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Ultrastructure Study on the T System and the Subcellular Localization of Calcium in Frog Skeletal Muscle

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Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1971 Ultrastructure Study on the T System and the Subcellular Localization of Calcium in Frog Skeletal Muscle Lawrence P. McCallister Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Medicine and Health Sciences Commons Recommended Citation McCallister, Lawrence P., "Ultrastructure Study on the T System and the Subcellular Localization of Calcium in Frog Skeletal Muscle" (1971). Dissertations. 1148. https://ecommons.luc.edu/luc_diss/1148 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1971 Lawrence P. McCallister ULTRASTRUCTURE STUDY ON THS T SYSTEM AND TH3 SUi3CELLUIAR LOCP.LIZA TION OF CALCIUM IN FROG SKELE.?AL MUSCLE by Lawrence P. McCallister A Dissertation Submitted to the Fac~lty of the Graduate School of Loyola Unive<rsity :tn Partial. Fulfilment of th~ Requirements f o~ the Degree of Doctor of Philosophy Juns. 1971 BIOORAPHY Lawrence P. McCallister was born on March 27, 19l~J, :ln Chicago, Illinois. He was graduated from Lake View High School in Chicago, in Fetruary, 1961; and continued his studies at Wright Jr. College, Chica.go, Ill., where he received the degree of Associa to of Ar.ts in Biolog~r in 1964·. He completed his undergraduate studies at the University f':f.' Illinois, Champtdgn, Urbana, and received the degree of Bachelor of Science in Zoology in I<\:ib:ru­ ary, 1966. In September of 1966 he began zraduate studies i.'1 the Dapartn:Gnt cf' Anatomy, l.oyola University, Str1.tch School of Hedicina, Chicago, Illinois ... He was awarded a National Defense Education Act Fellowship in the Dep:1rt­ ment of Anatomy from 1967 to 1970; and he was admitted as an associate men:ber in the Society of Sigma Xi in }fa.y, 1969. During the writing of his dissertation he w.;.s a.wn:r·ded a o. s. Public Health Fellowship :..n the Departments of Hodicine and Cardiology at the University of Chicago, Chicago, Illinois. ii ----------------------------------.....·------- ACKNOWLED:J 2:1-ENTS The author will always be indebted to his advisor, Dr. Robert F.adek, for unfailing support, encouragement, and scientific criticism throughout the preparation of this manuscript. Special thanks are also due to the Department of Pharmacology of Loyola University, and Dr. L. c. Blaber, who provided pharmacology equip-~ ment. A major portion of this work would not have been possible '!lrithout. the cooperation of Engis Equipment Co., Morton Grove, Illinois, which supplied the Cambridge Stereoscan used in the experiments. iii TABLE OF co~~T Ei~T s Page I. INTRODUCTION • • • • • • • • • • • • • • • • . • • • 1 A. PAin' I: THZ T SYSTEM • • • • • • • • • • • • • • • • 1 B. PART II: THE SU.DCELLULAR LOCALIZATIOJ.'l OF CALCIUM • • 3 II. REVI.d;'d OF THE P.EIATED LITERATURE • • • . • • • • • • • 6 A. THEORIES OF CONTRACTION. • • . • • • • • • • . 6 1. THE ROLE 01? IACTIC ACID AND ORGAiUC PHOSPHATES • 6 2. STRUCTURAL PROTZINS FOUND Di MUSCLE. • • • • • • 7 3. SLIDD~G FILA.Mi.i;NT TH.:!:ORY OF co.~TRl\.CTio:~ • • • • • 8 B. CALCIUN ION r:~ cmTRACTIOl'l • • • • • • • • . 8 c. EXCITATIO~I-CONTrtA.CTIOri COUPLING. • • • • • . 10 D. A?FR.OACHES TO TliE STUDY OF MUSCLE CALCIUH. 11 1. EARLY WORK • • • • • • • • • • • • • • • • • • • 11 2. RADIOCALCIUH EFFJ,UX. • • • • • • • • • • J2 J. CALCIUM i:HNDrnG DYES • • • • • • • • • • • • l'.3 #. FP...4.GME:?~TED SAHCOP:I..t-<SlHC REl'ICULU,;:·i. • • • • • • • lJ 5. AUTORADICDRAPiiIC STUDI&S • • • • • • • • • • • • 14 6. OXALATE PR3CIPITATIO.:f. • • • • • • • • • • • 15 7. LOC:\LIZ~_TION a:;· CALCIUM BY PO'l'ASSIUM PYROANTirl~ONAT E • • • • • • • • • • • • • • • • • 15 III. MAT ER.IAI.S AND M.:!:rHODS . 18 A. TIE MUSCLE •• • • Cl • • • • • • . 18 B. E,{?EBDrEi~TS Oi'l' T:E T SYSTEM: . • • • • • • • • • • 18 l. FIY.ATIO:~ AND DS:tYDT&"..TIO~~ • . 18 2. THA3SNI.$SIW ELEc1·:rw:i HICROSCOPY • • • • • • • • 19 J. SCA2{Hr~J ~LECfROi~ ViICROSCOPY • • • • • • • • • • 19 C. THE PHYSIOLOGY OF THZ C1iF'FEINE-INDUCED CWTP.ACTURE • 19 1. MUSCLE; PR~?AFWI'IC:I AND RECORDING APPARA.TUS • • • 20 2. SOl.UTIC.JS • • • • • • • • • • • • • • _. • . 20 J. PRCCLDURS . 21 iv Page D. SUBCELLUIAR LOCALIZATION OF CALCIUM • • • • • • • • 21 1. MUSCL~ EXPOSED TO l 0 -RINGER 1S SOLUTION, Cl1LCIUM F.RIB RING.i:R' S SOLU'l.'IO!~, OR RL'IJGE:cl' S SOLUTION CONTAINING CALCilr.·1 CHEIJ.Til'~G AG.GNTS • • • • 22 2. MUSCLES IN WHICH THE CAFFEINE-INDUCED CONTRti.CTURE WAS ARHS.::il' ED • • • • • • • • • • • • 22 IV. RESULTS: THE T SYST Ei·1 • • • • • • . 23 A. TRANSMISSION ELECTRON MICROSCOPY • • • • • • • • • • 2J 1. GEJ.'l.B;RAL MORPHOLOGY • • • • • • • • • • • • • • • 23 2. TJE SARCOPUcSHIC R3'.l'ICULUM • • • • • • • 24 J. ASSOCIATION OF THE T SYSTEM WITH THE F:GER SU~1.FACE • • • • • • • • • • • • • • • • • • • • 25 4. THE ZNTRA.NCE OF IANTH.fl..NUM INTO THE T SYST3M • • 26 B. SCANNING ELECTRON ¥.ICROSCOPY •• • • . 27 1. GENEP.AL MORPHOLCGY • • • • • • • • • • • • • • • 27 2. THE M1JSCLE SURI•'ACB OF EXT.RAFUSAL FIBERS • • • • 28 IV. RESULTS: THE SU.t3CELLUIAR LOCAJ.J:ZATION Oli' CALCIUH PY:KOA.NTDfONATE • • • • • • • • • • . A. PHYSIOlffiY OF THE CAFF~~JNE:-INDUCED CC'NTRA.CTURE IN CALCitJN-FRIB RINGER 1 S SQ1,ljI'IOl'~ • • • • • • • • • • • 30 B. ELECT RON MICROSCOPY OF I~USCIE.3 FIXED IN Os04 WTrHOUT POTASSIUN PYiWA.NTIEO.N'ATE • • • • • • • • 32 c. MUSCLES FIX3D Ii'l 1% Os04 CWTAIIUNG A 2;.o co:1Ci1NTHA- Tim~ OF POl'ASSIUH PYROANTIHWATE • • • • • • • • • • J2 D. MUSCLES £XPOSED TO CALCIUd-FiGB RINGER'S SOLUTIO~, AND RINGZR Is SOJ_,UTION co2nAINING CALCIUM CIBLADG AG E~~fr S • • • • • • • • • • • • • • • • • • • • • • • 34 E. NUSCL&S Ii~ ~·iHICH THE CAFFEINi-ItWGCED CONTR~CTURE WAS ARREST E:D IN CALCIUli-FRIB R.Il'lG ~P. 1 S SOLUTION • • • J4 F. MUSCLES IN WHICH THE CA!i'FEINE-TIWUC:SD co~~TrlAC:'l'URE WA.S A.:1RES'l'.2D Ill CALCIU~·l-fR.2:~ ?..Il-JGEP. 1S SOLUTION 1 I''LJr. I/. ,-,"'~A. A:"D '1 r;Y"_TA CONr· a 1\l ·\ u 5 m. i r... 11 r.. ...11.. ...., 5 tr.J."' .u..;i • .. • • • .. • • • 35 v p Page v. DISCUSSIO~~ • • • • • • . • • . A. GENER.\L MOR?HOLOGY . • • • • • • . :a. THE T SYSTEA ••• . • • • 1. ASSOCIATION WITd TH~ FIBER SURFACE • • • • • • • J6 2. FACTORS INFLUE;;:~CL'iG THE CO'.\TINUITY OF THE T SYST2l•1 WITH 1'1E EXTRAC2LLUL'\.R SPACE IN CON­ VENTIONAL ELECTrW~~ NICROORAPP.S • • • • • • • • • 38 c. THE JUNCTro:~ 3EI'iJEEi~ TIE T SYSTEM AND Tffi: SAR.CO- PLASMIC HErICULUl•I • • • • • • • • • • • • • • • • • 40 D. SPI~DLE-LI.t<3 STftUCIUP3S . 42 E. THE LOCALIZA'l'IOi1 OF CAlCIUM BY POTASSIUM PYROANTL\10- l~TE • • • • • • • • • • • • • . 43 1. D~·rRACZLLUL\R PRECIPITATE . 4J 2. EXTRACELLU.I.AR PR3:CIPITAT Z • • • • • • • • • • • 46 J. PRECIPITATE I~~ HUSCL2S t:XPOSED TO CALCIU11I-FREE SOLUTIOi~S Aim SOLUTIO~~s CWTAI2UNG CALCIUM C:iIElA.TING AG&.~TS • • • • • • • • • • •••••• 47 4. PRECIPITATE IN ~cHJSCLES IN '.'i'HICH THE CAFFEINE­ INDUC!i:D CON'l'&\CTUitE WAS ARREST3D •••••••• 48 VI. SU:•J·1ARY' Ai.rn CQ;:JCLUSIO:~s . 53 l3IrlLIOGR.\Pifi • • • • . • • • • • • . 56 PLATES AND FIGU;tES • . • • • • • • • • • • 66 vi • LIST OF TABLES Page TABLE I. EXPERil1ENT S ON THE T SYST El1 • • • • • • • • • • • • 19a TABLE II._,,,~PEK[l1E.NTS ON THE SlJBCELLUIAR LOCALIZATION OF W\.il;li.h·1 • • • • • • • • • • • • • • • • • • . • • 22a vii • r, LOSS;\l~Y OF TER~IS ADP adenosine dirhosphate ATP a<lenosine triphosnhate EDTJ\ ethylencdiaminc tetraacetic acid EGTA cthyleneglycol bis tetraacctic acid Os0 osmium tetroxide 4 viii • 1 I. IN'TRODUCTION This study had two goals; (~) to establish whether a direct continuity exists between the T system and the surface membrane in frog skeletal muscle; and (B) to observe and study the localization of calcium in skeletal muscle cells using potassium pryoanti.~onate as a reactant. .. .:l. • PART I: T'I:z T .SY7!'Z·: Transmission electron microscopy has demonstrated the continuity between th'3 membrc.nes of the T systero and the sarcolemma in a number of striated muscle cells (Franzini-Armstrong and Porter, 1961.~; Jasper, 1967; Rayns et al., 1968; Smith, 1966). A not<:d. exception, ho·:raver, is adult frog skeletA.l ::rnu:;cle in which the T system fo:r.ms complex terminations at the f.i'.:>ar surface, obscuring any direct co~tinui..ty between the wall of the T tubules and the sa.rcolemma (Peachey, 19~5). On the other hand, several stu::lies have demonstrated the free diffusion of substances between the e:Ktracellular space and the central elements of the triads in at>1phibian rn.wcle. For example, 2ndo ( 19:S!t-) traced the diffusj.0:1 of a fluorescent dye, Lisa.mine Rohda:irl.ne B 200, in isolated fibers of the semitendi- nosus muscle of the frog. In his experiments the dye was consistently otserved in the cente1• of the I band, without appea.rinz in the major portion of t".-le .r. l i. ioer. Using aul:.o~adio:-:,raphy, Hill (1964) localized tho uptake of trit:i.at.ed sertt:n a.lburnin to the central compo::ient of the triads in amphibian muscle; and both Huxley (19&'+) and Peisc~ (199~) '!:ave demo:1strated the uptake of ferritin :i.n T tubnles of frog sartc::--ius r:iuscle.:;. All indicatin6 a possibility of direct continu.i.ty between the T tuhulBs aZ'ld the muscle cell sm.•face. p • 2 Despite this indirect evidence, only two investigators, L.D. Peachey (1965) and R.I. Eirks (1965), have published transmisston electron micro­ gra.phs of frog skeleta.l muscle showine terminations of T tubules which could be interp1·eted as having a possible continuity with the extracellular space. However, due to the complicated termination of the T tubules, and their infre­ quent occurrence along the cell membrane, such evidence has been subsequently regarded as inconclusive (Bianchi, 1968; Eisenberg and E:tsenberg, 1968; Smith, 1966; Fra.nzini-Armsb~onc, 1970).
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    Frog Skeletal Muscle Contraction Experiment Is Ajai Hitlerite or cheliferous when swage some cubist daut capriciously? Martyn remains Neozoic: she herald her waterfront militates too iniquitously? Lyle remains piscatorial after Tibold disavows loud or mazes any clubbing. Curare is also known among indigenous peoples as Ampi, Woorari, Woorara, Woorali, Wourali, Wouralia, Ourare, Ourari, Urare, Urari, and Uirary. The secondå°• major difference between plant and animal cells is the cell wall. In B, note the half pillar protruding from the TC, also indicated by a line. You seem to have javascript disabled. Neurophysiology of the neuromuscular junction: overview. The influence of activity on some contractile characteristics of mammalian fast and slow muscles. What was the smallest current required to produce each of the following? Kullberg R, Owens JL. It often resolves without treatment. This electrical signal is communicated to the myofilaments inside the fibre in the following way. Connective tissue cells, fibers and intercellular medium are present in these tissues; The cells of these tissues are widely spaced and forms intercellular matrix. When the current reached the following stages, what proportion of fibers in the muscle were contracting? Most land animals, including humans, depend on angiosperms directly or indirectly for sustenance. These cells are actively mitotic, producing new cells that get pushed upward into the overlying layers. Shaded bars represent periods of strain heterogeneity. Now, gradually flex your wrist back to a straight or neutral position. Summation of twitch responses Recoding a double contraction. Since it was too expensive to be used in warfare, curare was mainly used during hunting. Tension responses to sudden length change in stimulated frog muscle fibres near slack length.
  • Calcium Antagonism of the Block in Excitation-Contraction Coupling Produced by a Urea Exposure- Removal Treatment

    Calcium Antagonism of the Block in Excitation-Contraction Coupling Produced by a Urea Exposure- Removal Treatment

    Jap.J.Physiol.,27,215-224,1977 CALCIUM ANTAGONISM OF THE BLOCK IN EXCITATION-CONTRACTION COUPLING PRODUCED BY A UREA EXPOSURE- REMOVAL TREATMENT G.B.FRANK and R.C.TREFFERS Department of Pharmacology,The University of Alberta, Edmonton,Canada Abstract Exposing frog's toe muscles to Ringer's solution made hyper- tonic with 400mM urea for 60min followed by placing the muscles back in Ringer's(urea-removal treatment)completely blocks the twitch without disrupting the surface openings of the T-tubules.The urea-removal treatment also increased the triadic junction width.Placing the muscles in Ringer's with an elevated calcium concentration(5mM)following exposure to the hypertonic solution prevented the block of the twitch response but not the increase in the triadic junction width.Exposing untreated muscles to Ringer's with 5mM calcium either had no effect on the twitch or reduced it by 30% or less.These results suggest the possi- bility that increasing the width of the triadic junction decreases the amount of calcium ions reaching the terminal cisternae during an action potential thereby blocking the twitch.Elevating the calcium concentration in the T-tubules would increase the amount of calcium which enters the triadic junction during an action potential and thus antagonize the above effects. Several years ago it was demonstrated that the T-tubular system plays an important role in conducting the electrical activity from the surface membrane of frog's twitch skeletal muscle fibres into the interior of the fibres(HUXLEY and TAYLOR,1958).Later it was found that reexposing skeletal muscle fibres to Ringer's solution following a 1 hr exposure to Ringer's solution made hypertonic by the addition of 400mM glycerol disrupted the openings of the T-tubules to the extracellular space of the muscle and simultaneously blocked excitation-contraction (E-C)coupling(GAGE and EISENBERG,1967;HOWELLand JENDEN,1967;EISEN- BERGand EISENBERG,1968).
  • Chapter 4 Calsequestrin

    Chapter 4 Calsequestrin

    Chapter 4 Calsequestrin DAVID H. MacLENNAN Banting and Best Department of Medical Research C. H. Best Institute, University of Toronto Toronto, Ontario, Canada KEVIN P. CAMPBELL Department of Physiology and Biophysics University of Iowa Iowa City, Iowa and REINHART A. F. REITHMEIER Department of Biochemistry University of Alberta Edmonton, Alberta, Canada I. Sarcoplasmic Reticulum ............................................................................. 152 II. Isolated Sarcoplasmic Reticulum Vesicles ................................................. 153 III. Isolation of Calsequestrin ........................................................................... 155 IV. Ca2+ Binding by Calsequestrin ................................................................... 155 V. Size and Shape of Calsequestrin................................................................. 157 VI. Amino Acid Sequence................................................................................ 159 VII. Carbohydrate Content................................................................................. 160 VIII. Stains-All Staining...................................................................................... 160 IX. Properties of Calsequestrin in Other Muscles............................................. 161 X. Phosphorylation of Calsequestrin ............................................................... 162 XI. Localization of Calsequestrin ..................................................................... 163 XII. Biosynthesis...............................................................................................