Durham E-Theses Biochemical and electrophysiological studies on muscle bres from Periplaneta Americana L. and Sphinx Ligustri L Wareham, A. C. How to cite: Wareham, A. C. (1971) Biochemical and electrophysiological studies on muscle bres from Periplaneta Americana L. and Sphinx Ligustri L, Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/8587/ Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in Durham E-Theses • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full Durham E-Theses policy for further details. Academic Support Oce, Durham University, University Oce, Old Elvet, Durham DH1 3HP e-mail: [email protected] Tel: +44 0191 334 6107 http://etheses.dur.ac.uk 2 BIOCHEMICAL AMD ELECTROPHYSIOLOGICAL STUDIES ON MUSCLE FIBRES FROM PERIPLANETA AMERICANA L. AND SPHINX LIGUSTRI L. by A. C. Wareham, B.Sc. Being a thesis submitted for examination for the degree of Doctor of Philosophy of the University of Durham, October, 1971. Grey College, Durham. (i) ACKNOWLEDGEMENTS I sun indebted to Professor C. J. Duncan and Dr. K. Bowler for their invaluable advice and encouragement throughout this work, and for their critical reading of the manuscript. I should also like to thank the following people: Professor D. Boulter for his per• mission to use the Liquid Scintillation counter in the Botany De• partment of Durham University; Mr. J. Richardson for his skilful management of the insect house; the technical staff of the Zoology Department for their frequent help. I am indebted to the Scientific Research Council for providing me with a Research Studentship and to Professor D. Barker for the use of laboratory facilities. I should like to thank Miss R. M. Hore of the Physiology De• partment of Birmingham University for her photographic assistance, and my mother, Mrs. K. M. Wareham, B.A., for her skilful typing in the production of this thesis. Finally I am deeply indebted to my wife, Kathleen, for her encouragement throughout this work and for her tolerance during the writing of this thesis. (ii) TABLE OF CONTENTS Acknowledgements (i) Table of contents (ii) Abstract (vi) CHAPTER 1: GENERAL INTRODUCTION 1 CHAPTER 2: GENERAL METHODS AND MATERIALS 1) Animal culture 7 2) Glassware 8 3) Chemicals 8 4) Radioisotope 8 CHAPTER 3 s THE BIOCHEMISTRY OP THE MUSCLE MEMBRANE Introduction 10 Methods 12 1) Preparation of microsomes 12 2) Estimation of enzyme activity 12 5) Estimation of inorganic phosphate 13 4) Standard phosphate curve 13 5) Estimation of microsomal protein 13 Results 17 1) Mg~+-dependent ATPase 17 2) Monovalent cation stimulation 17 3) Treatment with deoxycholate 21 4) Substrate specificity 21 5) Effect of metabolic inhibitors 24 6) Oxygen consumption of homogenates 27 7) Effect of guanidine-HCl 29 8) pH sensitivity 31 9) Thermal sensitivity 31 Discussion 38 (iii) CHAPTER 4: IONIC DISTRIBUTIONS BETWEEN INTRACELLULAR AND EXTRACELLULAR PHASES Introduction 44 Methods 48 1) Haemolymph analyses 48 2) Myoplasm analyses 43 3) Atomic absorption spectrophotometry 49 4) Tracer methods 49 5) Determination of muscle space volume 50 6) Temperature 52 Results 54 1) The ionic balance between haemolynmh and muscle 54 2) Extracellular suaoe 54 3) Saline composition 56 4) Effect of pharmacological agents 56 5) Effect of varying external pH 6l 6) Effect of temperature 6l 7) Movement of 2zNa 65 Discussion 71 CHAPTER 5: THE RESTING MEMBRANE POTENTIAL Introduction 77 Methods 80 1) Preparation 80 2) Temperature regulation 80 3) Microelectrodes 80 4) Electrode resistance 81 5) Tip potential .... 81 6) Resting potential measurements 85 (iv) Results 84 1) Effect of saline composition 84 2) Contribution of potassium and chloride 87 5) Contribution of sodium 9° 4) Effect of temperature 90 5) Action of pharmacological agents 94 6) Effect of external pH 106 7) Effect of divalent cations 106 Discussion 109 CHAPTER 6t THE ELECTRICAL RESPONSE TO EXCITATION Introduction , • 128 Methods 133 Results 134 1) The EPRP and the EER 134 2) Effect of divalent cations 137 3) Effect of sodium ions 140 4) Effect of lithium ions 145 5) Effect of potassium ions 149 6) Effect of chloride ions 149 7) Effect of temperature 151 8) Excitation and metabolic inhibitors 153 Discussion 154 CHAPTER 7: A COMPARISON WITH SPHINX LIGUSTRI Introduction 172 Methods 173 1) Membrane preparation 173 2) Ionic distributions 173 3) Electrophysiology 174 (v) Results * 175 1) ATPase activity 175 2) Ionic distributions 175 3) Electrophysiology 175 Discussion 180 CHAPTER 8 s DISCUSSION AND CONCLUSIONS 184 Summary (viii) Abbreviations (xii) References (xiv) (vi) ABSTRACT The permeability properties of skeletal muscle from Periplaneta americana and Sphinx ligustri have been studied by several methods and in each the effects of pharmacological agents and of temperature have been investigated. The properties of the microsomal preparation of skeletal muscle were characterized. Compared with preparations from many other types of excitable cell, although probably not muscle, the microsomal pre• paration was atypical in lacking an enzyme synergistically stimulated by monovalent cations and in being insensitive to strophanthin G. Normal trans-membrane inorganic ion distributions of skeletal muscle were determined. The ratios were similar in the two animals and not markedly dissimilar from those of vertebrate muscle / blood ratios. The results obtained with Sphinx muscle differed from those of previous workers. Based on the results obtained a new saline was developed for bathing the muscles of Periplaneta and Sphinx which proved to be more suitable than salines used by other workers. Intracellular microelectrode recordings were made from resting and active skeletal muscle of Periplaneta. The relative contributions to the resting membrane potential of actively and passively distri• buted ions were determined. A considerable hyperpolarizing contri• bution to the resting membrane potential was made by a metabolically dependent constant current generator. Similar results were obtained using Sphinx skeletal muscle. An electrical model was derived to account for the phenomena observed. The individual contributions of inorganic ions to depolarizing electrogenesis were determined. Sodium was found to make a major contribution. The possible role of electrogenic pumps in the development of end-plate potentials was discussed. The excitatory postsynaptic potential of Periplaneta (vii) skeletal muscle was compared with the end-plate potential of vertebrate skeletal muscle and the possible nature of the biphasic response of Periplaneta muscle to excitation discussed. Possible mechanisms of excitatory and inhibitory electrogenesis were related to the model developed to describe the resting membrane potential. CHAPTER 1 GENERAL INTRODUCTION. -1- The literature contains several very good reviews of the current state of knowledge concerning insect muscle neurophysiology (Hoyle, 1962$ Aidley, 1967} TJsherwood, 1967a & 1969). These reviews have been concerned with neuromuscular transmission, the excitatory mechanisms of muscle and the effect of inorganic ion distributions on membrane potentials. These papers aim to draw together information concerning many different insect species. One point which is evident from a study of the literature is that, although the mechanisms underlying the pro• duction and maintenance of bioelectric potentials are different from those in vertebrate muscle, they are not clearly understood. The pur• pose of this work therefore, was, by making a detailed study of one species, to make a contribution to the understanding of bioelectric phenomena in insect muscle. The anatomy and histology of Periplaneta americana has been well documented (Guthrie & Tindall, 1968). The muscles used in the present study were the skeletal muscles of the coxae of the cockroach. The fibres of these muscles are striated and normally contract and relax quickly, usually one twitch lasting less than 100 msec (Usherwood, 1962b). Histologically the muscles are very similar to those of ver• tebrates. Periplaneta muscle fibres are about 100 pM in diameter. The contents of the fibres are bound by the plasma membrane or earcolemma and include mitochondria, sarcoplasmic reticuli, sarcoplasm, nuclei and fibrils which contain the two contractile elements. The fibrils consist of interdigitating thick (myosin) and thin (actin) filaments, character• istic of vertebrate skeletal muscle. They are regularly striated with A- and I-bands and divided transversely into sarcomeres by dense Z-lines. The plasma membrane of the muscle fibres is normally about 7.5 urn thick with the characteristic "unit membrane" structure. It is invagi- nated to form the transverse tubular system with two morphologically distinct elements, tubules and sarcolemmal invaginations (Cochrane & Elder, 1967)- The sarcolemmal invaginations are circular in cross section but the tubules are flattened (Hoyle, 1965). Sarcolemmal in• vaginations enter at the Z~lines and give off vertical and horizontal branches. The tubules enter opposite the overlap regions of actin and myosin filaments. Branches of the transverse tubular system make