THYMOPOIETIN AND MyoD: THEIR EFFECTS ON
THE MUSCLE NICOTINIC ACETYLCHOLINE RECEPTOR
by
RuIa S. Odeh
Department of Pharmacology and Therapeutics
McGill University
Montreal, Quebec
Canada
May, 1993
A thesis submitted to the Faculty of Graduate Studies and Research in partial
fulfillment of the requirements for the degree of Master of Science e· \C) RuIa S. Odeh, 1993 TITLE: THYMOPOIETIN AND MyoD: THEXR EFFECTS ON THE MUSCLE NICOTINIC ACETYLCHOLINE RECEPTOR
SHORTER VERSION OF TITLE: (LESS THAN 70 CHARACTERS)
THYMOPOIETIN AND MyoD: EFFECTS ON THE NICOTINIC ACETYLCHOLINE R'ECEPTOR A)lSTRAC"f
The pr(:sent study was do ne to determine whether the muscle IllCotllllC aceiylchoI1ne T\.< '{Hor (nJ"Cl l ,{) functlOn and expression could be reglll.lted by lwo differenr r'-4~" l' thymü,101I.t l n (l'PO)", a l1Ieotil1le i:IntagolllsI .l'Ild h) M yoD, a myogenk .... " ;)\. faclor.
EXr)osure nf \ .~<.matal Illuscle ccUs 111 cult -c to TPO 011 a long ·Icrm (days) .md short-h'Tr Short-term p'rctr~tlll' 1 TrO .1Iso \ui to a dccreasc in ..:arhachol-~lllllulalcd "Na uptake; huwc\_ ... "110S11f," resulfed in an enhanœd carhachol-slllllulale development. l', j '.'. '" ,:st that TPO n~;gulate'5 the nAChR and excrls tmpill:': effects on myotuht! mor. ,,,~, ;'1. As another approach to sludy factors th~it affect nAChR expression, non-musc'e cells were transfected with MyoD cDNA. After transflxtlon, saturahle, I1lgh aflïnily 11\1_ œ-BGT binding was readily detectab\e, a'i W2S carbacho\-stllnulatcd llNa uptakc. BOlh these parameters developed in para\k\ over timc and werc 1Il1llbtted by nicotlllic antagonists. These results suggest that th~ transfectlOn 01 a non-lTIl1~clc œil !tIlC wlth MyoD cDNA results in the expression, at the cell sUïface, cl a flJlr1ctlonal mll~dc-ty!)C nAChR. This work shows that the nAChR function and expressIOn can be regulat,!d through a) the chronic interaction of TrO al the nAChR at the ccII surt;lcc and b) tl1C action of MyoD at the gene level. • As stated in the addendum to Ihls the~I!>, rt:Cent work hy QUik ct al. 19CjJa ha .. :-.hown thal tlle preparation pre~umed to be TPO, contained a-cobratoxlO; Ihe cfft:el .. ob.,ervoo JO the pre:-.enl IhcMs mu.,t therefore now be altnbuled 10 the prel.cnce of a-cobratoxm contammant. Ri:SUMÉ L'étude actuelle a été effectuée pour déterminer si la fonction et l'expression du récepteur nicotinique de l'acétylcholine (nAChR) de type musculaire pourraient être contrôlées par deux facteurs différents: a) la thymopoïétine (TPOr, un antagoniste nieotinique et b) la MyoD, un facteur de transcription myogénique. L' exposi tion à la TPO des cellules musculaires de rats nouveau né pour des périodes de longue durée (jours) et de courte durée (minutes) a résulté en une inhibition de l'attachement de la 125I-ex-bungarotoxine (BGT). Un prétraitement de courte durée avec la TPO a également conduit à une diminution de la captation du 22Na stimulée par le carbachol; toutefois, un prétraitement de longue durée a résulté en une captation accrue du 22Na stimulée par le carbachol. Un traitement chronique a également résulté en un plus grand développement morphologique des cellules musculaires. Ces résultats sugpèrent que la TPO contrôle le nAChR et exerce des effets trophiques sur la morphologie des myotubes. Une autre approche pour étudier les facteurs qui affectent l'expression du nAChR a été de transfC'çter avec de l'ADNc de la MyoD des cellules non-musculaires. SUite à la transfection, un attachement de haute affinité et saturable de la 125I-a-BGT a été détecté immédiatement; nOlis pouvions également détecter la captation du 22Na stimulée par le carbachol. Ces deux paramètres se sont développés parallèlement dans le temps et ont été inhibé par des antagonistes nicotiniques. Ces résultats suggèrent que la transfection • Td que sllpull! dans l'addendum de ce mémOire, Quik et al. 1993a ont démontré, dans le cadre d'une étude recente, la présenœ de a-cobratoxine dans une préparatIOn présumée ~tre du TPO pur; par con~équent lel! effets rapportés danll le cadre de cd ouvrage dOIvent ~tre attribué à cette contanunatlOn par \'a-cobraloxme. ii d'une lignée cellulaire de type non-musculaire avec \' ADNe de la MyoD résulte en l'expression, à la surface cellulaire, d'un nAChR fonctionnel de type musculaire. Ce travail montre que la fonction ct l'cxpressHm du nAChR pClIvcnt être contrôlées par l'entremise de a) l'interaction chronique de la TPO avec le nAChR à la surface cellulaire et b) l'action de la MyoD au mveau du gène. III To Nabil and my parents "Wisdom is supreme; therefore get wisdom. Though it cost you all have, get understanding. " Proverbs 4: 7 IV ACKNOWLEDGEMENTS 1 would like to express my deepest gratitude and appreciatlOl1 to: Maryka QUlk, my research sllpervisor, for ail her valuable gllldancc, patlencl' and support. Moshe Szyf, for his valuable advice and guidance on the MyoD pmJl'ct. Dr. Collier, for his helpflll comments and suggestions concerning thc thesis, as weil as timely encouragement. Dr. Cuello, for the opportumty to study in the encouraging env Irllll mcnt 01 the Department of Pharmacology and Therapeutics. Dr. Capek, my advisor, for ail hls help and advice. Dr. Robaire and Dr. Hales, for their understanding and encouragcment whlle servlllg a~ Chairmen of the Gradllate Committcc during my stay in the departll1cnl. Dr. Padjen, for his vaillable computer help. AIl my professors in the Department whose teaching was nourishlllg for thc Illliid. 1 wish to extend very warm and special thanks to: Jacynthe Philie, for bemg a true fnend as weil as a supcr-techmcian in the lab. Her hdp in teaching me most of the techl1lques 1 needcd, prooflcadll1g this lhc'il~ and translating the abstract mto French are very m lIch appreciated. Ronith Afar, for always being there wh en 1 necdcd moral !!lIpport and encouragement as weil as for her val uabJe advicc concermng the thesis. Susanne Geertsen, for her friendship and hllmorous adVICC Jennifer Chan, for aU her positive encouragement and fricndshlp v AmalIa Issa, for being a great pal and for proof-reading the thesis. Juhc Rouleau, for her guidance in the molecular blology techniques 1 needed Johannc Théberge. for her tcchnical assistance in certaIn parts of the MyoD projecl Ali my dcpartmcntal fnends, too numerous to mentIon, who contributed in severa1 ways to my success and enJoyment of the time 1 spent 111 the department. André Côté, for translatmg the abstract II1to French. Alan Forster, for the photography work. Words cannot express my indebtedness to my husband Nabll, for an his love, patience and moral support as weIl as to my family for all their encouragement. VI PREFACE Note on the format of the theSlS This thesis consists of two ~tudles. The tirst. prescnlt'd 111 \cctlOn 3. 1. descnhc~ thl effects of TPO on the nAChR in rat neonatal muscle ccll~ \11 Cllltur~ as weil Ils TP()'" effects on the morphology of the neonatal muscle cells. Ali the work (kscnht'd 111 tl\l~ study was done by R.O. The second study, presented in section 3,2, is based on the followlI1g milllll ... cn pl. M. Quik, R. Odeh, J. Philie and M. Slyf: Functional nicotmlc receptol cxprc-;SIOIl III mesodermal cells transfected with tvtyoD cDNA. Neuro.\('/eI1ce 57: 7'K7-795. 19l)~. The transfection of C3H lOTl/2 cel1s wlth MyoD cDNi\ wa~ done hy M.S. Thc~c l'C1I~ were subsequently subcloned by 1, P., reslIlting 111 the dcvclopmcnt 01 t WO C10IlC~, callet! clone 8 and clone 19, These clones were analyzed by northern analy\ls and irnmllnostaining by M,S. Full descriptions of these procedures arc provldcd III the Materials and Methods to explain the origins and characteristics of the cdb. I~xpcnmcllt\ relating to clone 19 were done by J.P,; the~e have bccn cllllllnatcd l'rom ~cclion J.2. Only the work performed by R.O. on clone 8 and the appropriatc control I~ prl;\cntcd III section 3.2. This workconsistsofbindingand functlonal assay~ as weil a ... lIlorphologleal assessments. VII TABLE OF CONTENTS Page ABSTRACT ...... " ...... RÉSUMÉ ii ACKNOWLEDGEMENTS ...... v PREFACE ...... vii TABLE OF CONTENTS ...... viii FIGURE INDEX ...... xi TABLE INDEX ...... xiii LIST OF ABBREVIATIONS ...... XIV SUMMARY OF CONTRIBUTIONS TO ORIGINAL KNOWLEDGE ...... xvi 1.0 INTRODUCTION ...... 1.1 MUSCLE-TYPE NICOTINIC ACETYLCHOLINE RECEPTORS ...... 2 \.2 FACTORS THAT AFFECT nAChRs...... 6 1.2.\ Induction of receptor c1usters at the developing nerve-muscle synapse . . .. 6 1.2.2 Neurotrophic Factors '" ...... 8 1.2.3 Suhmernhrane Machinery for nAChR Clustering ...... 11 1.2.4 Muscle Activity ...... 12 1.2.4.1 The MyoD Farnily of Myogenic ReguJatory Factors...... 13 1.2.4.2 The Effects of Agonists and Antagonists on Receptors ...... 15 1.2.4.3 Thyrnopoietin. a Thymie Polypeptide that Potently Interacts at V 111 the Muscle Nicotinic AChR...... 17 STATEMENT OF THE PROBLEM ...... 20 2.0 MATERIALS AND METHODS ... " ...... 22 2.1 Materials ...... 23 2.2 Methods ...... 23 2.2.1 Transfection of C3H 10TI12 cells with MyoD cDNA 23 2.2.2 Subc\oning ...... 24 2.2.3 Cell Cultures ...... 25 2.2.4 Measurement of I:!.~I-a-BGT hinding tll eclls in cultur\! .. .. . 26 2.2.5 Measurement of ~2Na intlux ...... " ...... 2H 2.2.6 Assessment of myotuhe length and hranch formation in rat mylltuhe cultures ...... 2H 2.2.7 Statistics ...... , 29 3.0 RESULTS ...... 30 3.1 LONG-TERM THYMOPOIETIN TREATMENT OF RAT NEONATAL MUSCLE CELLS IN CULTURE LEADS TO ENHANCEMENT OF NICOTINIC RECEPTOR FUNCTIONAL RESPONSE AND MORPHOLOGY '" ...... 31 3.1.1 Characterization of nAChR hinding and function 111 rat nt!onatal mu!->c\t! cells in culture ...... 32 3.1.2 Effecl of thymopoietin on 125I-a-BGT hinding amI carhachol- ... timulated ~2Na uptake ...... " ...... '" ..... 33 3.1.3 Effect vf thymopuh;tin on the morphological dt!vdopmt!nt ot rat nconatal muscle cells in culture ...... " ...... 36 3.2 FUNCTIONAL NICOTINIC RECEPTOR EXPRESSION IN MESODERMAL STEM CELL~ TRANSFECTED WITH MyoD cDNA .... " ...... 44 IX 3.2. ) MorphologicaJ deveJopment of C3H 10Tl/2 cells transfected with MyoD cD NA ...... " ...... 45 3.2.2 Nicolinie rceeptor hinding in eeJJ!. transfected with MyoD cDNA . . . . 46 3.2.3 Nicotinic reœptor-mediated function in cells transfected with MyoD cDNA ...... 47 4.0 DISCUSSION ...... " 57 4.1 LONG-TERM THYMOPOIETIN TREATMENT OF RAT NEONATAL MUSCLE CELLS IN CULTURE LEADS TO ENHANCEMENT OF NICOTINIC RECEPTOR FUNCTIONAL RESPONSE AND MORPHOLOGY ...... 59 4.2 FUNCTIONAL NICOTINIC RECEPTOR EXPRESSION IN MESODERMAL STEM CELLS TRANSFECTED WITH MyoD cDNA ...... " 68 4.3 CONCLUSION ...... 72 5.0 REFERENCES...... 74 x FIGURE INDEX FIGURE PAGE 1 (A) Time course of development of specitic 125I-a-BGT bindlllg in rat neonatal muscle cens in culture. 38 1 (B) Time course of development of nicotlllic response. .18 2 (A) Saturation curves of specifie mI-a-BGT binding to rat nconatal lIlusclc cells after 4 and 9 days in culture. 2 (B) Dose-response curves of carbachol-stimulated 22Na uptakc \Il sisler rat neonatal muscle cells after 4 and 9 days in culture. 39 3 Inhibition curves of specifie J25I-a-BGT binding to rat nconatal muscle cells after exposure to TPO for either 60 minutes (IInmcdiatcly prior to assay) or over a period of 4 (Fig. 3A) or 7 days (Fig 3B). 40 4 Inhibition curves of specifie earhaehol-stimulated 2lNa uplake 111 ral neonatal muscle cells after exposure to TPO for clthcr 60 mll1l1lcs (immediately prior to assay) or over a period of 4 (Fig. 4A) or 7 days (Fig. 4B). 41 5 Effect of TPO (4 day exposllre) on the nllmber of myotubes 0.5 mm or longer at 320X magnification. 42 6 Effeet of TPO (7 day exposu·:e) on nllmber of branehpoints in rat neonatal myotubes. 43 7 Morphological deveJopment of control (CON) and clone 8. 50 8 Time course of receptor development of clone 8. SI 9 Saturation curve of specifie mI-a-BGT binding to clone 8. 52 XI JO Inhibition curv(!S of specifie ml-a-BGT binding to clone 8. 53 l' Stimulation of nNa uptake by carbachol (lQ4 M) in clone 8. 54 12 Inhibition curves of carbachol-stimulated 22Na uptake in clone 8. 55 13 Time course of development of 125I-a-BGT binding and carbachol-stimulated 22Na up'Lake in clone 8. 56 XIJ TABLE INDEX TABLE PAGE Effect of transfection with MyoD cDNA on nicotinic receptor binding to C3H lOT1I2 cells. 49 XIII LIST OF ABBREVIATIONS AChR acetylcholine receptor ACh acetylcholine a-BGT œ-bungarotoxin Bnu,x maximum number of binding sites cDNA complementary DNA CON control CNS central nervous system DMEM Dulbecco's modified Eagle's medium DNA deoxyribonucJeic acid EC~ll concentration at which the functional response is reduced to 50% FCS fetal calf serum GABA 'Y-aminobutyric acid HEPES 4-(2-hydroxyethy!)-I-piperazineethanesulfonie acid hr hours IC Sll concentration required to reduee binding or functional response to 50% K kilodaltons KI) dissociation constant mAb monoclonal antibody MI-M4 transmembrane segments 1 to 4 - min minutes xiv rnRNA messenger ribonucleic acid nAChR nicotinic acetylcholine receptor TPO preparations presumed to be thymopoietin° S.E.M. standard error of the mean • As stated in the addendum to this tht:sls, rt:Cent work ~)y QUlk t:t al. 1993a has "hown that the preparation presumed to he TPO, contained a-cobratoxtn; the effects ob!.erved ln tht: prc!>t:nt thesÎs mu!.t therefore now he attnbuted to the pre!.ence of a-cobratoxtn contammant. xv SUMMARY OF CONTRIBUTIONS TO ORIGINAL KNOWLEDGE Studies were undertaken in this thesis to determine thle effects of the factors, TPO· and MyoD, on the nicotinic acetylcholine receptor in order to assess whether these factors can regulate the receptor. The novel findings of this thesis are summarized below. la) The long-term (days) pretreatment of rat neonatal mU'Yde cells with TPO, a nicotinic antagonist, resulted in a potent inhibition of l25I-ex-BGT bmding as has previously been shown for short-term (minutes) TPO pretreatment, suggesting that TPO interacts at the nAChR under both of these conditions by binding to the ex- BGT recognition site. b) Short-term pretre.atment with TPO also led to potent inhibition of carbachol- stimulated 22Na uptake; however, chronic exposure of the muscle cells resulted in an enhanced uptake. This suggests that long-term TPO pretreatment may up- regulate the levels of the nicotinic acetylcholine receptor resulting in changes in the functional response. Alternatively, the functional response might be affected as a result of changes in the conformation or state of functional activation of the nAChR. c) Chronic TPO exposure of rat neonatal muscle ceUs from an early stage of their development in culture led to the enhancement of morphological characteristics such as muscle cell length and branching. • As !.htt~d m th~ addendum to this thesls, recent work by Quik et al. 1993a has shown that the preparation pre!.umed to he TPO, contamed a-cobratoxm; the effects observed ln the present thesis must therefoœ now he attnhutcd to the presence of a-cobratoxln contaminant. XVl These findings suggest that thymopoietin, an endogenously oeeurring thymie polypeptIde. has the potential to affect the nAChR as we1l as myotube morphology through an interaction at the a-BGT site of the l11eotinic reeeptor. 2a) A non-muscle mesodermal stem cell line C3H 101'112, which does Ilot express any muscle-specifie genes, was transfected with the cDNA of a single myoget11l' transcription factor MyoD. Following transfection, saturable, high aftïnity \!~l-(~ BGT binding was readily deteetable. This bindmg IS potently inhlhllcd by I1Icotinu.: agonists and antagonists but not by muscarinic agolllsts and antagontsts. Thcse results indicate the development of a receptor with the pharmacologlcal protïlc of a muscle-type nAChR. b) Carbachol-stimulated 22Na uptake was also present aftcr transfection wlth MyoD cDNA. This flux was potently inhibited by nicotinic antagontsts but not a f11uscarinic antagonist, indicating the presence of nicotinic reccptor-mcdlated functional response. c) Paralle1 alterations in '25I-a-BGT binding and carbachol-sti mlilatcJ llNa lIptakc occurred during time course studies. Taken together, these findings suggest that the observed functional rcsponsc occurs as a result of activation of the a-BOT recognition site which appcars to have becn mduccd as a result of MyoD transfection. Thus, the transfection of a non-muscle ccII linc wlth MyoD cDNA can lead to the appearance of a functional cell surface musclc-t~/pc nicotinic acety 1choline receptor. XVII 1.0 INTRODUCTION Ligand-gated Ion channels represent a superfamily of receptors that can transduce chemlcal neurotransmitter signaIs released from one cell into an electncal signal that propagates along the target cell membrane. Members of this superfamily of ligand-gated receptors include: the nicotinic acetylcholine receptors (nAChR) of neuromuscular and neuronal origm, the kamate type glutamate-activated channels and the serotonin (5HT3) rcccptor which ail conduct Nr-,' and K + resulting in depolarization of the cells; and the neuronal GABA and glycme activated channels which conduct chlonde Ions leading to hyperpolarization. Also, in this superfamily is the N-methyl-D-aspartate (NMDA) glutamate-gated channel which has a high Ca2 + permeability controlled in a voltage dependcnt manner by extracellular Mg2+; the influx of Ca2+ into the postsynaptic celI, accompanying the rel case of Mg2+, is beheved to underlie activity-dependent changes in synaptlc strength and to be Important for memory function. Although these receptors differ in size and sequence, their subunits share the following features: four putative membrane spanning regions called M I-M4; strong sequence homology in these hydrophobic transmembrane sequences, especially in M2, the hydrophobic stretch that is hypothesized to line the ion-conducting channel; and a probable quasi-symmetric pentameric arrangement of similar or identlcal subunits. A homologous cystine-bridged loop is found at the amino terminal extracellular region as are several glycosylation sites. Finally. the subuOlts possess a large intracellular region extending between the third and fourth transmembrane domains (Changeux et al. 1984; Wan and Lindstrom 1984; Ratnam et al. 1986; Barnard et al. 1987; Grenningloh et al. 1987; Schofield et al. 1987; Stroud et al. 1990; Galzi et al. 1991; Peters et al. 1992; Unwll1 1(93). Several nicotimc receptors have been identtfied to date. These include the muscle-type nAChR present in electric organ and on skeletal muscle, and the nAChRs round III nervous tissue which can be fllrther slIbdivided into a-bungarotoxin «\'-13(;1') senSitIve and insensitive subtypes. 1.1 MUSCLE-TYPE NICOTINIC ACETYLCHOLINE RECEPTORS The muscle-type nicotinic acetylcholine receptor IS present \11 elcctroplax and neuromuscular tissue. Initial success in Identifying the nAChR was possible III large part to the availability of a specific, hlgh aftimty ligand a-BCiT as weil as tlSliliC sources nch in receptors, such as the electnc organ of Torpedo and Elcctrophoru~ (Changcux ct al 1970; Miledi and Potter 1971; Miledi et al. 1971; Karlin 1974; Cohcn and Changcux 1975; Rang 1975; Fambrough 1979). The essentially irreversible interaction of a-BGT with the nAChR and Ils abtlity 10 block receptor functlon have rendered it an excellent probe for the study of lhl~ rcccptor. ln addition, the a-toxin greatly facIlitated receptor purification. SDS gel c1cctrophorcslli showed that the Torpedo receplor consisted of five subllnits with apparent mo!cclIlar masses ranging from 40-65 kD (Weill et al. 1974; ClaudIo and Raferty 1977). These subunits are arranged in a pseudo-symmetric manner with a ~tO\chlOmctry of fxli'Yo (Lindstrom et al. 1979a; Raftery et al. 1980; Conti-Tronconi ct al. 19H2; Karl i n et al. 2 1983) In the ca~e of Torpedo and Electrophorus electroplax as weIl as fetal muscle. In adlllt muscle, the 'Y subunit is replaced by a similar but distinct E subunit (Takai et al. 1985; Mishina et al. 1986). Immunologically, the Torpedo, Electrophorus and mammalian muscle nAChR have been shown to be sllnilar (Lmdstrom et al. 1979b). The cDNA for each of these subunits has been cloned. The predicted ami no acid sequences yield calculated molecular welghts of 50-57 kD (NoJa et al. 1982, 1983b; Claudio et al. 1983). The difference between the ca1culated molecular weights and those dctcnmncd by electrophoresls is due to post-translatlOnal modIfications of the peptides. MlcromJcction of the mRNAs for the a, {J, 'Y, and ô subunits in frog oocytes resulted in the formation of functlOnal nAChR channels (Mishina et al. 1984, 1986). The use of cDNA probes from Torpedo receptor subunits led to the isolation of the full length cONAs and gcncs for the muscle subunits (Noda et al. 1983a, Takai et al. 1984; Tanabe et al. 1984; Kubo et al. 1985). These muscle-denved subul11ts were found to be homologous to the Torpedo-denved subunits. Certain regions whlch are important for ligand binding and for the channel properties of the receptor have been characterized. Studies using affinity labelling demonstrated that the acetylcholine bmdmg site resldes on the a subumt of the receptor (Weill et al. 1974; Damle et al. 1978). Subsequent reconstitution studies in mouse fibroblasts showed that the omissIOn of a-subunIts led to the loss of acetylcholine (ACh) sensitivity as weB as (X BGT bindmg suggesting that the binding sites of both these agents are found on this SUblll11t (Claudio et al. 1987). a-BGT was found to block acetylcholine binding, 3 suggesting that it bound to a :'lte near or at ACh b11ld1l1g sitl: (Gl:r~holll I:t al. \9~D). Thus, the nAChR contams two acetylcholine/a-BGT blllding sttl:S wlllch have hccll observed to interact in a positive cooperative manner (Jackson 1988). Whlk the Iwo IY subunits in Torpedo are encodcd by a single genc, they ,tn.~ nOlHXjlll\'aiL'nt 111 Ihal thl'y have differing aftinities for acetylcholllll: 111 assel11bl~d receptor prolelll"i. Morl'ovcr, monoclonal antibodles (mAb) to the cholinergIe bllldmg ..,Ite do Ilot always hind 10 lhe receptor with a stoichlOmetry of two IllAb per receptor (M Ihovllovlc and Rlchman 1087) In sorne cases, only one mAb will bind to each reccptor i\l(hcatlllg that thcre arc conformational differences betwecn the two ligand btnding reglons. TllIS may he duc tn the fact that each a subumt is slirrounded by differcnt :-.uhllnits wlllch may contnbute domains to the ligand binding site and thus affect the aftïmtics al thcse sites for cholinergie ligands (Oswald and Changeux 1982). The reœnt IdcntllÏcallon 01 a :-.econd a-subunit in Xenopus dlstmct from that origInally Idcntlflcd (Hartman and ClaudiO \090) raises the possibihty that the difference In observed bmdll1g aftil1\tles may he oue 10 the co-expression of two distinct a-subumts wlthm the same n:ceptor. Further characterization of the ligand bindtng site of the nÂChR led to the IdentificatlOll of a unique pair of adjacent cysteme residues at positions 192 and 193 that are hnkcd hy a disulfide bond in the native reeeptor (Kao and Karhn 1986; Mmcovill and (;er~holll 1988). Other stlldles involving a-BUT bmdmg to the a-'illbumt fragmcnts ((;cr~h()111 1987) as weil as to synthetic peptides (AronhellTI et al. 1988; Contl-Tronconl ct al. 19(0) showed that these adjacent cysteines do indeed constitutc part of the ligand bÎndlllg site. Further evidence that acetyJcholine binds to these reslducs was provldcd by ~tlldie.., 4 involving deletion mutations (Barkas et al. 1987) and site-directed mutagenesis of the a subumt (Mlshina et al. 1985). The adjacent cysteine residues are found only in the a subumts and nol the other subumt!) as was later revealed by comparisons of the cDNA sequence!) of the various subunits. The region where a-BGT binds has becn extensively invc~tigatcd. Neumann and colleagues (1986) sho\\ed that a synthetic peptide corrc~ponding to residues 185-196 binds to a-BGT direcUy. This bindmg could be inhlblled both by d-tubocurarine as weil as by a mAb (5.5) for the cholinergie binding site. Tzartos and Remoundos (1990) have further localized the major a-BGT site to resldues 189-195 of the Torpedo nAChR. The mapping of the acetylcholine binding site was achieved with the use of the photoaffinity label p(N ,N ,)-dimethyl-amjnobenzene diazonium fllloroborate (DDF) which acts as a reversible competitive antagonist of the acetylcholine response. When photoactivated, DDF covalently binds to amino acids in the acety1choline binding region. The following residlles were labelled: two tyrosines at positions 90 and 190, a tryptophan at position 149 and the two cysteines at positions 192 and 193 (Denms et al. 1986). Both a-BGT and carbachol blocked the binding of this label, further implicating these amino acids in ligand binding. These sites are only conserved in the a-subunits and not the other subunits of the electric organ or muscle nAChR (Dennis et al. 1988; Duvoisin et al. 1989). The nAChR channel has also becn characterized. Convergent results from several studies point to the hydrophobie M2 segment of each of the ftve subunits as a critical component of the channel. The non-competitive blocker 3H-chlorpromazine was found to bind to the serine residllc al position 262 of the ô-subunit of the Torpedo nAChR (Giraudat et al. 5 1986). This amino acid is located within the M2 hydrophobie region of the subunil. Similarly, in other subunits, several other amino acids were labelled that also OCClU in the M2 membrane spanning region (Giraudat et al. 1989; Revah et al. 1990). In otlter studies, molecular biology techniques were used to investigate the role of the M2 segment in determining the channel properties of the receptor. Chimcric rcceptors were proollccd using different componellts of the Torpedo and bovine muscle () subllnits in variolls combinations. It was found that a region comprising the M2 segment and the area immediately adjacent to it played a critical role in determining the rate of ion transport through the channel (lmoto et al. 1986; Leonard et al. 1988). Site-directcd mutagencsls studies involved introducing point mutations into the various subunits leadlllg lo a change in the net charge of the transmembrane segment. lt was found lhat tluee cllIsters of negatively charged ami no acids adjacent to the M2 regions of each of the four SUblllllt~ affect ion transport (lmoto et al. 1988; Hucho and Hilgcnfcld 1989). Thesc ncgatlvcly charged residues tine the ion pore and promote ion conductIon through the porc. Thc lugh degrec of conservation in the area surrounding the M2 region of each of the four subliOlts isolated to date is indicative of their functional importance in dcfining the channel properties. The nicotinic acety1chohne receptor has becn cxtcnslvely studlcd and several factors that can affect it have becn identified. 1.2 FACTORS THAT AFFECT nAChRs 1.2.1 Induction of receptor clusters at the developing nerve-muscle synapse The nerve plays an important role in the control of junctional and extrajunctionaJ 6 acetyJcholine receptor (AChR) density. AChR clusters form rapidly after the arrivai of the motor nerve (Blackshaw and Warner 1976; Kullberg et al. 1977; Braithwaite and Harris 1979; Chow and Cohen 1983, Kidokoro et al. 1980; Creazzo and Sohal 1983; Frank and Fischbach 1979; Role et al. 1982, 1987). Sorne areas of high AChR density are seen prior to innervation (Fischbach and Cohen 1973; Sytkowski et al. 1973; Anderson and Cohen 1977; Jacob and Lentz 1979). However, the ingrowing motoneuron does not seck out pre-existing c1usters but rather induces a new aggregate of receptors (Anderson and Cohen 1977; Frank and Fischbach 1979). Once transmission begins the number of extrajunctional AChRs declines steadily (Diamond and Miledi 1962; Bevan and Steinbach 1977). The growing nerve terminal is capable of release of neurotrophie factors before the formation of AChR clusters (Blackshaw and Warner 1979; Kullberg et al. 1977; Bevan and Steinbach 1977; AndersOlI et al. 1979; Frank and Fischbach 1979; Cohen 1980; Role et al. 1982, 1987; Kidokoro 1980; Kidokoro and Yeh 1982; Hume et al. 1983; Young and Poo 1983). These neurally-released substances therefore have the potential to exert an inductive influence on c1uster formation (reviewed by Dennis 1981). It appears that only cholinergie neurons have the capacity to induce AChR clusters. When cholinerglc motoneurons from the spinal cord and parasympathetic neurons contact myotubes in vitro, similar numbers of neurite-associated receptor c1usters are induced (Role et al. 1985, 1987). Other cholinergie neurons also c1uster AChRs (Nelson et al. 1976; Schubert et al. 1977; Nurse and O'Lague 1975), whereas non-motoneuron spinal cord cells or sensory neurons do not, even when their processes overtie the myotubes for hundreds of micrometers (Cohen and Weldon 1980; Kidokoro et al. 1980; Role et al. 1985). 7 1.2.2 Neul'otrophic Factors The possible significance of soluble neuTotrophic factors comes from carly observations that both the total number of AChR and number of AChR clusters increased on myotubcs close to spinal cord explants in vitro (Cohen and Fischbach 1977; Podleski ct al. 1978). Several factors have since been identified that differ in their lIlolccular wcighl, biochemistry and effects on the AChR number and c1ustenng (Chnstian et al. 1978; Podleski et al. 1978; Jessel et al. 1979; Salpeter et al. 1982b; Connolly ct al. 1982; Buc Caron et al. 1983; Neugebauer et al. 1985; Usdin and Fiscbach 1986). Many aspects of AChR regulation during synaptogenesis can be mimickcd by bralll derived factors. When these soluble brain extracts are applied to myotubes ;1/ vllm thcy lead (within a few hours of their addition) to the mobtlization of pre-exiliting AChR, the stimulation of AChR synthesis and the generation of AChR c1usters wlth ncar-junctional site densities (Connolly et al. 1982; Buc Caron el al. 1983; OIek et al. 1983; Salpctcr ct al. 1982a; Usdin and Fischbach 1986). As In the case of synaptogcncsls, factor-mduccd clusters form mini-aggregates that increase in size and coalescc to form a more compact structure (Olek et al. 1983). These clusters are in sorne cases associatcd with other synaptic specialization such as basal lamina antigens (Sanes et al., 1984). One of the factors purifie (acetylcholine receptor-inducing activity) which may regulate the accumulation of AChR at developing chick neuromuscular junctions (Usdin and Fischbach 1986; Falls et al. 8 1990). This protein selectively increases the level of rnRNA that encodes the AChR a subunit (Harris et al. 1988, 1989) and the € subunit (Martinou et al. 1991). A full-length cDNA clone was isolated using information from the available amino terminal amino acid acid sequence of thls clone was homologous to the mammalian PrP (a glycoprotein member of the family of prion-Iike proteins, found in normal brain but whose function is unknown) suggesting that the cD NA clone isolated may encode a chicken homolog of the mammalian PrP or a member of a fami]y of prion-like proteins. A modified form of the prion protein (PrPSC) is thought to be involved in the pathogenesis of scrapie in sheep and rodents and in severa] human degenerative diseases (Prusiner 1989). Thus, it has been speculated that an AChR-inducing activity may possibly play a crucial trophic role in the brain and that neuronal degeneration results from the accumulation of an inactive form of the protein (Falls et al. 1990). Agrin, a component of the synaptic lamina, is another factor thought to be responsible for the aggregation of preexisting AChRs beneath the nerve at newiy formed synapses (McMahan 1990). Agrin induces AChR clusters in cultured myotubes in the absence of protein synthesis (Godfrey et al. 1984); it also induces accumulations of acetylcholinesterase, laminin, and heparan sulphate proteoglycan in the basal lamina associated with these c1usters (Wallace 1986,1989). The proposed source of agrin at the neuromuscular junction is the motoneuron. Agrin is expressed in embryonic motoneurons (Magill-Solc and McMahan 1988; Rupp et al. 1991) and transported to the nerve terminaIs (Magj])-Solc and McMahan 1990), where it may be released into the synaptic basal lamina to organize the postsynaptic components of the developing neurornuscular 9 junction (McMahan 1990). Agrin gene expression has also been found in embryonic muscle and throughout the spinal cord and brain (Rupp et al. 1991). Cohen et al. (1992) recently demonstrated the externalization of agrin-like molccules by cmbryonic neurons in sufficient quantities to account for nerve-induced aggregation of AChR at the nervc muscle synapse. Agrin added to cultured myotubes induces tyrosine phosphorylation of the AChR. suggesting that agrin may act through a tyrosine kinase to clustcr AChRs (Wallace et al. 1991). The clustering of AChRs may also involve proteoglycans, as exogcnolls heparin and heparan sulphate inhibit agrin and nerve-induced receptor c1ustcnng (Hirano and Kidokoro 1989; Wallace 1990) and a muscle cel1line that IS defcctivc 111 proteoglycan synthesis fails to c1uster AChRs spontaneously (Gordon et al. 1992; Gordon ct al. unpublished data). RNA splicing of agnn transcripts has been shown to affect the aggregating activity of agrin; proteoglycans appear to be Involved in determining which forms of agrin generated by these transcripts is active (Fems et al. 1992). Another protein is the 43K protein, a peripheral membrane protein associated with the AChR clusters on the cytoplasmic si de of the cell membrane (Wallace 1989) which has been implicated in receptor aggregation (Froehner et al. 1990; Phillips ct al. 1991). Calcitonin gene-related peptide (New and Mudge 1986; Fontaine et al. 1987) and basic fibroblast growth factor (bFGF) (Peng et al. 1991) are factor~ that have also becn shown to affect the synthesis and localization of the AChR. JO 1.2.3 Submembrane Machinery for nAChR Clustering Several studies point to a role for the 43K protein in AChR clustering (reviewed by Froehner 1991). AChR and the 43K protein are present in equimolar concentrations and share a common distribution in the postsynaptic membrane and in AChR c1usters. Injection of 43K protein mRNA into Xenopus oocytes led to the formation of c1usters consisting of 43K protein. Coinjection of AChR subunit mRNA and 43K protein mRNA resulted in the expression of surface AChR in a clustered distribution; these c1usters also contain the 43K protein. AChRs are distributed uniformly when their subunits are expressed in oocytes in the absence of 43K protein expression. These results suggest a model in which the key event is the regulation of 43K protein clustering, and that the distribution of AChR is governed in large part by its interaction with the 43K protein (Froehner 1991). Several proteins are concentrated at the neuromuscular junction (NMJ) but are probably not directly involved in clustering. These include: vinculin, talin, paxillin, filamin, a actin, ankyrin and dystrophin. Desmin, laminin Band tubulin are concentrated in the general synaptic region but are not localized at the AChR sites. Anchoring proteins (other than the 43K protein) incJude: actin, ~-spectrin, 58K protein (found complexed with the torpedo dystrophin) and 87K protein (reviewed in Froehner 1991). Cytoskeletal clements probably play a role in maintaining and re-establishing AChR c1usters, but their function is less weil established (Schuetze and Role 1987). 11 1.2.4 Muscle Activity Muscle activity is known to play a role in regulating AChR number and distribution. although probably through very different mechanisms from those employcd by the neurotrophic factors. Early studies demonstrated that denervalion led 10 superscnsitivily. Agents that block action potentials (e.g. tetrodotoxin) or neuromuscular transmissIon (e.g. a-BGT) were found to increase the rate of AChR synthesis and the number of surface AChRs, suggesting that activity could be responsible for the dcvclopmcntal 1055 of extrajunctional AChRs that occurs after innervation (revicwed in Fambrough 1979). In fact, Burden (1977) showed that the loss of extraJunctional t\ChRs in dcvclopmg chick muscle was inhibited by trcalment with the nAChR antagonist curare. In contrast, direct electrical stimulation of the muscle prevents or reverses the increase in cxtrajunctional AChR in denervated muscle (Lomo and Westgaard 1975). Recent studies suggest that activlty regu/ates receptor synthesis via transcriptlOnal control. Activity-dependent regulation is reversibJe, since denervation of adult skeletaJ muscle leads to the reappearance of the AChRs throughout the muscle fiber's surface «(ioJdman et aJ.198S; Merlie et al. 1984; Shieh et al. 1987). Furthermore, tetrodotoxm (whlch blocks action potentials) increases message levels in chlck muscle (Klarsfeld and Changeux 1985). It was subsequently shown that the 1055 of rcceptor~ from the extrajunctional regions is largely due to nerve-induccd activity supprcssmg gene expression (Goldman et al. 1988; Merlie and Kornhauser 1989); howevcr, the molccular mechanism by which muscle electrical activity controls nAChR genc cxprc~si()n remains unclear. 12 1.2.4.1 The MyoD Family of Myogenic Regulatory Factors. The control of nAChR gene expression has been an active area of investigation. Studies on the 5' flan king regions of the a., 'Y and {j nAChR subunits led to the discovery of control eJements that conferred responsiveness to electrical activity as weIl as tissue specificity and/or developmentaJ control of the expression of these subunit genes (Klarsfeld et al. 1987; Gardner et a1. 1987; Wang et al. 1988; Piette et al. 1989; Wang et al. 1990; Chahine et al. 1992). At around the same time that the above studies were being conducted, the search was on for myogenic regulatory factors that could activate muscle-specific genes and hence, turn on a muscle-differentiation program. A family of genes coding for factors that commit mesodermal stem cells to the myogenic lineage, presumably by activating transcription of skeletal muscle-specifie genes, has been identified (reviewed by Weintraub et al.1991). Complementary DNAs (cDNA) coding for four of these factors have been described in higher vertebrates: MyoD, myogenin, Myf-5 and Mrf-4 (Davis et al. 1987; Wright et al. 1989; Braun et al. 1989; Rhodes and Kozieczny 1989). Pro teins of the "MyoD family" contain a consensus sequence predicted to conform to a helix-loop-helix structural domain necessary for dimerisation and a stretch of basic amino acids necessary for DNA binding (Murre et al. 1989). It has been shown that these nuclear proteins bind to and transactivate the promoter-enhancer regions of the muscle creatine kinase (Lassar et al. 1989), myosin light chain (Wentworth et al. 1991), troponin 1 (Lin et al. 1991) genes. 13 The discovery of these rnyogenic regulatory factors led to active investigation into thcir potential role in the control of muscle nAChR gene expression. Outhned bclow arc severa! pieces of evidence that point to such a role. Firstly. MyoD bindmg sites occur in the regulatory or control elements of the a, {j and 'Y subullIt gencs. Whcn MyoD hinds to these sites, it can transactivate the expression of a reporter enzyme gcnc whcn il IS placed under the control of these regulatory clements (Piette et al. 1990; Gilmour et al. 1991; Prodyand Merlie 1991; Prodyand Merlie 1992). Secondly, devclopmcnt.t1 studlcS show that MyoD mRNA appears shortly before nAChR (Y subunit mRNA, suggesting a role for MyoD in inducing nAChR gene expression durillg carly muscle dcvclopmcnl (Piette et al. 1992). Thirdly, reeent evidence sllggests that rcpression of MyoD expression (as weIl as that of other members of the MyoD family) may account for the down regulation in nAChR genes by innervation of the differentlated multlllucJeatcd myotubes (Buonanno et al. 1992). MyoD rnRNA levels were found to dectine during the period thal coincides with innervation; nAChR gene expression is also known to decreasc during this period (Merlie and Sanes 1985). When muscle was denervated. an accumulation of MyoD transcripts preceded the increase of the nAChR a subunit transcnpts. Furlhcrmorc. electrical stimulation of the denervated muscle prevented thesc increascs in MyoD and nAChR ex sllbunit transcripts (Buonanno et al. 1992). Finally. Asher el al. (1992) showcd that blockade of the nAChR with a-BGT in rat led to an IIlcrease ln the transcript level of several nAChR subunit genes as weil as that of myogcnin expression. This cVldcncc lends further support to the notion that the state of muscle actlvity, mcdiatcd through the nAChR can regulate the mRNA levels of myogenic regulatory factors which in turn control nAChR expression. Recent work by Chahine et al. (1992) indicates that cAMP 14 and calcium are capable of regulating AChR subunit mRNA levels as well as MyoD mRNA levcls. Acetylcholine has prevlOusly becn shown to induce a voltage-dependent increase of cytosollc calcium in mouse myotubes (Giovanelli et al. 1991). Thus, cAMP and calc\l1m are potential second messengers that may be an important Iink in the putative chain of events described above. J.2.4.2 The Effects of Agonists and Antagonists on Receptors According to c1assical receptor theory, chronic exposure to agonist (or conditions that incrcase the synaptic concentration of the natural transmitter, such as blockade of inactivation mechanisms) results in a downregulation of the target receptor. Conversely, chronic exposure to an antagonist (or conditions that decrease the synaptIc concentration of transmitter, including denervation) produces an upregulation of receptors. Studies demonstrating receptor changes in response to drug treatments (notably catecholamine receptor changes following chronic treatment with antidepressants and neuroleptics have sllpported this wldely held dogma (Overstrect and Yamamura 1979; Creese and Sibley 1981; Wonnacott 1990). Self-regulation of membrane receptors by the endogenolls ligand constitutes a homeostatic regulatory mechanism (reviewed in Wonnacott 1990). Exogenolls ligands can also modulate receptor nllmbers. thereby contribllting significantly 10 the development of drug tolerance as weIJ as to the withdrawal syndrome that frequently accompanies the end of drug usage. However, there are limitations to this theory as demonstrated by the paradox of nAChR 15 upregulation by nicotine (Wonnacott 1990). As reviewed by Wonnacott there IS a growing body of evidence that chronic admimstration of nicotinic agonists upregulatc IlIcotinl\: receptors in the central nervous system (although irrcversib\c anticholtnt~~terascs have Icd to nAChR downregulation (Schwartz and Kellar 1985; Costa and Murphy [48J)). Although agonist-induced desensitizatlOl1 may play a fOie III tllIs phenol11cnon, the mechanism remains unc\ear; a rccent study by Marks et al. [l)l)2 round that RNA Icvcls encoding nicotine receptor subunits are not affected by chrol1lc nicotine lrcalment. In contrast to the central effects of nicotine, carbachol and IllcotlnC downregulatc nicotine receptors on muscle cells (reviewed in Wonnacott 1990), sympathctlc ncurons (PC 12 cells) (Robinson and McGee 1985) and autonomie gangha (Berg ct al. 1989) ln the (;8 mouse muscle cell line, a 30-50% decrease ln wl-a-BGT bllldll1g and a 75 % (\ecrease in functional activity occurs after chronic (24-48 hours) carhachol cxposlIfe (Nohic ct al. 1978). A reeent study by Lukas (1991) showed that chronic (3-72 hour,» cxposurc of the TE671 human clonaI cell line (which expresses a muscle-type nAChR) to nlcotme or carbachol led to complete loss of functional nAChR respOI1SCS, referrcd 10 a,> 11 functlOnal inactivation". Recovery of function occurred with half tunes of 1-) days (a timc cOIlf~e which is much slower th an the recovcry From nAChR descl1sltlzatlOn whlch IS a rcvcrslble process that occurs 011 shorter term (0-5 mmutes) exposurc of cells. Jntcrc,>tingly, "functional inactivation" was prevented by treatment wlth the nicotinlc antagonisl.'t pancuronium and alcuronium (these antagonists alone had no effcct on the Icvch 01 expression of functional receptors). 16 Recent evidence for antagonist-induced up-regulation by Hogue et al. (1992) showed that chronic infu~ion of d-tubocurarine into rats leads to the tolerance and upregulation of nAChRs. Doses were chosen such that immobilization or paralysis did not occur since immobilization itself can lead to increases in extrajunctional nAChRs and resistance to competItIve antagonists. An endogenously occurring polypeptide, thymopoietin, has reccntly been shown to antagonize the muscle nAChR. To date, the effects of this endogcnous antagonist on upreguJation have not been examined. 1.2.4.3 Thymopoietin, a Thymie Polypeptide that Potently Interacts at the Muscle Nicotinic AChR. Thymopoietin (TPO), a thymie polypeptide, isolated from the thymus gland, is involved in immune system functions. It enhances the differentiation of prothymocytes to thymocytes, through a cAMP-mediated mechanism (Basch and Goldstein 1974; Basch and Goldstein 1975) and also regulates mature T cell function via aIterations in cGMP (Sunshinc et al. 1978). TPO has been shown to be present in the circulation (Twomey et al. 1977); TPO-like immunoreactivity has been detected In brain (Brown et al 1986; Quik ct al. 199Ia). Recently, TPO mRNA expression has been detected in several tissues including striated muscle, heart, small intestine, Jung and cerebellum (Zevin-Sonkin et al. 1992). TPO was initially identified as a result of investigations on the association of thymic abnormalities to myasthenia gravis (Goldstein 1966). Since myasthenia gravis was 17 characterized by muscle weakness and/or paralysis, the possiblltly that a Ihymil: blod.lI1g agent could affect neuromusclIlar transmission cxisted. Admlll1slration of TPO 10 1ll1ù' led to a small decrease In nellromuscular Iransmisslon which occurnxl wlth a delay~d onset (24 hr) (Goldstein 1974; Goldslem and Schlesinger 1975) TPCr was subscqllcnlly found to interact at the a-BGT site. Receptor binding sludics show cd (hal TPO plllcntly (nM) inhibited l2'I-a-BGT binding in electroplax (VcnkataslIhramalllan et al Il)Hb), human muscle (Morel et al. 1987,1988), rat muscle (Quik ct ai. 1990) and muscle ccii lines (Lukas et al. 1990; Quik et al. 1991). Furthermorc, TPO has bccn shown III hlod neuromuscular function in isolated rat phrenic nerve hemidiaphragm preparatwns (Qulk et al. 1990) as weil as blocking nicotlJ11c rcceptor-me(hatcd ion flux 111 nconalal 11lu~cJe cell lines (Lukas et al. 1990; Quik et al. 1991b). Rcvah et al. (1987) "howcd thal TPO, wh en applied (for only a few seconds) to C2 muscle cclls in culture sl\1lllltancollsly wlth ACh. resulted in the appearance of long channel clo~cd tlll1e~ scparatlllg grollp~ 01 channel openings, suggesting that TPO is Involved ln nicotinic reccptor dc-,emltl/ation. Nicotinic receptor activation has been shown to result ln damage to the ~ndplale rcgloll of skeletal muscle. Initial evidence for thlS came from studics WhlCh 'ihowt.:d thal acetylcholinesterase inhlbitors produced a progressive myopathy of van()u~ 1Il1l~clc groups. The severity of the myopathy wa~; decrca~cd hy pnor nervc ~cctlOlI, hemicholinium which depletes acetylcholine wlthm the nervc tcrnl/nal, and d- • As ~tat~d in th~ addtmdum to thl~ the""" r~t:nt work oy QUlk cl al J 993a ha ...... hown thal the preparatIOn prt!~umed 10 ht! fPO, contmnt!d a-çohratoxm; the dtC'-t.., oh~ervcd JO Ihe prc"cnl the.." .. mu..,t theœfore now be attnhuted to the pn::<,enLe ot o-whratoxtn wntallunanl Il now appear .. Ihat ail .. tUUII·'" quot~ JO Ihl~ the~l'" conCernmg TPO puhh~heu trolll 1986 and onwan.l., JlIay have necn (,.ontaJlunalcù w'lh a-cobraloxJO. 18 tubocurarine, a nicotinic receptor blocker (Ariens et al. 1969; Fenichel et al. 1972; Hudson et al. 1978). Further studies showed that nicotinic receptor blockade with a-BGT or d-tubocurarine prevented muscle damage caused by the application of carbachol (Leonard and Sai peter 1979, 1980, 1982). Recently, the action of TPO at the nicotinic AChR was examined as an antagonist to determine whether it might exert a trophic role on muscle cells possibly with an action opposing that of acetylcholine. TPO was shown to moduJate nicotinic agonist-induced degeneration in neonatal muscle cells in culture (Quik et al. 1992). These results suggested that TPO, an endogenously occurring polypeptide, has the potential to modulate muscle cell morphology through an interaction at the nicotinic receptor. 19 STATEMENT OF THE PROBLEM As detailed in the introduction, the muscle nAChR is one of the most extensively studied receptors. The objective of the present study was to determine whelher the nAChR function and expression is regulated by a) thymopoietin, a ni~otinic alllagonist and b) MyoD, a myogenic transcription factor. TPO was studied 10 delefl1une whcthcr the interaction of this thymie polypeptide at the nAChR on a long-tenu basis cOllld affect the nAChR or morphology of rat muscle cells in culture. MyoD was investigated to determine wh ether the expression of this myogenic transcription factor COli Id result illl the appearance of a functional muscle-type nAChR at the ccII surface. Short-term (60 minute) exposure of rat neonatai muscle cells to thymopoietin has previously been shown to potently inhibit nicotinic receptor binding and fllnction al the muscle nAChR. The objective of the tirst study was to dctcrmine whcthcr lhere was any difference between the effects of long-term (days) and short-term (minutcs) exposlIrc of neonatal muscle cells to TPO, with respect to nAChR binding and fllnctional rcsponse. The effect of TPO on morphology was also examined. PreVlOlIS studies have shown that TPO could prevent nicotinic agonist-induced reduction in myotllbe branching and MZC. However, TPO alone did not have an effect on morphology. The objective of the morphology experiments was to determine whether TPO alone could have atrophie cffccl under the appropriate conditions. In the present experiments, the neonatal muscle œlls were exposed to TPO at a much earher tlme point (from the tllne of platmg, as oppo:)cd to 5 days after plating) and for a longer time period (4 to 7 days, as opposcd to 3 days 20 as previously used). The muscle nAChR was also studied with respect to MyoD, a myogenic transcription factor. Previous studies have shown that MyoD binding sites occur in the regulatory regions of the various subunits of the nAChR and that MyoD transactivates thes~ regions in the case of the Ci, {3 and 'Y subunits of the nAChR. The objective of this study was to determine whether the transfection of a non-muscle cell line wjth MyoD cD NA would lead to the expression of a functional muscle-type nAChR at the cell surface. This was approached using 125I-a-BOT binding assays to determine whether a population of nAChRs was present. Carbachol-stimulated 22Na uptake studies were used to assess whether a nicotinic receptor-mediated response had developed. The effects of various nicotinic agonists and antagonists were determined in both the binding and functional assays to determine whether the receptor had the pharmacological profile of a muscle-type nicotinic receptor. Tlme course studies to assess wh ether nicotinic receptor binding and functional responses developed in parallel were done in order to provide correlative evidence that the observed flux occurs as a result of activation of the nAChR. Thus, the main objective of my work was to investigate the effects of the factors, TPO and MyoD, to gain a better understanding of how the nicotinic acetyJcholine receptor is regulated. 21 2.0 MATERIALS AND METHODS 22 2.1 Materials Thymopoietin was isolated and purified from bovine thymus (Audhya et al. 1987) and was provided to us by the Immunobiology Research Institute, Annandale, New Jersey·. Ot-BGT was purified as previously described (Quik and Lamarca 1982) from Bun~arus multicinctus venom, obtained from Miami Serpentarium Lab. (Salt Lake City, Utah). 125I- Ot-BGT (10-20 p.Ci/llg) and 22Na (900-1000 Ci/g) were purchased from New England Nuclear (Boston, MA). d-Tubocurarine, carbachol and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO). Media, Geneticin-418 and reagents for cell culture were obtained from Gibco-BRL. Ail other chemicals were purchased from standard commercial sources. 2.2 Methods 2.2.1 Transfection of C3U IOTI/2 ceUs with MyoD cDNA C3H IOTl/2 cells (1 x 1 • As stated 10 the addendum to thls thesis, recent work by Quik et al. 1993a has shown that the preparatIon presumed to he TPO, contamoo o-cobratoxm; the effects observed m the present thesis must therdore now he altnhutoo 10 thl! prl!sl!nce of o-cobratoxm contammant. 23 adding 0.2 mg/ml Geneticin-418 to the cells. Geneticin-418 resistant ceUs were cloned in selective medium. 2.2.2 Subcloning Cells that had been stably transfected with the MyoD cDNA were plated al a density of 10 cellsllOO mm dish in growth medium (DMEM supplcmented wilh \0% FCS, 50 units/ml penicillin and 50 Itg/ml streptomycin) containing Geneticin-418. Aner 2 wceks colonies representing the original cells were individually blotted with a small square of tilter paper soaked in 0.02% trypsin and 0.15 mM ethylcnediamine tctraacetic aCld (trypsin.EDTA). The tilter paper was then removed and placed in a culture weIl containing 500 Itl of growth medium that lacked Geneticin-418. When the ce1\s reachcd confluency, the medium and tilter paper were removed and 100 Itl of trypsin.EDTA added to the culture wells (3 min). The trypsin was inactivated by the addition of 900 Itl of growth medium; a 750 Itl aliquot of the resulting 1000 Itl ccII suspension was platcd in a new weil for a 125I-a-BGT binding assay while the remaining 250 Id werc platcd III a 35 mm dish for the purpose of propagating that particular ccII clone for future use Clones, to which 125I-a-BGT binding was observed, were further subcloned with the aim of obtaining clones with the greatest number of nAChR. Thrcc rounds of subcloning wcrc done. The final round of subcloning yielded 48 clones, of which J was selected for further studies, 23-8-8 (clone 8). 24 2.2.3 Cell Cultures C3H IOTI/2 cells. For the morphological studies, 125I-a-BGT binding experiments and 22Na uptake assays, 18,000 celIs/well were plated in a 24 multiwell dish under a humidified 95 % oxygen/5 % carbon dioxide atmosphere at 37°C and grown to contluency in growth medium containing Geneticin 418. To promote differentiation, the growth medium was subsequently replaced by fusion medium (same as growth medium but containing 2% instead of 10% FCS) and Geneticin-418. Rat neonatal muscle cell cultures. Rat myotube cultures were prepared as previously described with sorne modification (Nelson et al. 1976; Schaffner and Daniels 1982; Braun et al. 1989). Minced muscle from the pectoralis and hind limb of 1 to 2 day old Sprague Dawley rats was washed with phosphate buffered saline containing 0.5 mM Mg++. The muscle tissue was dissociated for 2 hr at room temperature in Hank's balanced salt solution (calcium and magnesium free) containing 0.25 % trypsin, with gentle mixing on a magnetic stirrer. After addition of an equal volume of culture medium, the cell suspension was centrifuged for 5 min at 750 x g at room temperature; this step was repeated once. The cell pellet was then suspended in culture medium, which consisted of25% Medium 199,65% minimum essentiaJ l,ediurn, 10% horse serum, penicillin (50 units/ml) and streptomycin (50 ug/mI). For the 22Na uptake studies, competition binding studies and morphological studies, cells were plated onto collagen-coated 24 multiwell dishes at a density of 0.25 to 0.30 x 1(16 cells per weil. Sister cultures were used in order to compare the results frorn the above 3 types of studies. Cultures were incubated in a 25 humidified atmosphere with 5 % carbon dioxide and 95 % oxygen. CeUs were no\ maintained for more than 10 to 12 days after plating because, with incrcasing myotubc development, enhanced contraction caused the ceUs to lift from the culture plates. ln the long-term pretreatment studies, TPO was added al the time of plating of the raI neonatal muscle cells. Four days later (day 4), one set of eclls was assaycd. The othel set received a change of medium including a fresh aliquot of TPO and was subsequently assayed on day 7. Thus, these experiments, a penod of 3-4 days had elapsed from the time of addition of TPO to the time of the assay. Sixt Y minutes prior to the assay, the cells received a change of medium. Thus, TPO was absent l'rom the medium dunng this 60 min period as weil as during the assay for long-tcrm prctrcatcd eclls. The assays used are described in sections 2.2.4 and 2.2.5. For the short-term studies, slster cultures of rat neonatal muscle cells received changes of medium and were assayed at the saille tillle points as the long-term pretreated cultures. However, the cultures only recclved 'l'PO pretreatment during the 60 min preincllbation period immediately prior to the assay. 'l'PO was also present during the assay. The assays llsed are described in sections 2.2.4 and 2.2.5. 2.2.4 Measurement of 12sl_a_BGT binding to cells in culture Before the assay, the cells in culture were washed twice wlth 1 ml DMEM containing 4.4 mM NaHCOJ , 2 mM HEPES (DMEM buffer) and 0.1 % bovine serum albumin. Ccll~ in culture were preincubated for 60 min at 37°C in the absence or presence of the 26 indicated agents, followed by incubation in the presence of 125I-a-BGT (1.5 nM for C3H IOTl/2, 0.8 nM for rat neonatal muscle cell, unless otherwise indicated) for 90 min at 37°C (Quik et al. 1991b). Binding was terminated by removal of the medium followed by three 1 ml washes with DMEM buffer. The cells were resuspended in 500 #LI of 1.0 N NaOH, with shaking, and the radioaetivity was counted using a 'Y counter. Non specifie binding was defined as binding in the presence of 300 #LM d-tubocurarine. Non specifie binding represented approximately 10 to 15% of total binding for clone 8 and approximately 5 % in the case of rat neonatal muscle cells. 27 2.2.5 Measurement of llNa influx 22Na uptake studies were donc as described previously (Catterall 1975. Stallcup and Cohen 1976). Immediately before assay. cells were washed twice with 1 ml of DMEM buffer (containing 156 mM sodium). In sorne of the experiments. the eclls in culture wcre preincubated with the indicated drugs for 60 min at 37°C. 22Na intlux mcasuremcnts (done over a 2 min incubation period) were initiated by replacement of the butTer with buffer containing 1 l'Ci/200 1'1 22Na, in the absence or in the presence of carbachol (100 l'M) and the other drugs as indicated. Uptake was ten1l1nated by aSpIratIOn of the medium, followed by 4 quick washes with DMEM buffer contatnl11g 100 p.M d tubocurarine. Cells in each well were removed with 500 J'lof 1.0 N NaOH, with shaking. The radioactivity was determined using a "Y counter. Non-speclfic uptake was defined as uptake in the absence of carbachol stimulation. 2.2.6 Assessment of myotube length and branch formation in rat myotube cultures Numerical analysis of myotube branching and length was done at various times after plating using phase contrast microscopy. To evaluate the number of myotubc branch points as weil as the number of myotubes 0.5mm or greater (as assesscd aftcr magnification), a diametric strip (approximately JO mm long) was countcd at 320X magnification for each culture weil. Each culture condition was tested ln triplicate or quadruplicate. 28 2.2.7 Statistics Statistical comparisons were done using one-way analysis of variance (ANOVA). The IC50s represent the concentration of drug required to inhibit 12.5I-a-BGT binding or carbachol stimulated 22Na uptake by 50%. 29 3.0 RESULTS 30 3.1 LONG-TERM THYMOPOIETIN TREATMENT OF RAT NEONATAL MUSCLE CELLS IN CULTURE LEADS TO ENHANCEMENT OF NICOTINIC RECEPTOR FUNCTIONAL RESPONSE AND MORPHOLOGY As detailed ln the introduction, prevlOus studies have shown that TPO, a thymie polypeptide involved in immune system functions, can bind to the muscle nAChR at the QI-BGT binding site and inhibit nAChR functional response. It was recently demonstrated that short-term (60 minute) exposure of rat neonatal muscle cells to TPO potently inhibited nicotinic receptor binding and function at the muscle nAChR (Quik et al. 1992). The question arose whether there was any difference between the effects of long-term (days) and short-term (minutes) exposure of neonatal muscle cells to TPO, with respect to nAChR binding and functional response. To assess this, long-term and short-term TPO pretreatment studies were undertaken. Nicotinic receptor activation has been shown to have an adverse effect on muscle morphology. As mentioned in the introduction, blockade of the nAChR with the antagonists QI-BGT and d-tubocurarine prevented muscle damage. Recent studies have shown that TPO could prevent nicotinic agonist-induced reduction in myotube branching and size (Quik et al. 1992). However, TPO alone did not have an effect on morphology. The question arose whether TPO alone could have atrophie effect under the appropriate conditions. To address this, the neonatal muscle cells were exposed to TPO' at a much • As l>latoo 10 the! adde!ndum 10 th!!. the!sls, roce!nt work by Quik e!t al. 1993a has shown that the! prl:!parallon pœsumoo to he TPO, contamed a-cobratoxin; thl:! I:!ffoct!> obst:rvoo ln the prt!st:nt tht!sis mu!>t thl:!retoœ now hl:! attnhutoo 10 the! pre!senœ of a-cohratoxin contammant. 31 ------. earlier time point and for a longer time period than was prcvlOusly uscd. 3.1.1 Characterization of nAChR binding and function in nit neonntnl muscle cl'lls in culture Experiments were initially done to follow the normal developmcnt of nAChR binding and function in rat neonatal muscle cells m culture. The tune coursc of the devclopmellt of the nAChR binding is shown in Fig. 1A which depicts the B",.x valuco; denved l'rom saturation curves determmed for cells cultured for 2, 4. 6. 9 and 12 days. These Bill" values of 12sI-a-BGT bmding reached a plateau at around 8 days Hl culture wlth half maximallevels being attained after 5 days. Subsequent expeflments were done 011 day 4 and 7 (unless otherwise indicated) since nicotinic receptor bll1dll1g wao; lound to he do"," to half-maximal and maximal, respectively, on these days. Fig. lB show ... the tllne cour'le of development of nicotinic response stimulated by 100 I-'M carhachol at the corresponding time points. Uptake levels appear to plateau after 6 dayo; III culture with half-maximal uptake occurring at around day 3. The saturation curves of 125I-a-BGT bmding for day 4 and l) arc shawn 111 Fig. 2A. Day 4 represents a time point when binding was approximately half-maxllnal; day 9, Itke day 7, represents a time point at the plateau phase of binding devclopmcnt (see Fig. 1A). The Bmax on day 4 is 24 fmol/culture weil compared to 76 fmol/culture weil on day 9. The dissociation constant, KI), is defined by formulas that assume a ligand bmd~ to a reccplor reversibly leading to equilibrium; however, a-BGT bind~ essenllally tn an Irrcvero;lblc 32 manner, hence the term "apparent Ku" is used. The apparent KD faIIs in the range of 0.7 nM to 1.2 nM for ail days tested and based on these observed values, subsequent binding assays were done using 0.8 nM 1251-a-BGT. Dose-response curves of carbachol-stimulated 22Na uptake were a1so determined in sister cultures al the above time points in order to monitor the time course of the maximal functional response (Fig. 1B). The dose-response curves determined after 4 and 9 days in culture are depicted in Fig. 2B. Carbachol appears to act more potently on cells that have been in culture for 9 days (ECso = 5 J.tM) compared to those that have a culture age of 4 days. However, it is difficult to draw any conclusions about the efficacy of carbachol on day 4 since plateau levels were not attained. Previous studies by Quik et al. (1992) had shown that nicotinic response plateaus at 100 J.tM carbachol in rat neonatal cells in culture, suggesting that the nicotmic response found at this concentration on day 4 is Iikely to be very close to a plateau level. Thus, the ECsu value could be approximated to be 24 J.tM on day 4. In subsequent experiments, a concentration of 100 J.tM carbachol was used since this led to uptake levels that approached plateau le,,'els. A lower concentration of 3 J.tM carbachol was tried but uptake levels were too low and showed a high degree of variability suggesting the Iimit of the assay's sensitivity was approached at such a concentration of carbachol. 3.1.2 Effect of thymopoietin on I%SI-a-BGT binding and carbachol-stimulated uNa uptake 33 Previous studies (Quik et al. 1991b; Quik et al. 1992) have shown that TPO ean potently inhibit 125I-a-BGT binding and carbachol-stimlilated nNa uptake al nM concentrations following a 60 min preincubation. In the present stlldy, the effeet of long-tcrm TPO exposure on nAChR binding and function was assessed to detcrminc whcthcr this would resuIt in any differences in the levels of 125I-a-BGT binding and carbachol-slimulalcd }lNa uptake that are attained following short-term pretreatment only. Long-tcrm prctrc,ltl11lCllt involved the exposure of neonatal muscle cells in culture to TPO from the lime of plating for a period of 4 or 7 days. TPO was absent from the medlllm only dllring the 60 min period immediately prior to the assay as well as during the assay; howcvcr the ahs,!ncc of TPO for this short period is unlikely to be significant. QlIik ct al. (1992) found that one hr of exposure of rat neonatal muscle cells in culture, followed by a 4 hr wash, resulted in an effect on 125I~(X-BGT binding essentially similar to that found when TPO was present in the assay. The results of these experiments wcre comparcd to the d'fecls of short-term pretreatment with TPO on cel1s of simllar culture age; thcsc cells wcrc exposed to TPO during a 60 min preincubation period immediately pnor to thc assay as well as dunng the assay itself (90 minutes). Control cultures dld not rcccive 'l'PO treatment at any time. The results depicted in Fig. 3A were taken from cells culturcd for 4 days. Thf.!sc show that there was no significant differenee betwccn the inhibition eurvc of IH'-a-BGT binding after a 4 day exposure to TPO and that found after a 60 min prcincubation with TPO a]so on day 4; both conditions resulted in an IC50 value of approximately 4 nM. Similarly, both a 7 day exposure to TPO and a 60 minute preincubalion with l'PO on day 34 7 led to ICso values of about 6 nM (Fig. 38). These results suggest that TPO inhibited 1251-a-BGT binding as potently after long-term exposure of the nAChR as it did after only a 60 min exposure. In contrast, long-term or chronic TPO treatment was found to have an effect on carbachol-stimulated 22Na uptake that was clearly distinct from that after a 60 min preincubation (Fig. 4A, 48). A 60 min preincubation with TPO prior to assay on day 4 and day 7 resulted in the inhibition of uptake with ICso values of 13 nM and 23 nM, respectively. These results were in line with previous findings by Quik et al. (1992) where a 60 min preincubation of 6-10 day cultured rat neonatal muscle cells with TPO yielded an IC~o value of 26 nM. However, when the cells in the present study were exposed to TPO for a full 4 days, the above inhibition of carbachol-stimulated 22Na uptake was not observed (ANOVA: main effect ofTIME: F=7.65, df 1,24, P In fact, exposure to TPO at varying concentrations for 4 days led to nicotinic responses that were close to those found in control ceUs that had not been exposed to TPO at ail (Fig. 4A); the uptake values ranged from 87 to 115% of control values. Interestingly, a 7 day exposure to TPO (Fig. 4B) actually led to a small increase in functional response compared to control with values ranging from 107 to 129% control). Again, the results obtained from a 60 min TPO preincubation on day 7 were significantly different from those found after a 7 day TPO exposure (ANOVA: main effect of TIME: F=34.5, df 1,24, p 35 dose-dependent inhibition of binding. These results indicate that there are diffcrenccs between the effects of long- and short-term TPO pretreatment on nicotmic response in neonatal muscle cells in culture. As was mentioned earlter. such a di ffercnce was not observed in the case ofbinding where TPO was able to potcntly inhibit binding both after short- and long-term treatment. 3.1.3 Effeet of thymopoietin on the morphological development of rat IIconalal muscle ceUs in culture Previous studies in our laboratory had shown that TPO could prevent agonist-it1duccd degeneration in neonatal muscle cells in culture (Quik et al. 1992). In those studics, l'PO alone did not appear to have an effect; however, it is important to note that the nconatal muscle cells had becn in culture for 5 days prior to a 3 day exposure to 'r;'o. Thus, the question arose whether the presence of TPO in the medium from the time of plating, as weil as for a longer duration (up to 7 days), could lead to atrophie effcct on morphologieal development. The results of this studyare depicted in Fig. 5 and 6. A significant dose-dependent increase (50-100% compared to control) in the number of myotubes of a specified length was observed after a 4 day exposure to 'l'PO (Fig. 5). The number of myotubes of a specified length represented a suitable parameter for asscssrnent at this time point sinee the myoblasts had begun to fuse into myotubes whose length could be easily measured. By day 7, it was no longer possible to count the number of myotubes using this method since the myotubes had developed into very long structures with a complex branching pattern. Therefore, the number of branehpoints was used as an 36 alternative parameter on day 7 (Fig. 6). Although somewhat qualitative, the quantitation of branchpoints serves as a useful method of comparing control cultures with respect to morphological development. Again, a signi ficant dose-dependent increase (95-160 %) was observed in the number of branchpoints after a 7 day exposure to TPO. These results indicate that TPO may exert a trophic mie on rat neonatal muscle cells that have been exposed to the polypeptide for several days from the time of plating. Another possibility is that TPO may exert a protective effect, for example, by preventing spontaneous necrosis in culture. 37 Fig. 1: (A) Time course of development of specific mI-a-BGT binding in rat neomltal muscle cells in culture. These values represent BRIA' values derived l'rom saturation binding isotherms determined for rat neonatal ceUs that had been in culture for 2, 4, 6, 9 and 12 days. Each value represents the rnean ± S.E.M. of Bmax values dcnvcd from 3 separate experiments done in quadruplicate (the value for day 2 represents the mean of 2 separate experiments done in quadruplicate). (8) Tune course of development of nicotinic response. Levels of22Na uptake stimulated by 100 ItM carbachol wcrc mcasured in rat neonatal muscle ceUs in culture. Each value represcnts the mean ± S.E.M. of 3-5 experiments done in quadruplicate. Sister cultures were lIsed for the binding and the uptake experiments. Where the standard error was not deplcted, it feH within the symbol. 38 ~ 75 A -Z ~~~- f-4 • 50 ~ Q) ~ I.,J8 ~~ .,~u ... 25 u -0 é='t-4- 8 u- ~ ~ CIl 0 0 2 4 6 6 10 12 DAYS IN CULTURE ~ ê; 0.4 B «1 Z T T BI_ 0.3 • • ~- ~~- • 0.2 ~iu CIl r~ ~u 0.1 :::z=-o ~ u 0.0 ~ 0 2 4 6 6 10 12 DAYS IN CULTURE Fig. 2: (A) Saturation curves of specific m(-a-BGT binding to rat neonatal muscle cells after 4 and 9 days in culture. Binding of '25I-a-BGT to the culturcs was done as described in the presence of varying concentrations of radiolabelled a-BOT. ~\ch valuc represents the mean + S.E.M. of 5 culture wells; the two sets of data points dcriv& .. d from 2 separate experiments are depicted in this graph. (B) Dose-rcsponsc curves of carbachol-stimulated 22Na uptake in sister rat neonatall1l11Sclc cells atkr 4 and 9 days in culture. Bach value represents the mean ± S.E.M. of 3-4 expcrimcnts. Whcre thl~ standard error was not depicted, it fell within the symbol. 39 o DAY 4 • DAY 9 A CJ BO TT ~ Q .. • 25- ~== • ~~ 60 CJQ) ~~ f~ 40 8,""'0 .. 1""'4 UO ~ 20 S~u- ~ ~ CIl 0 1 1 1 1 0 2 4 6 B 10 12 [t2llI-a-BGT] nM B 0.3 0.2 0.1 O.O~++------~------~------'------6 -5 -4 LOG [CARBACHOL] M Fig. 3: Inhibition curves of specifie mI-a-BGT binding to rat neonataI muscle cells after exposure to TPO for either 60 minutes (immediately prior to assay) or ovcr a pcriod of 4 or 7 days (as described in the Methods. section 2.2.3). The bindmg assays wcrc donc 4 days (A) and 7 days (B) after plating. Control specifie binding on day 4 \Vas 6.9 ± 0.7 fmol/culture weIl (n=36 determinations). Control specitic bindmg on day 7 was IH.5 + 1.4 fmol/culture weil (n=36determinations). Each value represcnts the Illcan + S.E.M. of 3 separate experiments done in quadruplicate. Where the standard crror was Ilot depicted, it fell within the symbol. There was no statistically significant diffcrenccs in binding between the control and TPO-treated cultures. 40 ~ 100 ~ ~ III 75 t; -0 -J.4 III ~ 1 tS 0 1 =CJ 1-4 50 Il.. ~ u - (A) 25 S • 4 DAY EXPOSURE U r.zl ll4 o 80 MIN PREINCUBATION AT DAY 4- CIl 0 -9.0 -8.5 -B.O -7.5 LOG [TPO] M ~ 100 T ~ Q ~ III E-4 .-4 75 t,:, -0 J.4 III ~ 1 tS =0 1 0 50 -..lQ ~ u - (8) é= 25 - • 7 DAY EXPOSURE u r.zl ll4 o 60 MIN PRE INCUBATION AT DAY 7 CIl 0 -9.0 -B.5 -B.O -7.5 LOG [TPO] M Fig. 4: Inhibition curves of specifie carbachol-stimulated 22Na uptakc in rat neonatal muscle ceHs after exposure to TPO for either 60 minutes i mmcdiately prior to thc 90 min assay period (when exposure to TPO continued) or over a penod of 4 or 7 days. As described in the Methods, section 2.2.3, in the case of long-tcrm prctrcatmcnt, l'PO was absent from the medium only during the 60 min period 1I11l11cdlatcly pnor (0 Ihe assay as weIl as during the 90 minute assay period. The uptake ass'lys werc pcrformcd on cells that had been in culture f(l~ 4 days (A) and 7 days (B) aftcr plating. Control spcctlil: uptake on day 4 was 0.15 ± 0.02 nei (n=36 determinatlons). Control SpCCltiC uptakc on day 7 was 0.16 + 0.01 nCi (n =36 determinations). Each value represcnts the Illcan ± S.E.M. of 4 separate experiments done in quadruplicate. Where the standard crror was not depicted, it feH within the symbol. 41 ~ 150 ~ 0 «S Z 125 T a l Cl-. 100 PIljbô ::> J:: 75 è! g T E-4~ fil -- 50 1 (A) ~ 0 • "DAY EXPOSURE ~ 25 ~ o 80 llIN PRE INCUBATION AT DAY" ~ ~ 0 () -9.0 -8.5 -8.0 -7.5 LOG [TPO] M ~ ~ 150 ~ 0 «S Z 125 • a T Cl_ 100 ~-0 ~b o § 75 ~~ fil - 50 1 (B) ....:l 0 • 7 DAY EXPOSURE :z:u 25 < o 60 WN PREINCUBATION AT DAY 7 0 ~u -9.0 -8.5 -8.0 -7.5 LOG [TPO] M Fig. 5: Effect of TPO (4 day exposure) on the number of myotubes 0.5 mm or longer at 320X magnification. The size range of Oj mm or longer representcd the most weil developed myotubes at this time point. Qllantitication of the Illllnber of myotllbcs 1J\ thi'i size range was a convenient way of assessing the effect of TPO on the morphologlcal development of the myotubes after 4 days in culture. The counttng of tllIS parameler was done using a diametric strip across the culture plate. The conlrol value for the numher of myotubes 0.5mm or longer was 28.0 + 2.4 (n= 13 dcterminations) per culture wdl. Each value represents the mean ± S.E.M. of 3 separale expenmcnt"i donc 111 quadruplicate. 42 100 T ri) rzl ~Qj' 75 o fil ~ f T ~ u o .S 50 ~~ rzl_ ~ :s 25 ::>z a -9.0 -8.5 -8.0 -7.5 LOG [TPO] M Fig. 6: Effect of TPO (7 day exposure) on number of branchpoints in rat nconat,,} myotubes. By 7 days in culture, the rat myotubes had developed extensive branching. The number of branchpoints at 320X magnification was therefore a convcnicnt paramcter in the assessment of the effect of TPO on morphological development al thts stage. The counting of this parameter was donc using a diametnc stnp across the culture plaIe. The control values for the number of branchpoints was 25.1 ± 0.2 per culture weil (11= 15 determinations). Each value represents the mean ± S.E.M. of 3 separatc expenmcnll. done in quadruplicate. 43 rn f-4 200 Z l 0 -~ ::t=- 150 U Q) ~ i al t; 100 ~ .8 ~ o~ ~-r:r:I al 50 ~ Z 0 -9.0 -8.5 -8.0 -7.5 LOG [TPO] M 3.2 FUNCTIONAL NICOTINIC RECEPTOR EXPRESSION IN MESODERMAL STEM CELLS TRANSFECTED WITH MyoD cDNA Previous studies have shown that the MyoD gene is able to eonvert non-muscle eclls from the C3H lOTI/2 mouse mesodermal stem cellline into cclls that posses~ a skcletal muscle phenotype (Davis et al. 1987). It has also been shown that the genc prodllct MyoD 15 ahlc to activate myogenesis in ccII lines derived from mesodermal, cctodcflmll and endodermal tissues (Weintraub et al. 1991). MyoD bmding sites occur \Il the regulatory clements of the a-, (3-, 'Y-, €- and ô- subunits of the nAChR. In fact, MyoD has bccn shown ln transactivate the 5' flanking reglons of the (X-, (3- and -y- subull1ts (plette et al. IlllJO; Gilmour et al. 1991; Prody and Merlie 1992). The question arose whether MyoD could (1) result in the expression of a cell surface muscle IllCotllllC acetylchol i ne rcccplor, as assessed by membrane binding assays and (2) whether slich a reccptor WOlS fllJ1ctional. To address this, C3H IOTI/2 cells were stably transfectcd with a plasmid conlailling MyoD cDNA by DNA mediated gene transfer. The C3H IOTl/2 cells wcre chmcn SIIH.:C they represent undifferentiated cells that do not express any I11USc\C-~pCCI fic tranSCription factors or muscle proteins. Other ccli lines, sllch as a tibroblast ccii linc or the HeLa ccII line could also have been used. However, the C3H 101'1/2 ccII linc wa\ choSCII ~tncc Il had been successfully used by others in past studies. The transtcclJ(m wa~ donc hy Dr. M. Szyf, a collaborator In this stlldy. Gcnetlcm-418 rC'II.,tant cl()nc~ wcrc I~olatcd, propagated and subsequently subcloned as described in the Method., The '\lIbcloning procedure was designed to select myogenic clones on the ba~is of thcir abllity lO bind 12S1_ 44 (X-BGT, a highly specifie ligand at the muscle nAChR, as weil as on the basis of their morphological characteristics. Development of the clones was done by J. Philie in the laboratory. The clones were shown to express MyoD mRNA by way of Northern blot analysls and werc positively immunostamed for the myosin heavy chain, a musclt.~ specifie protein. In contrast, control cells that had been transfected with a plasmid lacking MyoD cDNA, showed no MyoD mRNA expression and did not exhibit any myosin heavy chain immunostainmg. The Northern blot analysis and immunostaining experiments to characterize the clonaI cell Hnes were done by Dr. Moshe Szyf. My experiments were foeused on a MyoD-transfected clone, called "clone 8" because mitial observations indieated that ml-a-BGT binding to this clone was parlieularly high in comparison with other clones of the sa me subcloning generation. Only one other clone of the same generation possessed higher mI-a-BGT binding; this clone also expressed more MyoD mRNA (Quik et al. 1993b) suggesting that the extent of 125I-a··BGT binding may correlate with MyoD mRNA levels. These findings ralsed the possibility that, as a result of MyoD transfection, this non-muscle cell line, expressed nAChRs. Experiments were therefore earned out in order to characterize clone 8 and determine whether the 1251_ a-BGT binding observed represented a functional nicotinic reeeptor. 3.2.1 Morphological development of C3H IOT1I2 cells transfected with MyoD cDNA The morphological development of clone 8 with time was followed (Fig. 7) to determine whether clone 8 exhibits the muscle-type morphological eharacteristics that have 45 previously been shown to occur after transfection with the MyoD cDNA. Clone 8 myoblasts began to fuse earlier than other clones of the saille gencration and devclllpcd an elongated muscle-type morphology after a 2 to 8 day exposurc to medIUm containmg 2 % FCS (fusion medium). In fact, the morphology of the cells rcsemhled that of the primary rat myotubes in culture used in the tirst chapter of this thcsis. Likc pnmary rat myoblasts, clone 8 myoblasts began to fuse, within the lirst 24 ho urs of cxposurc lo fusion medium, into multi-nucleated myotubes that werc c1ongated. they dld 1101, however, exhibit spontaneous contraction or extensive branchmg, wlllch were ob~crved in primary rat myotubes. In contrast, control cells rctained the tibrohlast-II le characteristics of mesodermal cells even after exposure to fusion l11edllllll for several days. 3.2.2 Nicotinic receptor binding in ce Ils transfected with MyoD cUNA. The developmental profile of mI-a-BGT binding with time was examined. As can he secn in Fig. 8, nicotinie receptor development was observed after an mitlal lag pCTlod. The receptors developed with a t1/2 of approximately 7 ~o 8 days, reachlllg a maxllnal levcl 11 days after the switch to fusion medium. In contralil, wl-Q'-BGT hllldlllg was nol detected in control cultures grown under Identieal conditu)Os Cfable 1). The pharmacologieal profile of the receptor ",as studled III deléll! ln suh~cqllent experiments. Binding of 125I_Q'_BGT was saturable (Bm". of 6 fmol/culture weil) and of high affinity (apparent K" of 1.5 nM) (Fig. 9). This indicatcd that the ml-o-BGT hinding 46 observed earlier represented an a-BGT receptor population as opposed to non-specifie binding (which would not have been of high affinity or saturable). The apparent ~ value is comparable wlth the apparent Kd values observed for 125I-a-BGT binding in rat neonatal muscle cultures in the first chapter of this thesis. as weil as those found for various muscle cell lines. The effect of nicotinic and muscarinic receptor ligands on 125I-a-BGT binding to clone 8 is depicted in Fig. 10. The ICso values. which represent the concentration of drug required to inhibit 125I-a-BGT binding by 50%. were: a-BGT, 1 nM; d-tubocurarine, 0.3 l'M; nicotine, 3 l'M and carbachol, 2 l'M. The muscarinic ligands atropine and muscarine had no signifïcant inhibitory effect. The above results indicate the presence of a saturable, high affinity receptor that has the pharmacological profile of a muscle-type nAChR. 3.2.3 Nicotinic receptor-mediated function in cells transfected with MyoD cDNA To determine whether the nAChR expressed in MyoD-transfected cells was functional, carbachol-stimulated 2lNa uptake was measured 10 the cells in culture (Fig. Il). Cells were stimulated with the nicotinic agonist carbachol at varying concentrations in the presence of 2lNa to determine whether this would result in an ion tlux. This uptake occurred in a concentration-dependent manner. Carbachol was used in subsf:quent functional assays, at a concentration of 100 JA.M, since this concentration was able to 47 maximally stimulate the cells. The dose response curve is very similar to that found for various muscle celllines, with an EC~ll value of approximately 25 #lM. Experilllcnts were subsequently done to determine whether this functional rcsponse was mcdiated by a receptor with nicotinic characteristics. Inhibition studles (Fig. 12) showed that the nicotinic antagonists, a-BGT and d-tubocuranne effectlvcly IIlhibited carhachol-stimulatcd 22Na uptake. The ICso value for a-BGT was 6 nM and that for d-tubocurarinc was O. 1 #lM. Atropine showed no significant effect. These IC~n values are very similar to those found in the 125I-a-BGT binding assays. To assess whether the cell surface receptors and nicotinic reccptor-mcdialcd functional response developed in parallel, time course studies were done. The results shown in Fig. 13, indicate that there was a good correlation between changes III wl-a-BGT binding and carbachol-stimulated 22Na uptake. Levels of 22Na uptake and 11~I-a-BGT binding both started at negligible levels (day 0), developed approximatcly in parallcl (day 2-6) and peaked at similar time points (day 8). The subsequent dccl\l1e 111 thcsc lwo paramctcrs presumably reflects the graduai morphological dectine of the cultures thal was observed at these later time points. 48 TABLE 1 Effect oftransfection with MyoD cDNA on nicotinic receptor binding to C3H IOT1I2 cells. After 8 to 9 days in fusion medium, binding of 125I-a-BGT was measured to control cells and cells transfected with MyoD (clone 8), using a submaximal (l nM) and maximal (6 nM) concentration of radiolabelled toxin. Each value represents the mean + S.E.M. of 6 to 8 culture wells. The results are representative of 2 experiments. CONCENTRATION SPECIFIe mI-Q'-BGT BINDING OF m'-Q'-BGT (fmol/culture weil) control cells clone 8 1 nM 0.04 ± 0.01 3.65 ± 0.10 6nM 0.00 ± 0.03 5.78 ± 0.35 49 L Fig. 7. Morphological development of control (CON) and clone 8 (8). Clone 8 was transfected with MyoD cDNA whereas control cells werc ilOt. Photographs \Vcrc takl'Il immediately prior to replacing the growth medium wlth fusion IllCdlll1ll (day 0) as wdl as after 2, 4 and 8 days in fusion medium (200X magnitication). 50 CON 8 • DAY o 2 4 8 Fig. 8. Time course of receptor development of clone 8. Specifie t2~I-a-BGT binding was measured immediately prior to replacing the growth medium with fusion medium (day 0) as well as after the indicated number of days in fusion medium. Each valuc represents the mean ± S.E.M. of 12 to 16 culture wells from 3 to 4 separatc experiments. Where the standard error was Ilot depicted, it t'ell within the sYl11bol. 51 c.!J Z 20 ~ - T z-_....-4 ~Q) • T E-t ~ 15 c.!J CI) ~ ~ J.t 1 :;j tS~ _ 1 :;jCJ 10 ~, ...... -4 U 0 -~'t-4 S 5 U --~ ~ fil 0 i i 0 2 4 6 B 10 12 DAYS IN FUSION MEDIUM Fig. 9. Saturation curve of specifie 125I-a-BGT binding to clone 8. After 7 days in fusllln medium, binding of 12~I-a-BGT to the cultures was donc as ctescrihed 111 the presel1l:c of varying concentrations of radiolabelled a-BGT. Each value rcprescnts thc I11can ± S.E.M. of 6 to 8 culture wells. These results arc Icprcsentatlve of .' scparatc experiments. Where the standard error was not depictcd, it fcIl within th4: symbol. 52 6 T 4 2 O+------r-----,.-----.------.------~----~ o 1 2 3 4 5 6 [125I-a-BGT] nM Fig. 10. Inhibition curves of specifie IHI-a-BGT binding to clone 8. After 8 to 10 days in fusion medium, cells were preineubated for 60 min with the indl\:ated drugs. The binding assay was then donc as deseribed. The results are exprcssed as percent contlOl specifie mI-a-BGT binding; control binding was 2.9 ± 0.2 fmol/culture weil (h experiments done in quadruplicate). Each value represents the mcan ± S.E.M. of J to 4 culture wells. Where the standard error was not depictcd, It fell within the symbol. 53 0 D-lubocurarlne «-BGT •6- Atropine  NlcoUne 0 llulcariD. • Carbachol T 0 100 1f-4 ...... CJ .... C:Q 0 lb tS ~ .....1 u0 !I N 50 u....., fi ê3 0 -10 -8 -6 -4- LOG [DRUG] Id Fig. 11. Stimulation of 22Na uptake by carbachol (100 ~M) in clone 8. At'ter 7 days in fusion medium, cultures were incubated in the presence of varying carbachol concentrations and 1 l'Ci 22Na for 2 min as described. E.-'lch value rcprescnts the mcan ± S.E.M. of 4 culture wells. The results are representativc of 2 experimcnts. Whcrc the standard error was not depicted, it feH within the symbol. 54 0.12 ~ p..::=E-4- ~ ~ 0.08 «S Q) Z ~ SI +' ...... u'3 u ~, ...... ut,)1"'4 0.04 rz:I 1=1 p..- fi) 0.00 -+--H---,..--..._---,---,..---r----,----r- -6 -5 -4 -3 LOG [CARBACHOL] M Fig. 12. Inhibition curves of carbachol-stillllllated 22Na uptake in clone 8. After 8 to 9 days in fusion medium, the cells wcre preinclIbated for 60 mm with varying concentrations of the indicated drugs and earbachol (100 J-tM)-stllllulatcd !lNa uptakc was subsequently determined over a 2 min incubation period. The results arc cxpressed as percent control specifie 22Na uptake; control specifie uptakc was 0.3) ± O.OJ nCi/cultlllc well (n = 6 experiments). Bach value rcpresents the mean ± S. E. M. of :3 to 4 culture wells. Where the standard error was not depicted, it feH within the symhol. 55 o D-tuboeurarine • a-BG! A AtroplDe T 6 J 100 is -n 50 O+-~~----~--~----~--~~--~------la -8 -8 -4 LOG [DRUG] M Fig. 13. Time course of development of 12~I-a-BGT binding and carbachol-stimlJlat~d 22Na uptake in clone 8. Specifie 12~I-a-BGT binding and carbachol( 100 JtM)-stil1llJlat~d 22Na uptake were assessed in sister cultures after the indicated number of days in fusion medium. Each value represents the mean ± S.E.M. of 4 or 6 culture wells for l.'Na uptake and 12sI-a-BGT binding, respectively. These rcsults arc reprcsentativc of J to 4 separate experiments. Where the standard error was not deplcted, it l'cil withm the symbol. 56 o SPECIFIC l-I-a-BGT BINDING • SPECIFIC -Na UPTAKE UJ ~ T t-J 0.3 12 n i~ e. i !~ }- Id 0.2 8 e. 1 Z~5 ,. ~ o~• -a ~ Cl~ 2 ~ 4 =tI:J r:.lB~ 0.1 11. -~ fil ~ 0 0 0 2 4 6 8 10 12 DA YS IN FUSION MEDIUM 4.0 DISCUSSION·· •• The dlseul>slon 10 section 4.\ rdatmg to TPO was wntten wlth tht.! pre"ulllpllOn Ihat what wa~ provlded as TPO wal> the thymie polypepltùt.! l',olat~ trom the thynuc glanù ... The obJcctlve ot Ihe~t: experiment!> wa!> formulated and tht! t!xpenmt!nl~ were dont! wlth the ~amc t.!xp~tallOn. Thl! preparalton presumed to btl TPO ha!. now hetln ~hown 10 conlam a-cobraloxJn ("ontammant (QUIk ct al )993a; ~œ addendum to thesll». Thu!>, the IOterpletatlOn provld~ 10 ~t!Çtt()n 4.1, although appropriait.! a~ a dl'>("U~~lon of rel>ult!. obtained wlth a OIcotimc anlagont~l, appear .. not appropnatt: 10 TPO a ....uch. 57 The present work was done to study factors that regulate the muscle nAChR. The effects of two factors, TPO and MyoD, were determined to assess whether they exerted any regulatory influence on nAChR functlOn and expression. The results of the present studies show TPO, a nlcotinic antagonist, caused an enhancement of nicotinic receptor functional response as weil as greater morphological development in rat neonatal muscle ceUs treated ehronically in culture. These results suggest that TPO exerts a regulatory effect on the nAChR as weil as a trophic effeet on muscle cell morphology. The effeet of MyoD, a myogenic transcription factor, on nAthR expression was also examined in order to study regulation of the nAChR at the gene level. This was approached by transfecting non-muscle cells MyoD cDNA then testing to determine whether this would result in the appearance of a functlOnal nAChR at the cell surface. The development of saturable, high affinity 125I-n'-BOT binding indicated the presence of a nAChR population. The presence of carbaehol-stimulated 22Na uptake suggested the development of a nicotinic functional response. These two parameters were found to develop in parallel over time and both were inhibited by nicotinic antagonists. These results suggest that the transfection of a non-muscle cel1line with MyoD cD NA results in the expression, at the cell surface, of a functional muscle-type nAChR. 58 4.1 LONG-TERM THYMOPOIETIN TREA Tl\IENT OF RAT NEONATAL MUSCLE CELLS IN CULTURE LEADS TO ENHANCEMENT OF NICOTINIC RECEYfOR FUNCTIONAL RESPONSE AND MORPIIOLOG\'. As detailed in the introduction, severa! studies have dCl110nstratcd that TPO intcracts at the nAChR at the a-BOT site in vanous tissues and blocks nicotinic functiona\ rcsponsc. The nature of the interaction of TPO at the nAChR has becil stll(lIcd in the C2 ccII IIlll' (Quik et al. 1991b) where TPO was found to bind to thc a-BGT 'lIte and .. flect thl: functlOnal response of the nAChR relatlvely slowly; at 10 K M 'l'PO, maxllnal IIll1lbltion of 125I-a-BGT binding and carbachol-stimulated 22Na uptake occurrcd alkr about 1 hour of exposure to the polypeptide. Recovery of (Y-BGT binding and functional rcspollsc to control values after exposure to lO-K M TPO occurred only afler 16 hours. The reversibility of thymopoietin's interactIon at the nicotinic reccptor was a1so studicd 11\ rat neonatal muscle cells in culture (Quik et al. 1992). Thesc studlcs showcd that one hr of exposure of the cultures to TPO, followed by a 4 hr wash, resulted III an cffect on l''\_('t_ BGT binding essentially similar to that found when TPO was present in the assay. Thc"ie studies suggest that TPO btnds to the rcccptor in a vcry slowly rcver~lbJc manllcr. However, the possibility remains that TPO binds Irrevcmbly. Sillcc the turnover time of the nicotinic receptor in cultured muscle cells is approximately 17 to 24 hr (SaI peter and Loring 1986; Steinbach 1989) the apparent recovery in binding aftcr 24 hr IS very likely due to the synthesis of new receptor. A recent study by Quik et al. (1992) demonstrated that short-term (60 mlllute) cxposure 59 of rat n(~natal muscle cells to TPO potently inhibited nicotinic receptor binding and function at the muscle nAChR (QUlk et al. 1992). In the present study, the effects of chrome 'l'PO trcatment on nAChR function and bindmg were examined by exposing rat neonatal muscle cells in culture to TPO for several days. Initial experiments followed the normal dcvclopment of nAChR binding and function in cultured rat neonatal muscle cells in the absence of drugs and thus established the most appropriate time points for subsequent experiments. m[-a-BGT bindi'1g and carbachol-stimulated 22Na uptake were found to dcvclop with similar time courses. '2'I-a-BGT binding re:ached a plateau at day 8 with half-maxllnal binding at day 5. Day 4 and 7 were seleeted as slIitable time points for subsequent experiments as tl1ey represent time points when binding was close to half maximal and maximal. Carbachol-stimulated 22Na uptake reached plateau levels at day 6 with half-maxllnal uptake oeeurring at around day 3 day. The affinity of the receptor for a-BGT was similar at ail time points; the apparent KIJ feIl in the range of 0.7 to 1.2 nM. Carbachol, on the other hand, acts more potentIy on the r.AChR on day 9 compared to day 4. Using the time points determined above, the effect of long··term TPO pretreatment on nicotinie receptor binding was assessed. It was found that after a 4 or 7 day exposure, potent inhIbition of 12'I-a-BGT binding oecllrred. This resu.1t was similar to that found after only a 60 minute exposure. It is important to note that in the case of long-term prctreatment, TPO was added at the time of plating. FOllr days later (day 4), one set of cells was assayed. The other set received a change of medium including a fresh aliquot of TPO and was sllbsequently assaycd on day 7. Thus, in ail experiments, a period of 3-4 60 days had elapsed from the rime of addition of TPO to the time of the a~say. The observation that under these conditions, TPO lcd to as potent an inllloitlOn of 1 "l-(\'-B(iT binding as that after a 60 minute exposure suggcsts that adequate concentration ... 01 'l'PO are mainta1l1ed over a 3-4 day period to cause such an InlllbltlOll. Thu .... allcr long-Ierlll (several days) exposure, TPO is able 10 IIltcract wlth the nAChR III a "llllllar 1ll.1I11ll'r .1 ... it does after a short-term (minutes) pretrcatmcnt. In contrast, the same long-term exposure described above, does not Glw,e the I\lhlhltlOtl of carbachol-stimulated 22Na uptake that is observcd alter a 60 minute pfl:lI1cuhatioll; a 60 minute preincubation on day 4 or 7, 1cacls to the potent 1II111hlti01i 01 C It was indeed puzzling that long-term TPO expo:-.urc Icd tn potent mhlbltion of hlllding, but no inhibition of functional respunsc. Although wc have no dcfinlllvc l!xplal1i1tlon, possibilities inc1ude the followlIlg. Long-tcrm cxpo~ure to l'PO may hav~ led 10 an up regulation of the nAChRs involved 111 medlatlllg the funcllonal rC'ipon"l: hut Ilot thc total receptor pool. The presence orTPO-ln'ien~Itivc carbachol-\tl Illulated uptakc after chronic treatment with TPO may indicate the II1ductlon or up-rcglliation 01 a rl:œptor population that does not bmd TPO or Q!-BGT wlth high aflinlty. Slilh a rcceptor population would not have been detected in the assay'i that uscd radiolahellcd ft-HG'/'. Howcvcr, it\ presence would have been revealed In the fllnctlonal a~:-.ay ~ ln the forrn of carbachol- fil stimulated uptake. As mentioned in the introduction. an antagonist slich as TPO might he cxpccted to IIp regulate nAChRs (Martyn et al. 1992). Recent eVldcncc for antagolllst-indllccd IIp regulation by Hogue et al. (1992) showed that chronic II1fll~lOn of d-tllhOl:lIrannl' IIlto lats leads to the to\erance and upregu\ation of nAChR~. Doses werc l'hoscn sllch th.11 immobilization or paralysls did not occur ~mce immobihzation Itsdf G\II k.u.\ to IIlcrca~l'''' in extrajunctional nAChRs and reslstance to competitive anlagol11sts. Thus, the pos~lblh!y exists that chronic TPO interaction at the nAChR may have rcsllltcd III the up-rcglliallon of functional receptors. An alternative possibility is tha! long-tcrm IIltcr.\ctlOll of TPO may have resulted in sorne allosteric modification of the reccptor sllch that l'PO l'an no longer exert its blockade of carbachol-sti mulated uptake and/or renders Ihe rcccptor mOI L' sensitive to carbachol stimulation. Carbachol may thereforc aet wlth greater potcncy as weB as higher efficacy eausing uptake levels that are hlgher th an thosc round 111 control cells. Another (although less likely) possibility is that by actIng on the Il/V:hR, 'l'PO 1'1 preventing endogenous ACh released by the myotubcs From bindmg. Previous studlcs hy Entwistle et al. (1988b) have shown that chlek myobla~t'l and myoluhc\ ln culture expressed choline acety\transferase (the enzyme which ..,ynthcsIl.c~ acctylchohne) ami stained positively with antibodies agamst acetylcholine. Both innervatcd and dcncrvatcd frog and rat muscle have been shown to synthesizc acetylcholinc (Milcdl et al. 1977, 1978, 1981) although the functiona\ rote of this muscle-dcflvcd ACh wa") not c1ucidatcd. 62 Lukas (1991) showed that chronic exposure to nicotinic agonists leads to loss of functional re~ponses in the TE671 cell line which expresses muscle-type nAChRs. Recovery of function occurred with half-tlmes of 1-3 days (a time course whieh is much slower than rccovery from nAChR desensitization which is a reversible process that occurs on a much shorter term of 0-5 minutes). Interestingly, the nicotinic antagonists pancuronium or alcuronium protected TE671 cell muscle-type nAChRs from "functional inactivation" on long-!erm treatment with agonists. These antagonists alone had no effect on levels of expression of functional nAChRs. Thus, it is possible that long-term exposure tu TPO prevents any potential "functional inactivation" caused by acetylcholine synthesized by the myotubes. Control eells which never receive any TPO treatment may be susceptible to acety1choline-indllced "functional inactivation" (although a certain proportion of nAChRs must remain active since upon addition of carbachol, 22Na uptake can be stimulated and measured) whereas long-term treated cells are more resistant. Previous studies by Quik et al. (1992) had shown that TPO could prevent nicotinic agonist-indueed reduction in myotube branching and size in a dose-dependent manner. The concentration of TPO required to reverse the action of the agonIst correlated very weil with those at which TPO inhibited binding and affected short-term function. This sllggl.!sted that TPO reversed the effeet of nieotinic agonIst on myotllbe morphology throllgh an interaction at the nicotinic rec-jJtoT. An interaction of TPO at the muscarinic receplor was considered lInlikely sinee both nicotine and carbachol gave similar 63 morphological effects; alsû, previous results had shown that TPO did not affect binding to muscarinic receptors (Quik et al. 1989). ln the earlier study, TPO on ItS own was Ilot found to have an effect on morphology. On the other haml, the eclls III thcsc studics hall been growing for 5 days in culture and were thercfore weil cstablishcd, wlth devclopL'(\ nAChRs, prior to TPO exposure. ln the present study, TPO was prc~cnt in the CUltUfl.' medium from the time of plating for up to 7 days and thus had a grcatcr potential to affect the ceUs as they are estabHshing themselves and as new nAChRs hegm 10 appcar at the cell surface. From a morphological point of view, the results of this study suggcst that TPO aets as a trophic factor since long-term exposure to the polypeptide leads to an cnhanccmcnt of morphological development. Long-term TPO exposure led to an inercasc in the nUlllhcr of myotubes reaching a certain length or a certain level of branehll1g complexity. ThiS study suggests that the mechanism by which TPO exerts the observcd trophic l'flcc\ appears to depend on the age of the neonatal mll(" l". cells in culture, mdicating that carly interaction at the nAChR is an important factor. It is possible, as disCllsscd carlier, that the main action of TPO is to block the II1teraction of muscle cell-dcrivcd accty1chohnc at the nAChR, thereby preventing the nicotinic-agonist induced degcncration ob~crvcd hy QUlk et al. (1992). Altematively, TPO may be exerting a protectivc cffcct by prevcntlllg spontaneous necrosis in culture. Interestingly, Entwistle et al. (1988 a,b) have also suggesled a role for the nAChR in myoblast fusion. In these studies, nicotinic agonists per se had no effeet on myoblast 64 fusion; howevcr, if prostanoid synthesis inhibitors were used to delay spontaneous fusion, earbachol was effective. In addition, a-BGT inhibited spontaneous myoblast fusion. Whlle this study suggests a negative effect of antagonists on morphology, there are several possible explanations for the discrepancy between this latter study and the present study. (l) The time course of the two experiments are very different. Entwistle et al. (1988 a,b) showed that the effeet of carbachol and lor a-BGT was observed fairly early after plating (44hr) and was no longer evident by 60 hr in culture, possibly beeause other fusion-inducing factors develop to mask effects mediated through nicotinic receptor activation. In the present experiments, long-term TPO treatment of muscle eeUs was initiated from the time of plating, and the effects of such treatment were assessed over a period of severa) days (4 and 7 days). By this time the effects observed by Entwistle were no longer apparent. (2) Another difference between the two studies is the species from which the myoblasts werc obtained. Chick embryo myoblasts were used by EntwistIe et al. (1988 J,b) while neonatal rat myoblasts were used in the present study. As noted previously (Neugebauer et al. 1985; Salpeter and Loring 1986) chick and rat myoblasts appear to be subject to different regulatory factors. The concentration-dependent enhancement of rnorphological characteristics may explain, in part, the absence of the concentration-dependent inhibition of carbachol-stimulated uptake that are normally seen after short-term TPO treatment. The morphological enhancement observed is concentration-dependent; increasing the TPO concentration leads to greater rnorphological developmeut. lt is possible that a culture weil with muscle cells that have developed to a more ad' anced stage with longer myotubes and a more complex 65 branching pattern (as a result of long-terïl TPO trcatment), could exhiblt higher kVl.'I" of uptake than muscle cells in control wells which had not rcccivcd an)' 'l'PO tlc.llml.'Ilt. In other words, the concentration-dependent enhancemcnt of morphologicdl charactcnst H:S may offset any coneentration-dependent dec1ine in carbachol-slimulated uptakc Ihat IS observed in the case of short-term TPO treatment. Past studies have shown that nicotinic antagonists have no efrect on the dcvclopmcnl of the neuromuscular junction or reinnervation after nerve section (Cohen 1972; Frccman et al. 1976; Jansen and Van Essen 1975; Obata 1977). Of course, aftcr aniagolllsi treatment over a period of weeks, alteratiuns in the muscle tissue wcre obscrved, presumably due to muscle inaeti vit y (SaI peter and Loring 1986). The pre~ent study th tTcl ~ from these studies in that it assesses the effeet of an antagonist 011 gro~s muscle œil morphologieal development in culture (rather than only focuslIlg on the ncuromuscular junction and reinnervation). Under these culture conditions, whcrc the isolatcd muscle cens were free from the influence of the nerve, it was possible to delermlllc whclhcr TPO, a nicotinic antagonist had any trophic effects on gencral morphologlcal development. Interestingly, antagonists have becn shown 10 inuucc pre~ynaptle cffcct ... , including enhanced motoneuron survival and/or sproutmg (Meriney ct al. 1987; PIUman and Openheirn 1978; Wines and Letinsky 1991). The effeel of TPO on such rc~ponscs were not evaluated in the current study. To conclude, the present results show that long-term pretrcatment wlth thymopoictin can lead to effects on earbachol-stimulated 22Na uptake that diffcr from thm.e round aftcr 66 short-term pretreatment. Uptake levels remain at controllevels or higher when long-term l'PO tr~tmcnt was administered, whereas a 60 minute preincubation leads to potent (nM) inhibItion of carbachol-stimulated uptake. Thyrnopoietin is able to bind at the a-BGT site since mI-a-BUT bindmg is inhlbited JO a similar manner whether short- or long-term thymopoietin treatment 1!. adm1l1istered. Interestingly, thymopoietm can induce an enhanccmcnt in morphological characteristlcs when apphed to neonatal muscle cells at an early stage in thelr development JO culture and for an extended treatment time. Althollgh the phystologJ(~al relevance of the effects of thymopoietin at the nicotinic receptor remam to be elucidated. these results indicate that thymopoietin has the potential to affect myotube morphology throllgh an interaction at the a-BOT site of the nicotinic receptor. The above stlldy suggests that the chronic interaction of a nicotinic antagonist at the nAChR can result in the enhancement of nAChR functional response and muscle cell morphological development. The molecular mechamsms by which this occurs w~re not examined in this study. The second study was undertaken to study nAChR regulation at the molecular level. The role of a myogenic transcription factor, MyoD JO the regulation of nAChR expression was examined. The MyoD family of myogenic transcription factors have bccn implicated In the regulation of the various nAChR subunits. Electrical aClIvity appears to regulate the expression of these factors raising the interesting possibility that they mediate the regulation of nAChR expression by electrical activity. Thus, the following work on the effects of MyoD on the expression of the nAChR was undertaken as another approach to the study of factors that regulate the nAChR. 67 4.2 FC~CTION H.J NICOTINIC RECEPTOR EXPRESSION IN l\1ESODER!\IAL STEM 'CELLS 'rRA NSliF.CTED \VITH MjoD cDNA. Previous sîu~; .... ",1,.' • )'1 ' Dl1strateod that MyoD binding sites are prescnt in the promot ..,1' enhancer reg/lnn::- 'cl. /\.1 ) "AChR subunits (Klarsfeld at a1., 1987; Wang ct al., Il}HH; Piette et al.. 1999~ l rody a.<1 Merlie, 19q 1; Gardner et al., 1987, B.1ldwin and BUHkn, 1989; Wang et aï / 199,0. N· lI.erger ct aL, lQQ1; Gilmour et al , 19tJI; Prody and Merlie, 1992) '"nd ~h~' ,~, Ih ;." ·'notes SUblllllt genc expressIon (Plctle ct a1., I~)l)(); Gilmour et al., l' ~'., p(~~"'t .'~-,14 \'; ;\' " '!, 19(2). The~e observations slIggcsted a roll' for MyoD in nAChR f' ':";'"\ ' Developmentally, MyoD mRNA appears shortly before nAChR ex. sllblllllt is cxprc\sed f?iette et al. 1992). Subsequently, MyoD mRNA levcls declinc during the pcriod th.11 coincides with innervation (Eftimie et al. 1991; ?ieHe et al. 1992; Buonanno ct al. 11)92). Interestingly, denervation le.'1ds to increases in MyoD mRNA that precede II1crease~ ln ex. subunit transcripts (Piette et al. 1992; Buonanno et al. 1992). Furlhermore, in \/11/ extracellular electrode stimulation was found to repress the illcrcases in MyoD tran~cn pIs associated with denervation (Buonanno et al. 1992). Thesc studlcs provlocd furthcr evidence compatible with a role for MyoD in nAChR rcglliation and romt to the possibility that MyoD may regulate a repertoire of skeletal muscle gcnc~ that arc down regulated by electrical activity. The present studies, uSll1g an alternative approach, demonstrate that functional ccII 68 surface nicotinic receptors develop as a result of the stable transfection of a non-muscle ceI1 line with MyoD cDNA. Clone 8, a myogenic clone with a characteristic skeletal rnusclr. phenotype, was shown to develop 12~I-a-BGT bmdmg that was specifie, saturable and of high affinity indicating the presence of nAChRs. The nicotimc pharmacological profile of thi~ receptor was further dcmonstrated by the effects of nieotinic ligands on '2~I-a-BGT bmding. The muscarinic ligands, atropine and muscarine, had no significant inhibJtory effeet, contirming that the receptor is not a muscarinic cholmergie one. The ab ove results are in line with those typically found for the skeletal muscle receptor (Quik et al. 1990; Luka~ et al. 1990), the electroplax receptor (Changeux et al 1987; Guy and Hucho 1987), the muscle-type nicotinic receptor in TE671 cclls (Lukas et al. 1990), the nAChR found in C2 muscle cells in culture (QUlk et al 1991 b) and at the neuromuscular junction (Quik et al. 1990). Functional ~tudies indicate that these a-BG r binding sites formed ligand-gated ion channels. Carbachol, as well as nicotine (data not shown), stimulated 22Na flux into the cells with dose response characteristics which were very similar to those observed for muscle cell lines and neonatal muscle cells in culture (Caterall 1975; Stallcup and Cohen 1976; QUlk et al. 1991b). Inhibition studies showed that the muscarinic antagonist atropine had no significant inhibitory effcet, in line with the binding results. Furthermore, it was found that the nicotinic antagonists, a-BGT and d-tubocurarine, potently inhibited 2lNa uptake assays with ICso values that are very close to those found in the binding assays, suggesting that the carbachol-stimulated flux is mediated as a result of the 69 activation of the a-BGT recognition site. Further evidence sllpporting the above conclusion, comes from the tindlllg t1mt parai leI changes in these two parameters occur with time 111 culture; the dcvc10pmcnt of futll'tlon.\1 response correlated weIl with the development of I1ICOtll1lC reccptor hllldIllg. Both 111l'!\l' parameters peaked at around the same time, day 8. Interestll1gly, the lllorphology 01 Ihe ceUs also reached a peak at this time point. The subsequent dcclll1c III hindlllg ,I\ld functional response most likely suggests that the cultures bcgan to gradually degener.ltc after day 8. During the initial experiments, 125I-a-BGT binding was found to peak at around day Il (Fig. 8) whereas dllring the later experiments, binding peaked at around day 8 (Fig. 13). The reason for this discrepancy is not clear, although it is possiblc tha! as a rcsult of passaging the ceUs during the course of the investigation, the devc10pmcnt of c10nc X evolved and became faster. For this rcason, sister cultures werc used 111 Fig. 13 to CIl'lurc that the results for receptor binding and functional rcsponse wcrc comparahlc. Taken together, the present studies suggest that the MyoD transfecled cclls devclop, al the cell surface, a functional muscle-type nAChR. Howcver, it IS important to t.akc II1to account the foIlowing data in considering the significance of t.his study. At present, other members of the MyoD family have been shown t.o have similar acti()n~ 70 and patterns of expw;slOn as MyoD. Moreover, although there are exceptions, each of these genes i~ capable of activatmg other genes wIthm the MyoD family (and the correspond mg copy of itself) after It IS transfected into a reciplent cell. This auto- and cross-activatIon networl< results in the generation of large amounts of active myogemc regulatory proteins, once any one of these genes is activated (Weintraub et al. 1991). Thus, it is possible, in principle, that the transfection of the MyoD gene in the present study, could have led to the mitiation of a cascade of events that leads to the activation of the nAChR genes indirectly. Alternatively, MyoD may have acted directly on aIl subunit receptor genes to activatt! transcription. 1'0 distinguish between these two possibilities, 1t would be necessary to monitor the levels of nAChR mRNA as we11 as the transcripts of the other MyoD family members. To track the initial appearance of the nAChR subunits at the cell surface, specl fic antibodies for the various subunits could be employed. It is important to note that the question posed in the present study was whether the activity of one myogenic transcription factor, MyoD, could ultimately lead to the assembly, at the ccII surface, of a fully functional receptor protein. The positive results obtained, white not providing answers about the precise molecular mechanisms, nevertheless add valuable inslght into the way MyoD can affect muscle nAChR expression. 71 4.3 CONCLUSION Tile findings of the fÏrst study show that the chronic mtcractlon of 'l'PO. il 1\1l'Ot 1III l' antagomst. at the nAChR can reslIIt 111 cnhanced nAChR functlonal rcspon ... c and gll"I!CI muscle cell morphologlcal developmcnt. The ~econd study reveakd that the expIC"''iIOn of MyoD, a myogel1lc transcription factor, can lead 10 the appearanœ of a fUIlCllOll.t1 muscle-type nAChR at the cell surface. Evidence from numerous studies suggest thal the state of activation or lIlactlvatloll 01 thl' nAChR (which mediates electrical actlvlty) can affect MyoD expres'iioll, whkh 111 Iurn. affects nAChR expression. The finding that the transfectlon of a non-mu~c1e ccllllllc Wltl! MyoD, results in the expression of a fllnctlOnal muscle-type nACR at the ccII surfacl', provides further evidence compatible with a role for MyoD 111 nAChR regulatlOll. AI~o described in this thesis, were the effects long-term 'l'PO trcalment on nA( ïlR functional response and rnorphology of rat neonatal muscle cells 111 culture The possibility exists that an antagol1lst such a'i TPO can aet to oppo~e ACh-IIH.llIccd clectncal activity; on a long-term basis, this could lead to changes in the cxprc<"'ilon of myogelllt' transcription factors such as MyoD and hence resuIt 111 changc\) III nAChR cxpreS~I()n, as weIl as other muscle-specific genes. 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Zevin-Sonkin, D., lIan, E., Guthmann, D., Riss, L., Theodor, L. and Shoham, 1.. Molccular cloning of the bovine thymopoietin gene and its expression in dilfcrent calf tissues' evidence for predominant expression in thymocytes. Immunology Lcttl!rs 31: 301-310, 1992 . • 98 ADDENDUM The studies described in this thesis concerning thymopoietin were done in the summer and faH of 1991. This work was subsequently organized in written format approximately a year later (faH of 1992). The study was conceived and the results were interpreted with the presumption that this thymopoietin was the polypeptide isolated from thymus tissue as described by G. Goldstein. This study, Iike the eartier on es concerning thymopoietin, was do ne in coHaboration with G. Goldstein who provided the purified thym ic polypeptide. Since completion of this thesis work, the following observation was made concerning the identity of the active component in the thymopoietin preparations. Because the amino acid sequence ofthymopoietin obtained from polypeptide sequencing data and that deduced from the cDNA sequence experiments did not entirely correspond and because of an interest in preparing synthetic thymopoietin, experiments were initiated by M. Quik (while on sabbatical in the laboratory of 1. Patrick) to sequence the thymopoietin in the preparations obtained from G. Goldstein. During the course of • the polypeptide sequencing studies, the very surprising and shocking discovery was made that thymopoietin preparations in fact contained a-cobratoxin and that all of the observed effects of thymopoietin could be accounted for by the presence of a cobratoxin. Studies conclusively identifying a-cobratoxin as the active component in the thymopoietin preparations have just been completed. The origin of this a cobratoxin in the preparations of thymopoietin provided to us by G. Goldstein remains unknown. March 12, 1993 (Maryka Quik, Supervisor)