provided evidence that the ICCs associated with the myenteric plexus facilitated electrotonic coupling between the two muscle layers across the myenteric plexus of the canine colon (Chapter 7). In the longitudinal muscle layer, no positive evidence for electrical coupling between smooth muscle cells has yet been presented. We thoroughly examined the properties of electrical coupling in the longitudinal muscle layer of the canine colon (Chapter 8). The properties of electrical coupling between longitudinal muscle cells were compared with that between the circular muscle cells. Three electrical coupling parameters were measured: (i) the input resistance, (ii) the space constant (determined by the method developed with a double-electrode technique (Chapter 6)), and (iii) the phase relationship of simultaneously recorded electrical activities. Furthermore, a detailed electron microscopic investigation revealed the absence of gap junctions in the longitudinal muscle layer; whereas, numerous close apposition contacts were observed. These observations put forward the hypothesis that the pathways for electrical coupling between longitudinal muscle cells are consituted by close apposition contacts. Communication between circular muscle (CM) lamellae is necessary to generate propulsive phasic contractions for facilitating peristalsis along the longitudinal axis of the GI tract. The submuscular ICCs are extensively coupled to the underlying branching smooth muscle (bSM) cells forming an ICC-bSM network covering the entire submucosal surface of the canine colon. There is another ICC network located in the myenteric plexus. The roles of the submuscular ICC-bSM network, the myenteric ICC network and the longitudinal muscle layer in mediating communication across the CM lamellae were studied by simultaneous recordings with surface electrodes using different types of muscle strip preparations (Chapter 9). Electrophysiological evidence demonstrated that, within the pure circular musculature, circular muscle cells were electrically coupled along a CM lamella, oriented circumferentially around the lumen, but electrically insulated across CM lamellae. The submuscular ICC-bSM network, but not the longitudinal muscle nor the myenteric plexus, was shown to be essential for mediating communication between CM lamellae such that co-ordinated motility can be exhibited with neig,hbouring CM lamellae through excitation-contraction coupling. In summary, employing a number of electrophysiological and microscopic techniques, this dissertation presents novel evidence (i) to substantiate the pacemaker role of ICCs in the GI tract; (ii) to put forward a hypothesis that the pacemaker-frequency regulatory mechanism is synchronized with the ER Ca²⁺ refilling cycle; and, (iii) on the heterogeneity of mechanisms through which intercellular communication occurs along the radial, circumferential and longitudinal axes of the intestinal musculature.Doctor of Philosophy (PhD
Interstitial cells of Cajal as targets for pharmacological intervention in gastrointestinal motor disorders.
No full textInterstitial cells of Cajal (ICCs) have recently been identified as the pacemaker cells for contractile activity of the gastrointestinal tract. These cells generate the electrical 'slow-wave' activity that determines the characteristic frequency of phasic contractions of the stomach, intestine and colon. Slow waves also determine the direction and velocity of propagation of peristaltic activity, in concert with the enteric nervous system. Characterization of receptors and ion channels in the ICC membrane is under way, and manipulation of slow-wave activity markedly alters movement of contents through the gut organs. Here Jan Huizinga, Lars Thuneberg, Jean-Marie Vanderwinden and Jüri Rumessen, suggest that, as ICCs are unique to the gut, they might be ideal targets for pharmacological intervention in gastrointestinal motility disorders, which are very common and costly.Journal ArticleResearch Support, Non-U.S. Gov'tReviewSCOPUS: re.jinfo:eu-repo/semantics/publishe
Mechanisms Underlying Rhythmic Activities of the Gastrointestinal Tract
No full textThe organs of the gastrointestinal (GI) tract display a variety of motor patterns, involved in grinding, mixing, enhancing absorption and propulsion of nutrients and waste products. Specialized motor patterns are generated by unique mechanisms inherent to the GI segment in which they are found. Rhythmic contractions are a feature of most motor patterns. Slow wave driven peristalsis is an acknowledged motor pattern associated with interstitial cells of Cajal (ICC) pacemakers, but propulsive motor patterns which are blocked by tetrodotoxin are seen to be exclusively generated by the enteric nervous system (ENS). This has not been proven, however, and the origin of rhythmicity of propulsive motor patterns needs further study, particularly related to a potential role of the pacemaker ICC found throughout the GI tract. The aim of this study was exploring the mechanisms which underlie various GI motor patterns, with particular focus on the origin of rhythmicity of these patterns. I have demonstrated with manometry and spatiotemporal maps that murine rhythmic propulsion requires a myogenic pacemaker which is evoked by acetylcholine and substance P; nitric oxide is not involved. Calcium imaging evidence suggests that the pacemaker is the ICC of the deep muscular plexus, as these cells rhythmically activate to substance P. I observed rhythmic contractility patterns in human antrum, pylorus and duodenum when stimulated with carbachol. The hypothesis emerged that the ENS modifies the pyloric pacemaker into unique rhythmic patterns. Colonic muscle strip contractility from the rat has a low frequency rhythmic pattern which is myogenic. This pattern is augmented by the conditioned media from the probiotic E. coli Nissle 1917 through a non-neural mechanism. The current explanation of entirely ENS generated motor patterns is not accurate. The ENS plays an important role in stimulating and regulating GI motor patterns in conjunction with myogenic pacemakers. It is only through acknowledgment of all GI cell types that we can understand the mechanisms governing motility.Master of Science (MSc
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