J. Physiol. Biochem., 57 (2), 89-94, 2001
MLISO, a computer simulation of experiments to illustrate the cellular basis of intestinal muscle activity M. Díaz, B. Martín and M. Rodríguez1 Lab. de Fisiología Animal, Depto. de Biología Animal and 1Lab. de Comunicaciones y Teledetección, Depto. de Física Fundamental y Experimental, Universidad de La Laguna, 38206 Tenerife, Spain. (Received on January 4, 2001)
M. DÍAZ, B. MARTÍN and M. RODRÍGUEZ. MLISO, a computer simulation of experiments to illustrate the cellular basis of intestinal muscle activity (didactic contribution). J. Physiol. Biochem., 57(2), 89-94, 2001. An interactive program illustrating a number of concepts on the cellular basis of the intestinal peristaltis and the regulatory actions of the autonomic nervous system (ANS) on the duodenal contractile activity is presented. The program shows the isometric mechanical responses elicited by the duodenum to a number of experimental conditions and pharmacological drugs in a way that is consistent with real laboratory practicals. A selection of experiments demonstrates the dependence of extracellular calcium for the smooth muscle activity, the effects of membrane potential changes, the effects of ANS receptor agonists and antagonists, the role of the Na+K+-ATPase and the effects of temperature or oxygen changes on the myogenic muscle activity. The software was developed to run on IBM compatible computers under DOS and Windows environments and makes use of high-resolution graphic simulations derived from real experiments which are displayed on a simulated oscilloscope and the students are expected to make qualitative assessments and to draw conclusions from the data collected directly from the screen oscilloscope. The cognitive aspects of the software makes it is suitable for undergraduate and graduate students from an assortment of biomedical sciences and have been designed to be used under a tutored framework but also as learning resource for individual study by the students. Key words: Intestinal peristaltis, Smooth muscle, Computer simulation, Computer assisted learning (CAL).
Correspondence to: M. Díaz (Fax: 34 922 318 311 ; e-mail:
[email protected]).
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The increased availability of microcomputers has led to the development of computer-assisted learning (CAL) materials in different biomedical sciences, including physiology, biochemistry and pharmacology (2, 6). Several CAL programs have been designed to demonstrate the pharmacological properties of the intestinal smooth muscle, including the classical Finkleman preparation (3) and the simulation of the effects of cholinergic or sympathomimetic drugs on the colonic motility (4). However, our experience is that besides the pharmacology of neurotransmission in intestine, many important concepts related to the smooth muscle cell physiology may be explored by analyzing the whole intestinal muscle activity. The primary goal of the software presented here, MLISO, was to develop a simulation tool that would facilitate the understanding of some of the cellular mechanisms that underlie the intestinal smooth muscle contractile activity. In this sense, the simulation presented here is novel, providing a link between cellular physiology and systemic organ physiology, an important aspect that has not been considered in the existing educational software. The program is based on the experience of several years of laboratorybased demonstrations requiring several hours and a considerable number of animals to illustrate a few relevant concepts on intestinal peristaltis and its regulation by the ANS. However, our experience on these laboratory-based practicals indicate that the learning objectives suitable for demonstration in this preparation are hardly achieved due to the number of hours and staff personnel required, but also because, in most cases, the in vitro intestinal preparations do not resist time enough to perform a long sequence of experiments. A demonstration of MLISO was presented at recent meeting of the Spanish J. Physiol. Biochem., 57 (2), 2001
Federation of Experimental Biology Societies (5). The Simulation Program The software source code was written using Microsoft QuickBasic 4.5 and Turbo Assembler 2.0. The DOS version runs under MS-DOS 5.0 or higher but has been satisfactorily tested under Microsoft Windows 3.X, Windows 95 and 98. The minimal hardware requirements for this version are a 286 IBM compatible computer equipped with 1 Mbytes free hard disk space, 640 Kbytes RAM memory, a VGA display adapter and a Microsoft compatible serial mouse. Although the potency of personal computers increases franticly, it was found very convenient and useful to develop this simulation using a DOS environment, taken into account the considerable stock of computers with low hardware capabilities in many educational centers and also between the students. A Windows version is under development. A single executable file offers a choice of Introduction, Methods, and the experimental section. The Introduction and Methods sections use a combination of several text windows and high resolution graphics to describe the features of the mouse duodenal preparation and the experimental and recording equipment used (Fig. 1). The different experiments are organized in five main groups, i.e. Intestinal peristaltis, Calcium Experiments, Autonomic Nervous System, Membrane Potential and Na+-K+-ATPase (Fig. 2). Once an experimental group is chosen a choice of all different available experiments is shown. For instance, if Calcium Experiments group is selected, a subset of four possible experiments are presented: 1) Remove extracellular calcium, 2) Change extracellular calcium concentration, 3) Effect of calcium channels block-
COMPUTER SIMULATION INTESTINAL MUSCLE ACTIVITY
A)
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INTESTINAL PERISTALTISM SPONTANEOUS PERISTALTIS
EFFECTS OF TEMPERATURE EFFECT OF pO2 CALCIUM EXPERIMENTS
Calcium replacements Calcium chanel blockade Calmodulin inhibition Na+-K+-ATPase
B)
Na+-K+-ATPase inhibition MEMBRANE POTENTIAL Changes in extracellular K+ Inhibition of K+ conductance
REGULATION BY THE AUTONOMIC NERVOUS SYSTEM PARASYMPATHETIC EFFECTS
Cholinergic stimulation Muscarinic agonists and antagonists Calcium dependence of ACh response
Fig. 1. Screenshot examples from the Methods section obtained from the English (A) and Spanish (B) versions showing the organ bath and the experimental setup used to measure the intestinal contractile activity under isometric conditions using a tension transducer.
SYMPATHETIC EFFECTS Adrenergic stimulation α -Antagonists β -Antagonists Effects of forskolin
ade, and 4) Effect of calmodulin inhibition. Each group of experiments is prefaced by an introduction where the basic information related to the experimental protocols used as well as the physiological and pharmacological basis of the experiments to be performed are displayed. The experiments are displayed on a simulated oscilloscope using the screen of 640x480 pixels. A number of useful functions are available in the default displaying mode. These J. Physiol. Biochem., 57 (2), 2001
Fig. 2. Overview of the experimental section of MLISO.
including a measuring tool which allows the user to measure the time and isometric tension values from the displayed traces using the mouse or a cross-hair cursor facility. However, In this preparation, the variable levels of spontaneous motility mean that it is difficult to quantify drug effects and, for teaching purposes, it is
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only necessary to record qualitative effects. The Experimental Section. Illustrated Concepts Intestinal smooth muscle cells undergo changes in membrane potential and tension in the apparent absence of external stimulation. This endogenous electrical activity accounts for the spontaneous peristaltis that consists of a cyclic pattern of contractions and relaxation. The experimental system designed to record this contractile activity, which is sketched in Fig. 1 (as described in the Methods section), includes an isometric lever that is connected to a DC amplifier, whose output is sent to a chart recorder. The first experimental section of the program presented here, Intestinal peristaltis includes several real-time experiments showing the peristaltic activity of the mouse duodenum and the effects of lowering temperature or oxygen partial pressure in the physiological bathing solution. The primary signal for smooth muscle contraction is an increase in sarcoplasmic free calcium concentration ([Ca2+]i). Calcium regulates this phosphorylation indirectly by combining to the calcium binding protein calmodulin. This triggers the activation of calmodulin-dependent myosin light-chain kinase, which catalyses myosin phosphorylation and activates crossbridge formation and the development of force of contraction of the muscle cell as long as Ca2+ is present. Restoration of resting [Ca2+]i deactivates the kinase; myosin is dephosphorylated by myosin light-chain phosphatase and the muscle relaxes (1, 7). The experiments presented in this simulation cover the involvement of extracellular calcium as a primary source for intracellular free Ca2+. These experiments are concentrated in the Calcium experiments option, which demonJ. Physiol. Biochem., 57 (2), 2001
strates the reversible effect of extracellular calcium replacement and also the effects of calcium channel inhibition by compounds such as verapamil or lanthanum, all of which prevent the spontaneous contractile activity. Moreover, the involvement of calmodulin on the activation of the phosphorylation step of myosin is illustrated in specific experiments where the actions of the calmodulin antagonist trifluoperazine (TFP) are assessed. An example of these experiments is shown in Fig. 3A. Given that depolarization of the cells makes the sarcolemma permeable to calcium through the activation of voltagedependent calcium channels, and that relaxation depends on the restoration of resting calcium levels, we provide different experiments that demonstrate how changes in membrane electric potential or electrochemical sodium gradient affect intestinal peristalsis (Fig. 2). One of the experiments makes use of increased extracellular potassium to depolarize smooth muscle cells. The magnitude of the change of the transmembrane potential by increasing the external KCl concentration can be derived from the Nerst equation. Intestinal smooth muscle cells can be activated in a variety of ways including autonomic nerves, hormones, local factors, and in some cases by stretching the muscle. All these sources of excitation act ultimately by increasing the intracellular concentration of free calcium ions. In the simulation presented here, several experiments illustrate different aspects of the regulation by autonomic nervous system. The experiments include the responses of intestinal muscle to sympathetic or parasympathetic nerve stimulation as well as pharmacological properties of these responses as assessed by the use of different agonists and antagonists. An example of these is illustrated in the screenshot shown in Fig 3B.
COMPUTER SIMULATION INTESTINAL MUSCLE ACTIVITY
A)
B)
Fig. 3. Screen display from the Calcium experiments (A) and Autonomic nervous system (B) sections captured from the English and Spanish DOS versions, respectively. The traces showed the time-course of isometric force (g) in a control period and after the addition of the calmodulin antagonist trifluoperazine (A) or the muscarinic agonist carbachol (B) to the bathing physiological solution. Note the horizontal bar on the top of the screen showing the display options.
Teaching and Learning Strategies MLISO was originally conceived to provide an alternative, but not exclusive, educational tool aimed at illustrating a number of experiments that could be performed on the isolated intestinal muscle in relation with the cellular basis of muscle contraction. These experiments were J. Physiol. Biochem., 57 (2), 2001
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selected according to the routine questions and suggestions often raised by the students during the real practical classes. Our experience indicates that MLISO can be adequately used under a tutored learning environment using the computer room facilities but also admits the individual study by the students independently of the tutor, mainly because the different experiments compare reasonably well with the observations from live experiments. Under tutored environments, the students are expected to draw conclusions from each experiment in relation to the physiology of the contractile activity of the smooth muscle cell. In our case, at the beginning of each simulation session (2 hours in the trial group), the students were provided with a questionnaire that had to be answered during the simulation session on the basis of their preadquired knowledge. The questions included were elaborated in a way that the students had to a priori deduce the effects of the experimental condition imposed on the peristaltic activity, and to a posteriori make an assessment of the experimental results. For instance, once the students realized that the contractile activity requires the presence of calcium ions, the students were asked to design a simple experiment to decide whether the myogenic activity was dependent on the existence of intracellular or extracellular calcium sources. Almost 80% of the students decided that the most simple way to assess this question was to make an experiment where the calcium ions were initially absent in the organ bath and then supplement the bathing media with calcium to 2.5 mM. Similar observations were obtained for the rest of the questions and experiments.
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Concluding Remarks The whole set of experiments illustrated have been found to be very useful as an active learning tool mainly because they promote, among the student groups, flexible discussions to explain the nervous regulation of intestinal peristaltis, the role of calcium, membrane potential and Na+K+-ATPase on muscle contraction, which finally lead to the formulation of a physiological model for the intestinal smooth muscle contraction with the supervision of the tutor. Availability MLISO is available in Spanish and English versions. A copyrighted copy of this computer package is available to any instructor upon request to the corresponding author by ordinary or electronic mail after the publication of the article. All instructions and rules for the operation of this simulation are included on the program disk. Acknowledgements We would like to express our gratitude to Mr. Neil Abrey for the correction of the English spelling and grammar usage of the manuscript and to our students and colleagues from Universidad de La Laguna for their co-operation and helpful comments in the trials of the learning effectiveness of this simulation. This work was supported by CYCIT/FEDER (ref. 1FD97-1065-C03-01) grant to M.D.
M. DÍAZ, B. MARTÍN y M. RODRÍGUEZ. MLISO, una simulación en ordenador que ilustra las bases celulares de la actividad del músculo intestinal (contribución didáctica). J. Physiol. Biochem., 57(2), 89-94, 2001. Se presenta un programa interactivo creado para ilustrar conceptos básicos de los mecanismos celulares subyacentes al peristaltismo intestinal y el papel regulador del sistema nervioso autónomo (SNA). El programa muestra las respuestas mecánicas isométricas produciJ. Physiol. Biochem., 57 (2), 2001
das por diferentes condiciones experimentales y agentes farmacológicos de forma realista y similar a las obtenidas en clases prácticas convencionales. Mediante una selección de experimentos, se demuestra la dependencia de calcio extracelular en la actividad del músculo liso, los efectos de los cambios del potencial de membrana, los efectos de agonistas y antagonistas del SNA, el papel de la Na+-K+-ATPasa y los efectos de los cambios de temperatura y presión de oxígeno sobre la actividad miogénica del músculo liso intestinal. El software ha sido desarrollado para ordenadores compatibles IBM bajo entorno DOS o Windows, utilizando experimentos reales que son presentados en un osciloscopio simulado que emplea imágenes de alta resolución. Esta simulación permitiría a los estudiantes realizar valoraciones cualitativas y obtener conclusiones directamente desde el osciloscopio en pantalla. Por sus contenidos, este software es adecuado para estudiantes y licenciados de diversas titulaciones biomédicas y ha sido desarrollado para ser utilizado en un entorno tutorizado de prácticas simuladas, aunque también puede emplearse como una herramienta para el aprendizaje individual. Palabras clave: Peristaltismo intestinal, Músculo liso, Simulación en ordenador, Enseñanza asistida por ordenador (EAO).
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