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" .‘ i'fv‘: " ‘ 'l ‘ n I‘ I6 :~ ' :- N n 2 A, # J, l a: ' _I 44- y 1“? "iii—[i ~33 ' 9,35? ‘: A Q 1 Nam". ”.16 , ‘4 u m 1 u 5'93 "we; 1 l "A ‘5‘. ~: . I, - , :5, : F» I n ; r A . 3 ‘ E u n a ‘ ’.'. f Mfr. IN .5 Q "1} n i v '4 L . Erin/4.. r 4 ‘ 1’ l ' 1 > v‘ J.r1'.._ “u" ('- (x "L” l’ f 2 fz/CJJfilZI/ 31293 00910 8600 This is to certify that the dissertation entitled Energy Metabolism in Skeletal Muscle: Effects of ATP Depletion presented by Jeanne M. Foley has been accepted towards fulfillment i of the requirements for ‘ Ph.D. degreem Physiology Zia/$344 fif/«x Major professor Date il/7/‘/6? MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 _ i LIBRARY Mlchigan State University D t L PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. 4| :l___ i W __i___l J IP_—. MSU Is An Affirmative Action/Equal Opportunity Institution c:\ctmmpma-p.t MWLISH III mew. ma: arrears 0! ATP alumna 3! Jeanne Marie Foley A nIssmmoa Sabin-d to Michigan State University in partial fulfill-ant of the requireueute for the degree of nocroaor PHILOSOPHY Depart-eat of Phyeiology 1990 IEJWBWM JfiTWliAH Kl YRItHHAT1M Y“9iVi WHITSJQEG QTA 30 STUSQQH yd gqiod qich aunts? VOlTATg33770 A o) be it innit-8 Ytia1evin” 91812 nuaidu‘? atnemefiiupe1 ed: 10 Jnemlliiiui [sittsq n? to neWQQb sdt 101 YHQOROJTHQ W0 HOTDOG vaoloiavdq lo :nemtwuqufl 0°91 U (l /a¢< a45- ABSTRACT BNBRGY HBTABOLISH IN SKBLBTAL WSCLB: EFFECTS 01' ATP DBPLBTION 8! Jeanne Marie Poley The specific problem addressed by this research concerned the validity of a kinetic theory of control of oxidative phosphorylation rate by snount of the phosphate acceptor ADP in skeletal mscle. Respiratory control by ADP concentration requires that, for a given rate, any reduction in ATP met be countered by a comensatory increase in the ratio Cr/PCr in order to maintain the ease ADP level, assuming equilibriu- of the creatine kinase reaction and no large increase in pli. This prediction was tested by depleting ATP in rat gastrocne-ius mscles and using phosphorus Nil to nonitor ATP, PCr, Pi and pi! during rest and contraction in situ. ADP was calculated fro. these para-etera and the creatine kinase equilibrit- constant. ATP was depleted by codining intense stimlation with blockage of the purine nucleotide cycle (PRC) with the specific inhibitor hadacidin (N-fornyl-N-ludroxyaninoscetic acid) to prevent resynthesis of adenine nucleotides fro. inosine nonophosphata. Initial experiments using the drug AICAr (S-a-ino-lt-i-idasolacarbosanide riboside) deconstrated syste-ic effects of this drug, countering previous claim that mscle dysfunction associated with AICAr infusion was attributable to selective PRO inhibition. When hadacidin was used to sustain ATP depletion, the decrease in PCr predicted by the kinetic control theory did not occur: in fact, the Cr/l rel: alsc cont to t pate ATP- atte teen is t Othe ‘PPG cont teta Jeanne Marie Foley Cr/PCr ratio actually dropped slightly in resting ATP depleted auscles relative to controls, and p8 decreased slightly. Calculated ADP levels also rose .101! less in response to a series of iaonetric twitch contractions in ATP-depleted mscles than in control mscles subjected to twitch atiaulation at the sane rate. Calculated phosphorylation potential (ln{[ATP]/([ADP]*[P1])}) was identical in resting control and ATP-depleted uncles, and the decline in this para-ater was only mildly attenuated in depleted Inscles during sub-axL-al stilulstion. These results suggest that phosphorylation potential, rather than ADP alone, is the cytoplaslic factor‘which regulates respiration in luscle cells. Other effects observed in association with ATP depletion included an apparent reduction in the energy cost of both twitch and tetanic contractions and a potentiation of peak force in successive trains of tetani. This dissertation is dedicated to four people whose affection and support has sustained and encouraged us throughout this work and to who- ! wanly acknowledge a lifeti-e debt of personal appreciation. To Sax, w best (and oldest!) friend. To Curt and Judy, for helping us keep :7 sanity and sense of perspective over these last five years. And to Jane, for having the patience of Sarah. (ae. too!) iv “HummpVab tenp teb' IDs-Ive ttthEtr-g 1‘1.“ Flcnl I would like to express w heartfelt appreciation to the many people who have assisted Ia professionally with the work culninating in this thesis. Pore-oat along these is w adviser, Ron Meyer, who has been an inspiration to Is as mch for his professional integrity as for his scientific insight. Joining your laboratory was one of the best professional choices I have ever Isde. because I benefited not only fro- your talents and experience in research, but also fro. your ability to teach difficult concepts on ny level. Secondly I would like to thank Dr. Wayne Van Hues, both for teaching as well during w naster's progre- a decade ago and for encouraging me to return to do this doctoral work. You provided la with not only the enthusiasm but also the freedo- that has made this degree possible. Special thanks are also due to Dr. To. Ads. for always taking the tine to listen and to talk. Those hours have Iaant lore to la than I can express. Thanks also for teaching as how to tinker in the shop - building and polishing w little brass knee doodad was a project I'll always role-bar! I'd also like to express w appreciation to the other were of n Guidance Co-ittee: Dr. Jill Pisher. Dr. Shelagh Perguson-Miller, Dr. Patrick Dillon, and, last but certainly not least, Dr. Jacob Krier. Jack, ny wish for you is that you will find eternal happiness in our great Midwest... I also owe a lot to acne fellow students. Special thanks are due to Susie Berke-a. who spent any a late night listening to w curses in the NI! lab and who also helped greatly in getting this nnuscript out. Rich Hayes and Greg Ada-s also deserve thanks for their help in the library and in the lab. I also wish I could thank Mike Flowers for all the tine he spent in the early days helping ea get the hang of the bench experiments and just talking about stuff. We never did get to go shoot baskets like I pro-ised you, Mike. but I'll always re-enber our big plans for the one-on-one contest. The financial burden of ny graduate studies was eased by fellowships fro- the Mildred B. Erickson Fund and the American Association of University lilo-en as well as an NIH Cardiovascular Training Progral traineeship. The research work reported in this thesis was supported by NIH grant “38972. And finally, thanks to Bobbi, Sharon, Barb, Esther, Shirley, Marilyn, Bonnie and Anylou. You always were ready to ad-ire u latest Ienuscript, u newest kitten, and each new addition to u sports shoe collection. I just want you all to know that any a day was nde by your interest in w doings. Thanks. LIST ' LIST I I. I] II. 1 TABLE OF CONTENTS LIST OP TABLES LIST OF FIGURES I. INTRODUCTION II. REVIEW OF LITERATURE EARLY HISTORY OF MUSCLE ENERGY METABOLISM RESEARCH: PROM LACTATE TO ATP The Lactate Hypothesis Mole Buffer Capacity Exaained The Discovery of Phosphagen Recognition Controversy Denise of the Lactate hypothesis The ATP Challenge BEYOND ATP: DEVELOPMENT OF CONCEPTS REGARDING ENERGY USE AND RBPLENISNMBNT The Citric Acid Cycle The Electron Transport Syste- The Che-ios-otic Hypothesis thacle Structure, Mechanics, and Energetics Application of Phosphorus m 31 Spectroscopy to mscle Studies vi ix ll 18 22 31 ’35 III . IV. III. IV. CURRENT CONCEPTS IN NSCLE ENERGY METABOLISM Control of Respiration Functional Role of the Cl System Circuit mdel EXPERIMENTAL MANIPULATION OF ADENINE MJCLEOTIDES EXPERIMENTAL DESIGN AND RATIONALE UTILITY 0F AICAR FOR METABOLIC STUDIES IS DIMINISNED BY SYSTEMIC EFFECTS IN SIN INTROIMJCTION METHODS RESULTS In Situ Rat Muscle. In Situ Rat Phacle in M Probe. Isolated, Perfused Cat Muscles DISGJSSION Operation of the PNC Systa-ic Effects of AICAr ACUTE ATP DEPLETION DECREASES PNOSPNOQEATINE USE AND ACID WON DURING HISCULAR WNTRAC'I'ION INTRODUCTION ME'lllODS Surgical Preparation and Stimlation. 108 Analysis Che-ical Analysis Statistical Analysis vii 43 44 47 49 53 55 59 59 62 65 65 69 71 73 76 78 83 83 85 85 88 89 89 RESULTS Effect of the ATP Depletion Regilen Response to Sub-ainal Twitch Stimlation Response to Tetanic Stilulation DISCUSSION Control of Respiration Energy Cost ' Other Effects of ATP Depletion V. SUMMARY LIST OF REFERENCES viii 89 89 95 101 104 106 106 109 112 115 TABLE TABLE TABLE TABLE TABLE 1. TABLE 2. TABLE 3 . TABLE 10. TABLE 5. TABLE 6 . LIST OF TABLES Typical metabolite concentrations (i-ol/g) in selected ma-alian mscles at rest Relative peak areas in 31PM spectra rat gastrocnemius miscle after 75 minutes recovery from tetanic stimlation protocol Metabolite contents estimated from 31P-M spectra of rat gastrocnemius muscle after recovery from tetanic stimlation protocol ATP and PCr contents from analysis of extracts of rat gastrocnemius msscles frozen after recovery from final twitch stimletion protocol ‘ Contraction characteristics of test twitches and 500 - tetani before and after tetanic stimlation-recovery protocol Characteristics of PCr changes during and after twitch stimlation as calculated from monoexponential fits ix 44 92 92 93 96 100 FIGURE FIGURE FIGURE FIGURE FIGIRE FIWRE FIGURE FIGURE FIGURE FIGlRE FIGURE FIME FIGURE FIGURE FIGURE 6. 5. 6. 9. 10. 11. 12. 13. 1A. 15. LIST OF FIfllRES Electrical circuit model Peak twitch force in rat gastrocnemius muscle in situ Mean arterial pressure in rats during infusion and stimulation Ill-M spectra from extracts of rat muscles 31m spectra from rat gastrocnemius muscle in situ 319-1“: spectra from isolated cat biceps muscle 31PM spectra from isolated cat soleua muscle Peak twitch force in isolated cat muscles Confor-rs of AICAr coqared to adenosine and guanos ine 31m spectra from rat gastrocn-ius muscles after recovery from rigorous tetanic stimulation Peak isometric force in rat gastrocnemius muscles during successive bouts of tetanic stimulation 31m spectra from control and ATP-depleted rat gastrocnemius muscles during isometric twitch stimulation Peak isometric twitch force, phosphocreatine, pll, ADP, and phosphorylation potential in control and ATP-depleted muscles during and after final twitch stimulation 31m spectra from control and ATP-depleted rat gastrocnemius muscles during final tetanic stimulation Peak isometric tetanic force, ATP, phosphocreatine, and pl! in control and ATP-depleted muscles during and after final tetanic stimulation 51 66 66 68 70 71 72 72 81 91 94 97 99 101 103 I . INTRODUCTION "In the muscle, nature has produced a machine, so startling and at the same time so perfect, that the explanation of its mechanism could give satisfaction not only to the searching mind, but also promise a rich harvest to the technical progress of mankind. " Otto Meyerhof, 1925 (115) Muscle energy metabolism and its regulation have been the subjects of intense research and debate throughout the history of physiological investigation. As early as 1880, a regulatory link was proposed between the mechanics of contraction and the underlying muscle chemistry (153). During the century following this speculation, the details of muscle structure, mechanics, and chemistry were brought into focus. The question of the precise nature of the signal linking metabolic energy production with contractile energy demand has, however, remained incomletely resolved. The advent of nuclear magnetic resonance (MR) spectroscopy and the burgeoning application of this technology to physiological studies during the past decade have generated new insights into this lingering question of metabolic regulation. The NPR method allows accurate tracking of the changing levels of several energetically inortant intracellular metabolites during contraction and recovery in living muscles. Although much of this information could also be coqiled by traditional chemical methods, the NPR technique offers the advantage of noninvasive study of ongoing metabolic processes in an intracellular environment that is virtually undisturbed, in contrast to the trauutic disruption required for conventional methods. The mm method also allows serial measurements within a single muscle, thereby imroving practically obtainable time resolution and increasing statistical power over the one-muscle-one-time-point restriction of the older methods. In addition, the new technology provides some novel capabilities, reviewed in the following chapter, that were out of reach of previously existing methods. The studies described in chapters three and four supplemented traditional chemical methods with the newer spectroscopic techniques to study the effects of experimental manipulation of muscle adenine nucleotide levels on metabolic responses to contraction. The general aim of this research was to examine the effects of adenosine triphosphate (ATP) depletion on energy metabolism in skeletal muscle. The specific question being addressed was: Is kinetic control (by amount of the phosphate acceptor ADP) of the rate of oxidative phosphorylation sufficient to explain this regulation in contracting skeletal muscle? The existence of the creatine kinase reaction in a near-equilibriim state in muscle (103) provided the specific hypothesis to be tested. The concentration of ADP can be estimated from the levels of the other reactants and products of the creatine kinase reaction and from the equilibrium constant ([99) of the reaction according to the equation (146): [ADP] = [ATP]. (ta-i/iro-n . (Vim) . m... If [ADP] controls respiration rate, then for a given rate any reduction in ATP must be countered by a cowensatory increase in the ratio Cr/PCr in order to maintain the same ADP level, assuming no large change in pH. This prediction was tested by depleting ATP in rat gastrocnemius muscles and using phosphorus NIB to follow the changes in ATP, PCr and pl! 3 during rest and contraction in situ. Review of the methods by which ATP depletion had been induced in previous studies led to the choice of a technique codining intense stimulation to deplete ATP with blockage of the purine nucleotide cycle (PNC) with the inhibitor hadacidin to prevent resynthesis of adenine nucleotides from inosine monophosphate. As reviewed in detail in the following chapter, this method was selected because it introduced the fewest confounding factors into the experimental analysis. Before these studies could begin, however, it was necessary to address the recent claim (42) that PNC inhibition per se imirs aerobic energy metabolism in muscle. Further review of the literature revealed, however, that the drug AICAr used in this (study is known to affect other enzymes outside of the PNC in vitro. AICAr treatment had also been associated with systemic effects in vivo, most notably hemodynamic abnormalities. Furthermore, previous studies with the PNC inhibitor hadacidin had demonstrated no adverse effects of P110 inhibition on either submaximel twitch or intense tetanic contraction capacity (113). This information led to the formulation of the hypothesis that the administration of AICAr caused systemic effects and that these systemic effects, not PNC inhibition, were the basis of the performance decrement observed in the study cited above. To test this hypothesis the cited experiments were repeated while trying to uumesk any possible systemic effects by monitoring arterial blood pressure with an indwelling catheter and phosphorus metabolites and pH with 319-“. The hypothesis was then tested directly by surgically isolating and artificially perfusing cat muscles with an AICAr perfuaate. If the aerobic performance decline was indeed due to systemic hemodynamic 1.. effects, then isolation of the muscle from the cardiovascular system in this preparation should eliminate these performance effects. The outcome of these experiments was the detection of profound suppression of blood pressure in AICAr-infused rats and failure to recover PCr stores after a twitch series. In the isolated, perfused muscles, there was no adverse effect of AICAr on twitch force, and PCr recovered non-ally following the contraction series. Therefore it was concluded that PNC inhibition per so does not adversely affect muscle aerobic metabolimm ((46), chapter three). The specificity for PNC inhibition of the drug proposed for use in our initial research plan has been amply demonstrated, as documented in chapter two. The findings of our initial investigation cleared the way for moving forward with these studies to test the kinetic respiratory control hypothesis described above. An infusion/sthmulation/recovery protocol was developed to consistently produce depletion of nearly half of the normal ATP stores in the rat gastrocnemius muscle in situ. The decrease in PCr predicted by the kinetic control theory did not occur: in fact, the Cr/PCr ratio actually dropped slightly in resting ATP depleted muscles relative to controls. Calculated ADP levels also rose much less in response to a series of isometric twitch contractions in ATP-depleted muscles than in control muscles subjected to twitch stimulation at the same rate. These results indicate that mitochondrial respiratory rate control cannot be satisfactorily explained solely on the basis of kinetic mechanisms, assuming that respective respiration rates at rest and during the stimulation period did not differ substantially between the two groups. These studies also produced several additional, unanticipated outcomes. First, the energy cost of twitch and tetanic contractions appeared to be reduced in the ATP-depleted muscles relative to controls, complicating the interpretation of the ADP changes observed in response to stimulation. Secondly, tetanic stimulation caused the accumulation of markedly more acid in control than in ATP-depleted muscles, suggesting a reduction in the rates of glycolysis and glycogenolysis in the depleted muscles. Finally, ATP depletion was associated with a staircase or potentiation of peak force during series of tetanic contractions. This phenomenon was observed in both control and hadacidin-treated muscles when subjected to intermittent tetanic stimulation immediately following an initial intense stimulation to deplete ATP. This staircase effect disappeared after recovery of ATP in the control group, but persisted in the muscles whose ATP recovery was blocked. The rationale for the development of these stuudies and the implications of the results are best considered in the context of the existing body of knowledge in this area. To this purpose, an historical review of the research on muscle energy metabolism and its regulation is presented in the following chapter. II. REVIEN OF LITERATURE "The extreme eimlicity of the fabric (of muscle) has been the cause of the obscurity that prevails in understanding how a small, soft, fleshy portion can produce such strong and amle motions." Baron Albertus Heller, 1786 (56) The 'fabric' of muscle and the mechanism underlying the activity of muscle have been subjects of intense scrutiny throughout the history of physiological inquiry. In the mid 1700's, William Croone proposed that muscle was the biological material upon which studies would be most likely to yield an understanding of "the energy discharges of living elements" (66). As the eighteenth century drew to a close, however, the "motive cause" of muscle action was still attribuuted to "a law imediately derived from God” (54). Technological advances during the 1800's spawned mre sophisticated studies of the mechanism of contraction and led to the development of the concept of muscle as a chemical machine . EARLY HISTORY OF MUSCLE ENERGY METABOLISM RESEARCH: FROM LACTATE T0 ATP In the late 19th century, Herman and later Pfltiger conceived of a comlex "lactacidogen" or "inogen" molecule, containing lactic acid and made irritable by the inclusion of oxygen. Anabolism was thuus viewed as the formation of "elaborate, unstable, oxygen-charged molecules" into an "irritable protoplasm" which could then be discharged as needed to provide energy for life processes (46). This view was challenged by Fletcher and Hopkins, who were able to demonstrate that the imediate supply of oxygen affected contraction (44). The inogen theory, based as it was on the prior inclusion of oxygen, was thus rendered unlikely. These two researchers also pioneered a new low-temperature method of killing and extracting muscles to minimize excess lactic acid production caused by the excision and extraction procedures themelves. They noted that, prior to their 1907 work using the new technique, "there is hardly any imuortant fact concerning lactic acid formation in muscle which, advanced by one observer, has not been contradicted by another” (65). The work reported in this paper by Fletcher and Hopkins was later hailed by Meyerhof as "the first bridge between the biochemistry of muscle and its performance of work" (115) . We "In the evolution of muscle it would appear that advantage, so to speak, has been taken of this acid phase in carbohydrate degradation, and that by appropriate arrangement of the cell elements the lactic acid, before it leaves the tissue in final coduustion, is assigned the particular position in which it can induce those tension changes upon which all the wonders of the animal world depend. " Fletcher and Hopkins, 1917 (46) Fletcher and Hopkins authored this "lactate hypothesis", wherein the hydrogen ions from lactic acid supposedly interacted directly with the wofibrillar surface. Lactate was thus viewed as the direct cause of increased tension, leading to the notion that lactate was ”part of the machinery and not part of the fuel.” Fatigue and rigor were seen as manifestations of lactate accumulation: "...fatigue is the expression, not of an exhaustion of the energy supply, buut only of a clogging of the machine." Oxygen's purported role was to 'unclog' the machine by removing lactic acid (44). In 1921, Hartree and Hill noted that the temerature coefficient of the rate of heat liberation from a muscular contraction was of the same magnitude as those of ordinary chemical reactions. This led them to conclude that muscle energy supply regulation in a prolonged contraction is a chemical process (58). In this report they also outlined a regulatory scheme in which a 'shock' or nerve impulse caused permeabilizetion of the walls of a lactic acid comartment within the muscle cell, thereby releasing lactate onto the fibrillar surface to directly cause increased tension in contractile structures. According to this model, relaxation was caused by neutralization or other mans of removal of the acid from its site of action. The role of oxygen was to facilitate this relaxation process, either by oxidizing lactate or by somehow causing it to be returned to the imermeable intracellular comartment. Although this theory seem preposterous in retrospect, at the time it was proposed it very nicely conformed to and explained the known facts. Hartree and Hill also related the mechanical efficiency (defined here as work done per unit of heat liberated per second of stimulus) of muscle. to its 'quickness', observing that unstriated muscles exhibited a very high efficiency in long term maintenance of force. They proceeded yet further in characterizing fast vs. slow muscles, stating ...it is well known to athletes that heavy and exhausting exercise is very bad training for sports, such as sprint-running and juming, where great quickness is required: the training which leads to quickness of contraction and relaxation is directly opposed to that which leads to econom, and i-unity from fatigue, in slow, prolonged, and heavy movements." (58). Thus was the principle of specificity of training, a basic tenet of modern exercise physiology, initially and most eloquently recorded. In 1926, a student of A.V. Hill reported a series of painstaking experiments demonstrating that muscle has the ability to vary its energy transformation rate according to the tension production and duration of a contraction (35). This phenomenon, which became known after its author as the 'Penn effect', initiated a search that continues even today for the signal linking energy demand to production. II I I if E 'l I . I Later the same year, Hartree and Hill again collaborated on a paper, this time concerning the apparent need for a large buffer capacity in muscle. With deft calculations using existing data, they concluded that a mere ten seconds of 'violent effort' by a human athlete would produce enough lactic acid to increase hydrogen ion concentration 18 fold. Given the buffers then known to be available to muscle, this would produce a pH of 5.75, lower than had ever been observed even in isolated muscles. The authors exclaimd that "it is difficult to imagine that such a change in c]! (sic) in the intact animl could be tolerated without disaster" (59). The experimental section of this article reported the results of the addition to .an isolated muscle of an amount of exogenous lactic acid approximting that expected from a ten second maximal contraction. The observation that intracellular [8"] increased only by a factor of 2.2 (i.e. pH decreased to 6.7) promted the authors to conclude that some heretofore unidentified but highly effective buffer must exist in muscle. According to the chemical data available at that tim, they speculated that this unknown substance was probably an ”alkaline protein buffer". The question of acid neutralization in muscle was further 10 addressed in 1925 by the Australian Tiegs. Although Hartree and Hill and earlier Meyerhof had claimed that the neutralizing base must be a muscle protein, Tiegs dissented. He proposed that the buffering base was actually produced simultaneously with the lactic acid (144). This view, though closer to the actual truth, seemed based on rather unconvincing histological evidence. Tiegs also surmised a link between the buffer production process and the compound creatine, noting that creatine content in muscles varied in proportion to their activity. Citing previous work, he remarked that "plain" (smooth) muscles were lowest in creatine content and "quickly contracting 'pale' muscles" (fast white skeletal muscles) were highest. Again citing earlier evidence that creatine increased with stimulation, Tiegs offered three possible mechanisms by which the "very feebly basic” creatine could undergo conversion to a base of sufficient strength to neutralize the lactate produced during contraction. The first two routes, conversion to the strong bases creatinine or dimethylguanidine, were ruled out by showing that these compounds were not present in stimulated muscles. Thus remained only the third of Tiegs' alternatives, namely that creatine was transformed to a more basic isomer upon stimulation of muscle. To accommodate this hypothesis, Tiega suggested that the 'normal' form of creatine in resting muscle was not the comonly-held open structure, but rather ~a.cyclic form. Upon stimulation, this cyclic molecule would undergo conversion to ”active creatine” by opening of the ring structure to expose a free amino group, thuue providing the necessary increase in effective base. Although his experiments on isolated frog muscle did confirm that creatine was indeed liberated from 11 exercising muscles, his explanation of the mechanism of increased buffer capacity was clearly contrary to the known chemistry of that time. The 'lactacidogen' theory which Tiegs held as "now quite certain" was decisively refuted by Meyerhof, also in 1925. The theory was based in large part on the claim of a lactic acid maximum due to a limited amount of precursor. Meyerhof showed that lactic acid production by isolated frog gastrocnemius could be increased to 501 beyond the supposed maximm by speeding removal of lactate from the muscle via added alkali in the bath (115). Despite this caveat, Meyerhof still subscribed to the 'lactate hypothesis' in which lactate directly caused the contractile event. WW Experiments conducted at Harvard University and at University College in London during the period 1925-1927 finally shifted the muscle energy focus away from lactic acid and towards organic phosphorus camounds, generating a "revolution in muscle physiology" as it was subsequently designated by A. V. Hill (60). In 1927, the American team of Cyrus Fiske and Yellapregada Subbarow and, working independently, the Britons Grace and Philip Eggleton separately reported the discovery of a labile organic phosphate commund whose stimulation-induced degradation released inorganic phosphate inside muscle cells. Most historical accounts of muscle metabolism research provide only a scant mention of this discovery. Most such essays also give primary credit for phosphocreatine (PCr) discovery to the Eggletons, despite the fact that Fiske and Subbarow were not only the first to correctly identify the labile phosphorus comound as phosphocreatine, but were also much more accurate in their speculations as to its 12 functions. For these reasons, the circumstances surrounding this breakthrough will be dealt with in some detail here. In 1925, Fiske and Subbarow published a detailed account of a new colorimetric assay for inorganic phosphate (P1) in biological samples. Their primary inrovement over the older Briggs method was the substitution of an alternative reducing agent which reduced the time to maximal color development from 30 minutes to less than five minutes (41). Following up on apparently erroneous results they obtained using this new method, the pair discovered that previous estimates of muscle inorganic phosphate content were mistakenly high due to excess orthophosphate liberated during the extraction and assay process. Since this excess phosphate release was virtually complete within 30 minutes, the longer~color development time required by the older assay method tended to mask the orthophosphate increase. In 1927, Fiske and Subbarow decisively and correctly identified the labile organic precursor in a terse abstract in the W, given here in its entirety: "Only a small fraction of what has previously been regarded as inorganic phosphate in voluntary muscle is actually inorganic. The bulk of it is an unstable comeund of creatine and phosphoric acid which is hydrolyzed upon stimulation and resynthesized when the muscle is permitted to recover." (40) In April of the same year, a more detailed account of their findings appeared in the American journal m. In this article, Piske and Subbarow showed that the "delayed color development” of their 1925 P1 assay when applied to muscle tissue was due to a "labile phosphorus" camouuud (38). The amouunt of unstable phosphorus was estimated from successive color intensity readings at short intervals uuntil full color development. Extrapolation back to zero time (muscle 13 excision) gave true 'inorganic' phosphate content, the difference between this and the maximal value being 'labile' phosphorus. They identified this precursor of Pi as a derivative of creatine (Cr). They also observed that pro-stimulation of the muscle prior to extraction and assay reduced the labile phosphorus by two-thirds compared with resting muscle. Additional experiments demonstrated recovery of the phosphorus- creatine compound in stimulated muscle that had been rested briefly prior to excision. The authors remarked that the release of Pi and Cr by fatigued.auscles, previously attributed to fatigue-induced alteration of membrane permeability, could now be explained singly by increased concentration gradients of the two compounds resulting fromtstimulation- induced hydrolysis of PCr inside the muscle cell. Thus their discovery not only introduced a new compound into the muscle energy scheme, but also began to weaken the underpinnings of the lactate hypothesis by casting doubt upon the reality of the permeability changes required in the Hartree/Hill model of contractile regulation (58). Regarding their observation of delayed color development and their suspicion that an unstable organic phosphorus compound existed in muscle, Fiske and Subbarow cemented, A "Although these facts have been in our possession now for more than a year, we have until this time refrained from placing them on record, inasmuch as the phenomena observed could not with any certainty be ascribed to the presence of an organic camound of phosphoric acid until the compound had been isolated, or at least until the organic radicle (sic) had been identified." (38) This co-itment to presentation of a complete story unfortunately was to cost these two men much of their deserved credit for this landmark discovery. In February of 1927, after submission but before puublication of 14 the Fiske and Subbarow report, there appeared in the WM], an article by Grace and Philip Eggleton announcing the discovery in muscle of an unstable organic phosphorus camound which theydesignated as 'phosphagen' (30). The Eggletons used the 1922 Briggs method for determining Pi, taking color intensity readings at two minute intervals for 30 minutes rather than a single reading at 30 minutes as had been done in previous applications of the mthod. The Eggletons noted that color development in P; standards and in extracts of dead muscle was exponential with a time constant independent of the phosphorus concentration. In extracts of fresh muscle, however, this time constant was much larger. Therefore, they concluuded, previous estimations of muscle inorganic phosphorus were erroneously high due to the acid hydrolysis of an unstable organic phosphorus compound during the color development period. Using a clever extrapolation procedure, they estimated that the 90-100 m of 'inorganic phosphate' per 100 g of muscle reported earlier by Briggs, Embden and others actually consisted of only 20-25 m Pi, the remainder being 'phosphagen'. They also noticed that the amount of phosphagen was lower in fatigued muscles than fresh, and practically nonexistent in muscles in rigor. The Eggletons speculated that this 'phosphagen' might be a phosphate ester of glycogen, or perhaps a combination of lactate and phosphoric acid, i.e. the 'lactacidogen' impugned by Meyerhof. In a subsequent letter to the British journal m, the Eggletons stated, "Whilst we have here at present no definite knowledge of the nature of this substance, it seem quite possible that it may be the unstable ('active') hexose monophosphate..." (32). Their next paper claimed that 15 "recent unpublished work on the isolation of phosphagen shows that it is a hexosemonophosphoric acid, though some doubt attaches to the nature of the hexose" (32). Based on this result, the authors proposed a reaction scheme in which phosphagen split to yield lactate and Pi , the latter recombining with glycogen to give a compound 'X' which "may be identical with-lactacidogen". The final reaction of this proposed cycle regenerated phosphagen from 'X' . Sumerizing this model, the Eggletons remarked, "These three reactions fonm a cycle of changes which, if properly balanced, leads simply to the conversion of glycogen into lactic acid." The proposal of this scheme can be viewed as a testimonial to the prevalence of the lactate hypothesis, inasmuch as it attempted to fit the new discovery into the lactate-centered model rather than recognizing the new compound as the key to an entirely new explanation of muscle energy generation. In February of 1928, the 'special articles' section of Science carried another succinct report by Fiske and Subbarow entitled "The Isolation and Function of Phosphocreatine” (37). This co-Iunication provided the correct empirical formula and chemical structure of PCr isolated fromtrabbit muscle. In what can certainly be in retrospect deemed a major understatement, the authors commented, "... the instability of the phosphnmic group marks it as one of considerable biological importance". Fiske and Subbsrow additionally observed that phosphocreatine was not only hydrolyzed during activity, but also resyntheaized during subsequent recovery. According to their calculations, the Pi liberated by PCr hydrolysis during stimulation was sufficient to neutralize much of the lactic acid produced. This prompted their assertion that l6 ”... the function of phosphocreatine in muscle - or one function, since there may be others - (is) that of neutralizing a considerable part of the lactic acid formed during muscular contraction." This fulfilled the 1924 prediction of Hartree and Hill regarding the existence of a novel, highly effective buffer in muscle (59). Later in 1928, the Eggletons reported some "Further Observations on Phosphagen" (29), in which they described the isolation of a labile compound of creatine and phosphoric acid, finally acknowledging the Harvard research by a footnote: "Confirming Fiske and Subbarow”. The British team still maintained, however, that "... it is not yet certain whether this substance is identical with, or is a breakdown product of phosphagen". The sharp contrast between the previously noted connitment of Fiske and Subbarow to present a finished research picture and the Eggletons' approach to publication is illustrated by the introduction to the 1928 Eggleton article: "The collection of experimental results presented in this paper is too inconsecutive to be used as the basis of any theoretical discussion of phosphagen, but the results are puublished in the hope that they will be of practical use to other workers in the field of muscle chemistry." (29) Without reference to their previous misidentification of phosphagen as a hexose phosphate and likely precursor of lactic acid, the Eggletons acknowledged in this paper that phospagen breakdown and lactic acid production were chemically distinct mechanism. One of the 'inconsecutive' results of this study that did prove to be of importance later was the correlation of rate of energy output with muscle phosphagen content. This relationship had been suggested in one of their earlier papers (32), based upon the observation that l7 gastrocnemius muscle contained more phosphagen than did cardiac muscle, and smooth muscle contained little or none (31). This correlation was presaged by Tiegs' earlier observation that muscle creatine content varied with muscle activity (144). The 1928 Eggleton report compared the phosphagen:Pi ratio in various skeletal muscles, noting a higher ratio in the "quicker" muscles, such as the gastrocnemius, than in "muscles intended for lower rates of energy expenditure", such as the soleus (29). The same paper reported the results of a comparative study in which the absence of phosphagen and creatine and the prevalence of arginine were noted in invertebrate muscle. In an insightful commentary, the Eggletons observed that both arginine and creatine gave the same "colour reaction" characteristic of a guanidino group and argued that this could be the basis for physiological function in both. The following year, now nearly two years after the identification of phosphocreatine by Fiske and Subberow, the Eggletons produced yet another paper in which they claimed that "The exact nature of phosphagen is not yet known" (28). They advised that the term 'phosphagen' be retained ”for the substance existing in muscle", as distinguished from the PCr isolated from muscle extracts. They went on to conclude that "... the 'breakdown' and 'resynthesis' of phosphagen in a living muscle are reactions involving very little energy". Later in this paper they reported their failure to replicate some published results of Fiske and Subberow, provided that "clean glassware and pure reagents are used". 18 E °| . E v In view of the preceding account, the position accorded the Eggletons in most historical records as the discoverers of phosphocreatine in muscle seem unmerited, particularly since Fiske and Subberow have received only secondary credit or mere passing mention in most muscle metabolism reviews. Fiske and Subberow were not only the first to isolate, purify, and identify phosphocreatine in muscle: they also proved more meticulous in their methods and much more accurate in their deductions of PCr function in muscle than did the more acclaimed Eggletons. Fiske and Subberow showed evidence of their awareness of this controversy in an article in the 1929 W. In this 50-page discourse, the two physiologists directly addressed the claim of the Eggletons in a most pointed manner: "Eggleton and Eggleton, using the Briggs method -- which we have shown to be inaccurate -- likewise have observed the slow development of color in the case of filtrates from ... muscle These authors, however, also without experimental evidence, chose the alternative assumtion, v13“ that the cause of the phenomenon is the presence of an unstable organic compound of phosphoric acid, and hazarded the guess that they might be dealing with a new variety of hexosemonophosphate, or 'e phosphoric acid ester of glycogen. ' Somewhat later, in a paper published several months after we had announced that the labile phosphorus in muscle is combined with 1 equivalent of creatine, Eggleton and Eggleton claimed to have M that muscle contains a hexosemonophosphate with the properties that have been described above, though admitting that 'some doubt attaches to the nature of the hexose. ' As a fitting accompaniment to this extraordinary assertion, for which they could not possibly have had the slightest evidence, Eggleton and Eggleton offered an elaborate hypothesis in which the attemt was made to incorporate this hexosemnophosphete into the series of chemical reactions (involving glycogen, lactic acid, m.) already known to occur during muscular contraction. Needless to say the theory, based as it was on a structure devoid of facts, is valueless, and has since been quietly abandoned by its authors." (39) The reminder of this extensive article was devoted primarily to 19 an exhaustive account of the extraction, purification, and assay methods used in obtaining the results reported in the two years prior. This paper represented the last published work by Fiske and Subberow on the topic of phosphocreatine. Nothing further appeared over Piske's signature until a 1935 study of 'depressor substance' (adenosine) in the blood, followed in the l9h0's by studies devoted to purine chemistry. Likewise, Subberow produced no additional publications until the late 1930's, when he reappeared as a secondary author on several studies involving nutrition, vitamins, and growth factors. The ngletons' perceived place in history was most likely the result of a 1932 article by the eminent British physiologist A.V. Hill. In the opening statements of this widely acclaimed review entitled "The Revolution in Muscle Physiology", the influential Hill gave full credit for the breakthrough discovery to the Eggletons: "The revolution to which this paper refers broke out on the last day of December, 1926, when Eggleton and Eggleton sent to the Biochemical Journal a paper describing phosphagen, a labile form of organic phosphate in muscle. In 4} years their discovery has changed our outlook on the relation between chemistry and the . energy exchanges of muscle ..." (60) Several pages later in the paper appeared the sole mention of Piske 5nd Subbarow, a brief paragraph remarking that "Shortly after, and independently of, the ngletons' publication, Piske and Subbarow reported the same discovery ..." Hill went on to downplay the role of the Harvard team in deducing the buffering functions of phosphocreatine by claiming that this conclusion had been suggested in the 1925 Tiegs article (144). It should be recalled that Tiegs had maintained that creatine itself provided the extra buffering power by conversion to a more basic isomer upon stimulation: the existence of a phosphorus- creatine cmd, much less its hydrolysis to release buffer phosphate, 20 was not even remotely suggested in this paper. This apparent oversight in an otherwise comprehensive synthesis of studies could perhaps be considered a reflection of Hill's position as a collaborator and colleague of the Eggletons at University College. Alternatively, Hill's apparent disregard of Fiske and Subbarow's work might be viewed as a reaction to their 1929 "Phosphocreatine" paper (39) in which they harshly criticized the work of not only the Eggletons, but also other European scientists such as meden, Lolmann, and Meyerhof. In any case, the outcome of Hill's pronouncement has been that later reviews of muscle energy metabolism concepts have tended to give primary credit for the discovery of phosphocreatine to the ngletons (e‘.g. Lundsgaard (98), Lolmann (93, 94), Huxley (69), Hill (64), Lipmann (91)). A notable exception is the monograph by Kalckar (72) who, perhaps not coincidentally, hailed from Harvard. I! . I I I I I . Credit controversy notwithstanding, the discovery of the presence, hydrolysis, and resynthesis of phosphocreatine in muscle did indeed lead to a 'revolution' in muscle physiology by finally turning the research focus away from lactate as the key chemical in muscular contraction. By 1930, Binar Lundsgaard had proposed a system of linked reservoirs in which 'phosphagen' was the final energy source for muscular contraction (99). In Lundsgaard's model, the phosphagen pool was fed in turn by a glycolytic reservoir involving lactate formation. He also envisioned a third "carbohydrate oxidation reservoir" which could either lead to lactate formation or directly augment the phosphagen reservoir without coincident lactate production. This model is new recognizable as the 21 first reasonable approximation of the modern paradigm of the muscle energy system. Lundsgaard was particularly prescient in his conception of the third, oxidative reservoir, since the citric acid cycle and electron transport system had not yet been proposed. The key experimental evidence leading to the development of this model consisted of the demonstration by Lundsgaard of "alactacid" contractions in muscles treated with iodoacetic acid (IM) to block glycolysis. Achievement of these results required an ingenious experimental design, since animals treated with 1M normally develop rigor in all skeletal muscles shortly following treatment. Lundsgaard discovered, however, that prior paralysis of a limb via nerve section served to protect the muscles of that limb from rigor development. He had previously observed that the rigid muscles of unparalyzed, 1AA treated frogs contained no acid in excess of that normally found in untreated, resting muscles; this was in great contrast to the large lactate accumulation normally associated with rigor development. Pursuing this clue, Lundsgaard electrically stimulated his IAA treated, denervated muscles and observed series of twitches in which no lactate was produced. Furthermore, the poisoned muscles fatigued faster than normal muscles, and exhaustion coincided with the near total depletion of phosphagen. This demonstration finally and conclusively struck down the lactate hypothesis by clearly dissociating lactate production from contraction, and lactate accumulation from fatigue. The linked reservoir model was suggested to Lundsgaard by two facts: unpoisoned muscles performing the same number of twitches as IM- treated muscles retained 70-75! of their phosphagen stores, and the energy liberation from the extensive PCr hydrolysis in poisoned muscles 22 approximated the combined energy of PCr hydrolysis and lactate formation in unpoisoned controls. Prom this evidence he deduced that lactate formation normally provided energy for the post-contraction resynthesis of the phosphagen utilized in contraction. This solved Hill's persistent problem of the source of the "delayed anaerobic heat" following muscular contraction. Lundsgaard also co-ented that the previous failure of researchers to correlate PCr breakdown with contractile work output was now most certainly due to the confounding effects of glycolysis. Although Lundsgaard's scheme represented major progress, his model still placed PCr in the position of direct energy source for contraction. He was certainly aware of the existence of ATP, or "adenylpyrophosphoslure" as it was named by its discoverer Lohmann in 1929 (92). However, Lundsgaard's observation that no appreciable ATP breakdown occurred until development of exhaustion and rigor misled him to conclude that PCr, not ATP, must be the direct source of energy for contraction. In 1932, Hill lauded Lundsgaard's work as "admirable in expression and execution", but in the same paper Hill prophetically mused, "I wonder whether we are still failing to see something which in 10 years will seem obvious" (60). W The missing piece to the muscle energy puzzle was indeed provided during the ensuing decade. In 1934, Karl Lohmann reported that PCr hydrolysis in muscle extracts occurred only in the presence of ATP and ATPase, whereas ATP hydrolysis was able to proceed even when PCr breakdown was inhibited by alkaline pH (94). He further noted that ATP exerted a catalytic influence on PCr hydrolysis in muscle extracts, with 23 one part ATP, in the presence of ATPase, capable of causing the splitting of 1000 parts PCr by "creatine-phosphoric acid-splitting enzyme". Lohmann proposed the following scheme, which became known as the 'Lohmannn reaction' (93): (A) ATP <===> AMP + 29‘ (B) M+2FO~<===>ATP+20~ (0) (Net) 21%:" <===> 20* + 2 P1 Although this model showed ATPase directly hydrolyzing ATP to AMP without the intennediate myokinase reaction and portrayed AMP rather than ADP as the phosphate acceptor in the creatine kinase reaction, Lohmann did correctly infer that ATP hydrolysis was the direct energy- supplying reaction in muscle contraction. He was able to explain Lundsgaard's observation that muscle ATP levels do not fall significantly until exhaustion (99) by showing that the creatine kinase reaction (B) proceeds much faster than the ATPase step (A). Therefore, Lohmann argued, PCr hydrolysis rapidly regenerated ATP so that little "adenylslure" (AMP) built up until PCr stores began to fail. Although Lohmann's proposal constituted the missing "something" of Hill's earlier speculation, it would be nearly 30 years before the presentation of decisive proof that ATP was indeed hydrolyzed during contraction of intact muscles. Throughout the 1930's and #O'e, physiologists continued to argue against Lohmann's theory, citing the undeniable fact that all supporting experimental evidence was derived from chemistry experiments on muscle extracts rather than from studies 24 of intact muscle. A 1937 paper by Sacks, Sacks, and Shaw was representative of the resistance to Lohmann's concept. In this article, the first to report on muscle contracting in a steady state of twitch force, the authors confirmed Lundsgaard's earlier observation (99) that ATP apparently did not undergo decomposition unless muscle was stimulated at a relatively high twitch rate until fatigued (131). Furthermore, this study demonstrated that although ATP was degraded under such circumstances, lactic acid also accumulated in large amounts. The authors reasoned, somewhat speciously, that these results were inconsistent with Lol'aaann's claim that ATP synthesis occurs at the expense of lactate formation. Despite lingering opposition, evidence began to mount in support of Lolmann's claim that ATP was the energy source closest to muscular contraction. Needham deduced this fact from a series of observations beginning with the apparently unique character of "adenylpyrophosphatase" (ATPase) in muscle: "Other phosphatases in muscle are, so far as it is known, very feeble and unimportant...Now experiments on muscle extract indicate that no enzyme is present for splitting creatinephosphoric acid into creatine and free phosphate, but only for catalysing its reaction with adenylic acid. . . If adenylpyrophospatase is really the only muscle phosphatase, it is clear that the phosphate must have come from this source..." (122) Additional suggestive evidence surfaced in 1939, when Bnglehardt and Ljubimowa established that muscle ATPase activity was so intimately intertwined with myosin as to be inseparable from it by any standard fractionation method (33). Hydrolysis of ATP was thus at least enzymatically possible at a site directly involved in the contractile 9:03.80 e In 1941, Fritz Lipmann proposed a novel chemical mechanism by 25 which phosphagen might store metabolic energy to be utilized not only for muscular contraction, but for many other cell processes as well (90). In this remarkable paper, Lipmann introduced the term 'high- energy phosphate bond' and 'group potential' and the symbol “ph (later rendered as *P). Despite the major advance represented by this idea, Lipmann persisted in identifying PCr as the primary direct energy source for contraction, hypothesizing that, " Pyrophosphatase (ATPase) operates rather like an outlet for the adenylic acid system to adjust the flow of ”ph in case of overproduction, much in the manner of a valve." Despite Lipmann's adherence to the notion of the primacy of PCr, Lohmann's earlier theory specifying ATP as the direct energy source for contraction gradually gained acceptance during the decade following Lipmann's phosphate energy transfer proposal. Lohmann's chemical experiments on muscle extracts (93, 94) were repeated by numerous others, and the results confirmed his reaction rate reports. This evidence was bolstered by the previously noted localization of ATPase activity to the contractile apparatus and by the apparent character of ATPase as the sole quantitative phosphatase in muscle. Eventually the growing body of chemical evidence convinced even skeptics such as Lundsgaard, who finally abandoned his earlier position (99), admitting that, "Most likely inorganic phosphate does not originate directly from phosphocreatine but from adenosinetriphosphate" (9B). Acceptance aside, the fact still remained that ATP hydrolysis in intact muscle had not yet been demonstrated during individual contractions or submaximal twitch series. This persistent doubt was rekindled by the venerable A. V. Hill in a 1949 letter to Nature (61). 26 Although Hill conceded the likelihood that "the energy of contraction is derived in the first instance from the breaking of the temminal energy- rich phosphate bond of ATP", he contended that the issue could not be finally laid to rest until ATP hydrolysis could be conclusively associated with contraction/relaxation rather than recovery. In this letter, Hill also offered some possible experimental approaches to achieving this goal. He advocated working at lower temperatures to slow reaction rates, and using tortoise muscles, noting that "... frogs' and rabbits' muscles are singularly ill-suited to the enquiry, they are much too quick ..." Hill elaborated on this theme the following year in his famous "Challenge to Biochemists", published in a special issue of fiigghg-iga g;_fligphygigg_¢g§g commemorating the 65th birthday of Otto Meyerhof. In this essay, Hill spoke of the new 'revolution' in muscle physiology in which ATP hydrolysis seized the role of the "fundamental change", supplanting the phosphagen which had engendered the revolution out of the "lactic acid era". Hill commented: "It may very well be the case, and none will be happier than I to be quit of revolutions, that the breakdown of ATT’really is responsible for contraction or relaxation: but in fact there is no direct evidence that it is." (62) Hill went on to speak somewhat disparagingly of in vitro work on muscle extracts, indicating his strong bias toward working on living muscle: "And I when I say muscle, I mean muscle: living muscle". Expanding on the suggestions advanced in his 1949 commentary (61), Hill put forth some ideas for new techniques involving metabolic poisons, different animal species, and other variations of method: "If one's instruments, or methods, are too slow, one can make them relatively quicker by using slower materials - tortoises, toads 27 or even sloths. That means, of course, that biochemists, like biophysicists, must also be biologists (as Meyerhof always has been and as Hopkins was) - but why not?" (62) Continuing on this topic later that same year, Hill stated, "Indirect evidence suggests that ATP occupies a key position in the chemical machinery of contraction: but it remains possible that its intervention is confined to the chemical process of recovery.” At this point, Hill conceded that even the modified chemical methods he had suggested earlier might be too slow and insensitive to resolve the question. As an alternative, he promoted the use of physical methods, particularly heat measurements. Lundsgaard (97) pointed out that labeled-phosphate experiments had verified that "ATP constantly is broken down and rebuilt in the intact muscle", although he did admit that it had not been shown that the rate of “P turnover in ATP was increased by stimulation. At a symposium chaired by Hill on the physical and chemical basis of muscular contraction, the Cambridge biochemist Dorothy Needham proposed a novel method for approaching the ATP 'challenge' . Crediting Herman Kalckar for development of the chemical techniques, Needham outlined in detail an 'enzymatic spectrophotometric' method for following ADP production rather than ATP loss for determining the extent of ATP breakdown during a single contraction or short series of twitches (121). The method depended upon a clever series of enzymatic conversions of the ADP released by ATP hydrolysis into a compound with a UV absorption maximum sufficiently distant from that of ATP so as to be differentiable from ATP even in minuscule quantities. Needham recognized that such a method held ”The great advantage that it aims at estimating a small increase from zero concentration”, in contrast to 28 attempts to measure a very small decrease in the relatively large muscle ATP concentration. Thus her method addressed the first of the two shortcomings, sensitivity and speed, of previous chemical methods as identified by Hill. Needham'was, however, clearly aware of the limitations of her proposed method, noting that its success depended upon the ability to instantly (and reproducibly) arrest the cellular enzymes at the height of the twitch and to minimize the effect of the "traumatic stimulus" on ATP breakdown. She also conceded that the rate of the creatine kinase reaction in vivo was unknown, and might exceed the time resolution capacity of the chemical methods. A variation of this experimental design was employed by Hommaerts and Rupp, who reported their results in a letter to HBSHIB in late 1951 (119). It is interesting to note that, although their methods adhered closely to the proposal of Needham, these authors failed to acknowledge ' her contribution even though their awareness of her work was evident from their citation of Hill's introduction to the symposium in which it was presented. In any case, Homerts and Rupp claimed to have shown using these methods that ATP was clearly hydrolyzed during contraction. However, close examination of their work reveals some serious problems which, taken together, render their results inconclusive. Pirst of all, application of simple statistical methods to the analysis of the raw data presented in this article shows that, although the ADP values showed a tendency to increase in the contracted muscle, this difference was not statistically significant (one way ANOVA, p >.05). Secondly, their assay methods were incapable of separating the unbound, 29 physiologically reactive fraction of the total ADP from the larger, bound portion. Furthermore, they did not measure PCr and therefore could not address Needham's concern (121) regarding the extent of rephosphorylation of ADP. Finally, their results suggested that ."precipitous cooling mobilizes the contractile apparatus to a somewhat greater extent than occurs physiologically in a single twitch.” Such an interaction between contraction and the freezing process would serve to reduce yet further the true contraction-induced difference in ADP levels between the control and stimulated muscle groups. ‘ The challenge posed by Hill in 1950 would stand without an ultimate solution for more than 10 years after Hoamaert and Rupp's efforts. Finally in 1962, Robert Davies and his stuudent D. F. Cain reported the results of a study using the recently discovered CK inhibitor 1-flouro-2,4-dinitrobenzene (FDNB), which had been shown by Kuby and Hahowald to completely inhibit creatine kinase in vitro (76). Previous work from Davies' University of Pennsylvania laboratory, incluuding Cain's 1960 doctoral dissertation, had provided the first conclusive evidence that PCr was ludrolyzed during a single contraction. Using the new inhibitor to block PCr breakdown, Cain and Davies were now able to finally and irrefutably demonstrate that ATP itself was also degraded within a single twitch ( 13). According to their results from - isolated, FDNB-treated frog muscles, 0.44 umol/g muscle of ATP was hydrolyzed per twitch. This figure agreed nicely with the 0.5 umol/g value predicted on the basis of an assumed thermodynamic efficiency (81) of 502 for the isotonic workload performed. The in vivo inhibition of CK by FDNB was confirmed by the fact that the PCr content of these stimulated muscles was the same as in resting, untreated controls. 30 Furthemmore, the importance of phosphagen's role in energy metabolism was illustrated by the 90! reduction in number of twitches produced before onset of fatigue. At last Hill had his proof, in "muscle: living muscle" that ATP hydrolysis was not confined to the recovery process of muscular contraction. BEYOND ATP: DEVELOPMENT OF CONCEPTS REGARDING ENERGY USE AND REPLENISHHENT The discovery of PCr in 1927 and of ATP in 1929, along with the 1934 proposal by Lohmann correctly identifying their relative positions in muscle energy metabolism, set the stage for the development of modern concepts of muscle physiology. The next three decades brought elucidation of the oxidative and glycolytic mechanisms by which the high energy phosphate pools are maintained and replenished, as well as precise characterization of the molecular mechanism by which ATP hydrolysis drives muscular contraction. These discoveries have been well chronicled: their treatment here will be limdted to a brief overview and citation of pertinent original studies and reviews. In E'I' l'lfll The regeneration of phosphagen in stimulated muscles allowed to recover in oxygen was described in the earliest reports on "labile phosphorus" (37, 38). A large step toward clarification of the mechanism for this oxidative regeneration of energy was made by Krebs and Johnson in the now famous 1937 m1; paper, in which "experiments are reported which ... allow us to outline the principle steps of the oxidation of sugar in animal tissues" (74). In developing 31 the concept of the tricarboxylic acid (TCA) cycle or Krebs cycle, as it came to be known, the authors keyed on the catalytic nature of the effect of citrate on respiration. Added citrate was observed to increase oxygen consumption, and this effect was "by far greater than can be accounted for by the complete oxidation of citrate.” The enhancement of respiration was even more pronounced if additional carbohydrate was added, prompting the researchers to ”presume therefore that the substrate the oxidation of which is catalyzed by citrate, is a carbohydrate or related substance." In an excellent integration of their results with previous reports by other researchers, Krebs and Johnson proposed a cycle involving citrate, malate, fumarate, oxaloacetate, succinate and o-ketoglutarate. Clever application of metabolic inhibitors illustrated the likely relative positions of the elements in this cyclic pathway. In addressing the question "from.which substance the two additional carbon atoms of the citric acid molecule are derived", the authors predicted that the answer was to be found in a trioae derivative such as pyruvate 01' acetate e We Kreb's citric acid cycle explained the mechanism of oxidation of the 2-carbon breakdown products of carbohydrate precursors, and of lipid substrate as well, but the connection between oxidation of foodstuffs and phosphorylation of ADP and Cr remained to be drawn. Finally in 1956, Britton Chance and G. R. Williams synthesized decades of results from a variety of sources into a coherent model joining oxidation of the ”dihydrodiphosphopyridine nucleotide" (DPNH, or nicotinamide adenine 32 dinucleotide (NADH)) produced by the TCA cycle to mitochondrial ADP phosphorylation (16). Citing the work on oxidases and cytochromes by Warburg, Keilin and others, Chance and Williams outlined the components of the electron transport system (ETS) and their likely relative positions in "the respiratory chain, the main pathway for the transfer of electrons or protons from metabolites to oxygen.” The authors recognized that: "Work along distinctly different lines has shown at least three properties of the respiratory chain to be of fundamental importance in then-etabolic and regulatory functions of the cell: (1) to transfer electrons or protons fromrsubstrates to oxygen and particularly to maintain the necessary level of oxidized DPN (NADI) within the aerobic cell: (2) to act as a sequence of three or more energy conservation steps by which ADP is converted to ATP so that the latter is available as a common medium for energy expenditure throughout the cell: and (3) to regulate the metabolism in accordance with the levels of control substances, for example, of ADP itself or of a hormone upon the rate or efficiency of the energy conservation process." (16) In addition to this characterization of the ETS, Chance and Williams reviewed the work on uncouplers of phosphorylation from oxidation in an attempt to uncover the mechanismnof the coupling of the ETS to ATPase activity. The results of these uncoupling studies led the university of Pennsylvania pair into ' ... postulation of new and presently unidentified high-energy intermediates which participate in the oxidative phosphorylation process." This interpretation, however, was to be rendered invalid by the work reported five years later by Mitchell. The Chance and Williams paper also reported extensively on their own work regarding steady states of respiration in isolated mitochondria. From these results, the authors defined five metabolic states of mfitochondria, each characterized by a distinct combination of substrate level, ADP level, respiration rate, and oxidation levels of 33 NAD and of specific ETS components. The experimental significance of these states was considered to be their utility as tools for examining ”the nature of comonents of the respiratory chain involved in oxidative phosphorylation." The remainder of this exceptional paper utilized work on transitions between the defined metabolic states of isolated mitochondria to formulate a theory of respiratory control. Chance and Williams proposed that control was achieved by reversal of an inhibition of respiration rather than by direct activation. The authors concluded that "ADP itself (is) probably the physiological substance responsible for the activation of respiration of biological systems following stimulation.” The evidence supporting this conclusion was, however, based almost exclusively on the results of experiments involving the addition of excess ADP to suspensions of isolated'liver mitochondria. As A.V. Hill might have cautioned, the direct application of the resulting model to intact, living muscle was perhaps premature. Chance and Williams did concede that "Several workers have suggested that the ATP/ADP ratio should determine the respiration rate of the mitochondria, but our experiments provide evidence that this is not the case." Curiously, none of the three papers cited as exemplifying the work supporting this opposing view did in fact directly address the issue of ATP/ADP as controller. Siekevitz and Potter reported on the establislment of experimental conditions in isolated mitochondria in which respiration rate increased despite lack of change in either ADP or ATP (125). These authors went on to suggest that ATP, ADP, and Pi all together "are able to regulate the oxidative rate in the mitochondria according to physiological need." They did 34 not, however, propose any mechanimm by which such control might occur. The Lardy and Wellman paper cited by Chance and Williams also proved devoid of arguments for respiratory control by ATP/ADP ratio, although it did claimythat "In general, rates of respiration vary inversely with the "P potential' against which the oxidative system must work (86)". The third paper offered by Chance and Williams as typical of this opposing view in fact dealt with oxidative phosphorylation in insect sarcosomes and primarily recounted methods of isolation and inhibition. No mention of respiratory control by either ADP or ATP/ADP was made, excepting this brief notation in an appendix: "Except at very low concentrations of ADP, (the rate of synthesis of ATP by oxidative phosphorylation) is independent of the concentration of ADP (89)". Even though these cited sources did not provide worthy examples of the exceptions taken to Chance and Williams' theory of respiratory control by level of phosphate acceptor, controversy regarding the nature of this control was indeed spirited. This debate persists even today, and provides one of the questions addressed by the research reported in later chapters of the present work. Details of alternative views of respiratory control are presented later in this chapter. Despite some continuing skepticism regarding the Chance/Williams concept of respiratory control, their work did undoubtedly greatly advance the understanding of electron transfer and oxidative pathways of energy production. A key issue remaining unsatisfactorily explained, however, was the question of how oxidation was linked to phosphorylation of ADP. 35 3 Q . I . I II . In 1961, Peter Mitchell proposed a novel mechanism whereby phosphorylation could be accomlished by either oxidative or photosynthetic electron transfer (116). This new concept was based on a comletely different approach than the orthodox view as chamioned by Chance and William, which postulated the existence of a 'high-energy intermediate' in order to reconcile oxidative phosphorylation with the known mechanism of substrate-level phosphorylation in glycolysis. In addition to the fact that these chemical intermediates were "elusive to identification", Mitchell listed a number of other shortcomings and inconsistencies of the chemical model. Among these points of contention were the failure of the prevailing model to explain the close association of phosphorylation with membranous structures or the shrinking/swelling phenomena associated with phosphorylation activity, and the fact that oxidation could be uncoupled from phosphorylation at three different sites and by agents possessing ”no identifiable specific chemical characteristic." To explain these inconsistencies, Mitchell proposed an entirely new mechanism based on the concept of group translocation: ”This type of mechanism differs fundamentally from the orthodox view in that it depends absolutely on a supramolecular organization of the enzyme systems concerned. Such supramolecularly organized syste- can exhibit what I have called chemi-osmotic coupling because the driving force on a given chemical reaction can be due to the spatially directed channeling of the diffusion of a chemical covenant or group along a pathway specified in space by the physical organization of the system." (116) The three basic features of Mitchell's model were a reversible ATPase system, an oxido-reduction system for transferring electrons and protons, and a charge-impermeable membrane across which these two systems 36 would be anisotropically located. The electron/proton translocator was proposed to create a proton gradient across this membrane. The resulting electrochemical potential would then provide the driving force for phosphorylation of ADP via a channeling mechanism resident in the ATPase component. This model yielded a number of testable predictions which withstood subsequent experimentation, as reviewed in 1979 by Mitchell in a lecture delivered on the occasion of his acceptance of the Nobel prize for chemistry (117). In sumaarizing the metabolic sylmetry inherent in his new model, Mitchell concluded his original paper as follows: "In the exact sciences, cause and effect are no more than events linked in sequence. Biochemists now generally accept the idea that metabolism is the cause of membrane transport. The underlying thesis of the hypothesis put forward here is that if the processes that we call cell metabolism and transport represent events in a sequence, not only can metabolism be the cause of transport, but also transport can be the cause of metabolism. Thus we might be inclined to recognize that transport and metabolism, as usually understood by biochemists, may be conceived advantageously as different aspects of one and the same process of vectorial metabolism." (116) W The molecular mechanism by which ATP hydrolysis drives muscular contraction was laid out in some detail by Davies in a 1963 treatise in Mn (22). Acknowledging the contributions of numerous other scientists, including Huxley's sliding filament hypothesis (development reviewed by its author (69)), Davies presented a molecular theory based on calcium-dependent interactions of "interdigitating filaments" of actin and main. The role of ATP hydrolysis in the development of tension was that of breaking the electrostatic bonds of actin-wosin crossbridges to permit "quantal contractions”. Muuscle studies throughout the remainder of the 1960's and 70's 37 dealt in large part with the long list of predictions enumerated by Davies in this paper. The results of the physical and chemical studies confirming and expanding upon the contractile energetics theory advanced by Davies have been recently reviewed in detail by Kushmerick (80). "It is usually open to the physicist or pure chemist to control and simlify the conditions of his experimental work, or wisely to avoid regions of comlexity uuntil collateral progress has made them siaple. In biology the complexities of the conditions are in the essence of the phenomena, and the experimentalist, when he tries to simlify them, is even viewed with suspicion. Thus even the operation of excising a muscle before studying its chemistry has been regarded with some prejudice..." Fletcher and Hopkins, 1915 (44) Until the mid-1970's, studies of metabolite changes in working muscle required sampling of discrete time points by the process of freezing, excision, and acid extraction of the muscles and completion of an individual chemical assay for each metabolite to be studied. With the development of biological applications of phosphorus nuclear magnetic resonance (31PM) spectroscopy, continuous monitoring of the intracellular levels of the energetically important metabolites ATP, PCr and Pi could be accomplished simultaneously in tissues and even in intact muscles in living animals and in human subjects. The words of A.V. Hill in heralding the development of the galvonometer at the turn of the century find a new and fitting application to the effect of NMR technology on modern-day muscle research: "(This new technology) has rendered fertile again a field of work which in (previous) days seemed barren by reason of poorness of methods” (63). The physiochemical phenomenon of NMR was first reported in 1946 by 38 Bloch (9) and Purcell (127), who shared the 1952 Nobel physics prize for their independent achievement of this discovery. Their work provided a new analytical tool for chemists, allowing the use of radio waves and magnetic fields to nondestructively study the structure and composition of chemical compounds. These initial chemical applications exploited the fact that a hydrogen nucleus placed in a strong magnetic field resonates between specific quantum energy states whose separation varies with the chemical environment surrounding that nucleus as well as with the strength of the applied field. Therefore a proton in a given molecular environment in a static magnetic field will absorb and emit radiofrequency energy of only the specific frequency corresponding to the energy quantumlseparating the resonance states. Each chemically distinct proton thus exhibits a characteristic resonance frequency by which the chemical entity within which that proton lies can be identified. Biological applications of this method surfaced with the refinement of NMR techniques directed toward the phosphorus nucleus. This method allowed the measurement of relative levels of phosphorus compounds present in millimolar amounts within the target sample. In muscle tissue, this includes the high energy phosphate compounds ATP and PCr, as well as inorganic phosphate but few other peaks to complicate the frequency spectrum. The first application of 31P-NMR to living cells was reported by Moon and Richards in a 1973 study of 2,3-diphosphoglycerate levels in erythrocytes (120). The following year Hoult, et a1, published the first 31P-NMR study of intact tissues (154). Descriptive studies of phosphorus metabolite levels in intact muscles were reported in 1976 by 39 Burt, Glonek, and Birany (11, 12). The first experimental studies of muscle contraction and recovery were communicated by Wilkie's group in 1977 (24), and scores of muscle studies followed. Entirely noninvasive studies of specific muscles became possible with the development of the surface coil technique by Ackerman, et al, in 1980 (1). Prior to this breakthrough, the restriction to a saddle- type coil configuration required excision or at least surgical isolation of the muscle for placement within the coil cylinder, or cross sectional studies of entire limbs affixed within such a coil. Although the muscle research field could not be properly characterized as "barren" prior to the introduction of this new method, the NHR technique does offer many advantages over classical chemical methods. The new method is not without its own drawbacks, however. Disadvantages of the NMR technique include the high cost and limited availability of the superconducting magnets, specialized probes, and computer systems required to collect and analyze NMR data, as well as the technical difficulties attendant upon operating equipment near such strong magnetic fields. The aforementioned sensitivity limitation precludes the direct measurement of metabolites present at concentrations Inch below 1 I14. Data analysis is also complicated by the need to standardize relative peak areas to some known concentration value in order to convert spectral integrals into absolute concentration units. The actual process of integrating the peak areas in the frequency spectrum also poses interpretative problems which have been addressed (109) but not yet entirely resolved. In surface coil experiments, care mat be taken in defining the location of the mscle volume from.which the sun signal arises (83). The standardization and 40 other technical difficulties have been sufficiently resolved to permit widespread use of the NMR technique for in vivo muscle studies (see Meyer (103) for review) but some caution must still be taken in evaluating and interpreting NMR studies in view of the analytical methods applied. Of primary importance among the advantages offered by the NMR method is the noninvasive nature of the technique. NMR studies provide information arising from.intact cells in a physiologically realistic setting, whereas classical chemical methods require gross disruption of the cellular membrane and substructures in preparation of the muscle sample for analysis. This shortcoming of the traditional method has long been recognized and was aptly described by Fletcher and Hopkins in 1917: "The inherent difficulty besetting the chemical examination of muscle lies, of course, in the fact that the necessary processes for extraction of the constituents cause in the moment of their application profound chemical change." (44) Needham also recognized early on the effect of the ”traumatic stimulus" of the freezing process on muscle phosphagen levels (121): the interaction of this stimulus with the contraction process itself has already been noted as a confounding factor in earlier experiments (119). Another major advantage of the NMR method is that it allows monitoring of metabolite levels at multiple time points before, during and after contraction of a single muscle. In contrast, the conventional method provides but one time point per animal. In addition to the obvious increase in practically obtainable time resolution and reduction in number of experimental animals required, this feature of the NHR method also substantially reduces statistical variability by allowing 41 comparison between successive time points within the same subject rather than the between subject comparison to which the classical method was limited. One practical consequence of this fact is that a muscle can be used as its own control in some NHR experimental designs (e.g. 107, 2). Time resolution on the order of 15-30 seconds is easily obtainable with existing an equipment and methods (e.g. 107, 101). Precision of timing can be increased yet further via gating methods which synchronize data collection to a cyclical event such as repetitive electrical stimulation of a mscle (24, 2). The m method also provides a very sensitive, accurate probe of intracellular p11, as demonstrated by the first biological application of phosphorus 101R (120). Because the resonant frequency of a peak in an M spectrum shifts with the association or dissociation of hydrogen ions from the molecule generating that signal, the position of the peak corresponding to a weak acid or base reflects the pH of the solution in which that comound resides. In 31PM spectra from muscle tissue, the position of the inorganic phosphate peak (pk. z 6.8) provides a very useful, noninvasive indicator of intracellular pH within the normal physiological range (83). Furthermore, the m method permits the simltaneous measurement of p11 and phosphorus metabolite concentrations. Although the free, metabolically active quantity of ADP in muscle cells is well below the threshold for 3lp-m detection in vivo (103), the free ADP concentration can be estimated from NPR-measurable parameters assuming equilibrium of the creatine kinase reaction (83). Chemical assays of ADP in mscle extracts measure a total ADP value on the order of 1 id, but this amount includes the large fraction of ADP that is bound tightly to macromolecules such as P-actin and is thus 42 metabolically unreactive. The free fraction of ADP represents less than 5% of this total (146). Estimation of free ADP via the CK equilibrium can, of course, be done using metabolite values obtained by traditional chemical methods (146), but such calculations generally overestimate [ADP] because of inflation of Cr values and reduction of PCr values (103) due to artifactual hydrolysis as noted above. Finally, use of magnetization transfer techniques allow direct measurement of the rates of exchange in chemical reactions. Whereas biochemical methods permit such measurements only in cell-free systems attemting to approximate intracellular conditions, the NMR method allows exchange rate analysis to be done within living cells in an intact organism (10). Numerous and extensive reviews have been made of the large body of metabolic information acquired over the past two decades using the m technique on living mscle. These include general reviews on M principles and biological applications (e.g. 49, 10), as well as reviews of specific areas such as applications to general tissue metabolism (151), metabolic control principles (15), and striated Insole metabolism (103). 43 CURRENT CONCEPTS IN MUSCLE ENERGY METABOLISM The modern mscle energy scheme incorporates all of the discoveries outlined in the previous sections. In this system, the direct source of energy for muscular contraction is ATP. Hydrolysis of ATP via actomyosin ATPase (reaction A below) provides energy to drive crossbridge movements resulting in sarcomere shortening. The inediate resource for regenerating the ATP pool is phosphocreatine, which acts via creatine kinase to rephosphorylate ATP (reaction B). The effectiveness of the creatine kinase system is such that, at all but the most intense workloads, ATP levels are maintained at an essentially constant level. The net reaction observed is therefore the hydrolysis of PCr (reaction C), as noted by LolmIann in 1934 (93): (A) ATP <====> ADP + P1 (ATPase) (8) Po“ + N? <====> Cr + ATP (creatine kinase) (c) (Net): PCr <====> Cr + P1 The total muscle phosphagen stores available vary somewhat by species and fiber type, but average roughly 5-8 m for ATP and 20-30 nil for PCr in mammalian skeletal Imiscle (e.g. 107, 83: see Table 1). Assuming an energy cost of 0.2 umol ~P/g‘muscle/t‘witch (68), the entire ATP/PCr pool would provide enough energy for roughly 100 twitches. unassisted, this pool could support the most intense workloads for only a few seconds, or a mild workload for only a minute or two even if the entire pool could be hydrolyzed. Augmentation of these immediate energy sources is accomplished by oxidative phosphorylation, involving the mitochondrial respiratory chain and mitochondrial ATPase and creatine 44 TABLE 1. Typical mtabolite concentrations (Incl/g) in selected mmmmelian.muscles at rest. Muscle Pi PCr Cr ATP free ADP rat gastroc 3 27 4 7.5 0.02 in situ (83) cat biceps 3 35 0.2 9 0.0003 isolated, perfused (107) cat soleua 10 17 8 5 0.014 isolated, perfused (107) kinase enzymes, and/or the cytosolic pathways of glycolysis/ glycogenolysis. The mechanisms by which the cell matches the rate of oxidative phosphorylation with changing ATP demand in working muscle has been studied extensively, but no firm consensus has yet been reached regarding the correct model of respiratory control. The experiments in this study have been designed to provide further information for discriminating between some of the proposed mechanisms outlined below. W The mass action ratio ([ADP]*[Pi])/[ATP] for reaction A above is maintained at a steady state level far from equilibrium.in the vicinity of myofibrils. In resting muscle, the reciprocal of this quantity, termed the phosphorylation state, is on the order of 1 X 106 11-1 (higher in fast, glycolytic fibers: lower in slow, oxidative fibers) (146). This comares to an expected value at equilibriu of 1.5 x 10'5. Consequently, a chemical potential of about 60 kJ/mole is available for conversion to mechanical work upon activation of the actomwosin ATPase. 45 Reaction B, on the other hand, is poised near equilibrium in muscle cells (103). Therefore the creatine kinase system can respond to any decrease in ATP/increase in ADP with a corresponding change in Cr/PCr. Response is very rapid because of the high catalytic efficiency and high muscle levels of creatine kinase, and very sensitive because of the steady state levels of the adenine nucleotides (see Table 1). In fast twitch muscle, for example, hydrolysis of just l/10 of 12 of ATP can cause nearly a 10 fold increase in ADP, resulting in a 90! drop in the ATP/ADP ratio. As mentioned above, the CK system on its own could serve to maintain ATP levels only briefly, particularly since it is apparently impossible for living cells to completely drain their phosphagen pools (143). The process of regeneration of ATP must then be supplemented by mitochondrial oxidative phosphorylation and/or anaerobic glycolysis and substrate-level phosphorylation. The question arises: what is the signal that tranmmits the myofibrillar energy need to the mitochondrial respiratory system? The simplest theory of respiratory control is perhaps the model in which availability of ADP to the mitochondrial ATP synthetase determines the rate of oxidative phosphorylation. According to this view, the rate of ATP synthesis would depend on substrate concentration in classic Michaelis-Menten fashion. This model, proposed by Chance in 1956 as noted previously, appears adequate to describe respiratory control in isolated mitochondria and has in fact been accepted by many as the mechanism operating in intact muscle. Indeed, this mechanism of respiratory control is presented as fact in many current biochemistry texts (e.g. 398). Some adherents of this kinetic theory postulate that 46 it is actually the diffusion of creatine fromxmyofibrillar to mitochondrial creatine kinase that carries the [ADP] information. This 'creatine shuttle' hypothesis (8) is examined in greater detail in the next section. Alternatively it has been argued that mitochondrial respiration is responsive to the thermodynamic entity [ATP]/([ADP]*[Pi]) (80). Although classical thermodynamics does not permit the prediction of reaction rates from concentration ratios, postulation of a near- equilibrium network of the reactions of oxidative phosphorylation and cytosolic energy metabolism allows use of non-equilibrium thermodynamic principles to provide a time factor link (14). According to this theory, metabolite ratios from other reactions in this network, most notably oxidation of mitochondrial MADE, will also affect the rate of rephosphorylation of ATP (73). Pluctuating intracellular calcium levels have also been implicated in respiratory regulation. Elevated free cytosolic Ca" levels link excitation of the muscle cell with crossbridge formation, ATP splitting, and contraction of the myofibrils. This 'calcium signal' can also affect the activity of enzymes such as mitochondrial dehydrogenases (56), thereby altering the rates of citric acid cycle flux and the nfitochondrial redox potential. Glycogen phosphorylase kinase activity is also increased by elevated Ca“, thus providing a link between contraction activation and glycogenolysis (100). 47 InnsIi9nel_lnle_2£_§hl_§E_Sllnll The‘most directly apparent function of PCr in muscle is to provide an energy store for maintenance of high ATP levels during periods of increased ATP turnover. This 'temporal buffering' effect is the result of thermodynamic considerations as previously discussed. It has also been posited that the CK system provides an essential diffusive link, or 'creatine shuttle' of ...P, between myofibrillar and mitochondrial adenine nucleotide pools. According to this view, the flux of ATP/ADP between these two compartments is limited by some diffusion barrier. Thus PCr/Cr diffusion is proposed to provide a "spatial buffer" between these two compartments and their localized CK and ATPases (8). A mathematical model developed by Meyer, Sweeney, and Kushmerick (112) demonstrated that the transport (spatial buffering) and classical (temporal) buffering functions of the CK system both could arise simply from the near-equilibrium state of the CK reaction. Physical compartmentation is therefore not required to explain the transport function of PCr/Cr. Thus the CK systemlis apparently not required for diffusive transport of ~P, but it can significantly increase the rate of this transport, thereby maintaining a given rate of diffusive flux with smaller spatial gradients of ATP and ADP than would otherwise be possible. This lessens the dissipation of the free energy of hydrolysis of ATP that would occur if the adenine nucleotides themselves carried all of the diffusive flux. The model equations demonstrated that this 'spatial buffering' aspect of the CK systemnwould likely be important only in relatively large cells with a low density of mitochondria (e.g. fast, glycolytic fibers). 48 In addition to energy buffer functions, the presence of a large store of PCr in muscle cells also allows the generation of a much larger increase in free inorganic phosphate for a given not drop in ATP. This feature of the CK system has several interesting implications. First of all, if the ATPase reaction occurred in isolation, a 1/101 decline in ATP would produce a decrease of about 892 in [ATP]/[ADP] and 901 in [ATP]/[ADP]*[Pi] as per the previous example using fast muscle metabolite parameters. With the creatine kinase system in place, however, even a very moderate workload might easily cause a 30! decline in PCr. In the fast muscle example under consideration, this amount of PCr hydrolysis could raise Pi 10-fold or more, resulting in a correspondingly larger reduction in [ATP]/[ADP]*[P1] than would have been possible without the CK system. ‘This lO-fold amplification of the thermodynamic signal could provide much finer control of respiratory rate if the near-equilibrium network theory is correct. The ability to generate large increases in Pi via the CK system could also affect metabolic regulation in several other ways. The CK reaction produces the divalent anion flP04", with a pK. of 6.8. At an intracellular pH of 7.0, this divalent Pi will take up protons until the ratio of mono- to di-protonated species reaches 1.6 (10(7‘5-3)). Thus for every mole of Pi produced via the CK reaction, 0.4 moles (l/(1+1.6)) of protons are consumed. The resulting drop in [8*] may be enough to relieve fl+ inhibition of PPK, thus accelerating the rate of glycolysis (80). The presence of this additional free phosphate also increases the effective buffer capacity of working muscle, as first noted by Piske and Subberow over 60 years ago (37). This increase serves to attenuate the 49 intracellular acidification that would otherwise occur when a high rate of anaerobic glycolysis causes significant production of lactic acid. Although numerous studies have demonstrated a correlation between intracellular acidification and fatigue (e.g. 132), more recent experiments have produced evidence indicating that it is diprotonated Pi rather than acidification per as that mediates fatigue (52, 152), perhaps by directly inhibiting the actoawosin ATPase complex (23). This is consistent with an earlier report that experimentally lowering intracellular pH to levels comparable to those produced by intense workloads did not significantly affect mscle force, provided that Pi was kept low (111). Recent NMR studies confirmed this dissociation of fatigue from acid accumulation, and also demonstrated fatigue generation independent of 82P04' levels (3). It has also been suggested that increased phosphate levels might induce Ca“ sequestration within sarcoplasmic reticulum (and mitochondria) by formation of a calcium phosphate precipitate within the organelle (75). This trapping of calcium could reduce the amount of excitation-induced calcium release, thereby reducing the contractile response. go ‘0! '1 "(Oxygen) is used presumably in some process whereby the molecular machinery of the Imuscle carries out oxidations for the storing of 'free energy'... analogous to the oxidation of coal in a steam engine in order to turn a dynamo and so to charge an accumulator - whereby free energy, the power of carrying out life-processes, is stored in the living tissue, which power, which free energy, may be utilised by the tissue whenever an appropriate stimlus is applied." ' A.V. Hill, 1913 (63) 50 Although the analogy suggested by Hill in 1913 referred to "the storing of potential energy ... in the conversion of lactic acid into glycogen" (115) according to the lactacidogen theory in vogue at the time, the "accumulator" concept was resurrected in later years and applied to the high energy phosphate scheme of muscle energetics. The first such application was outlined by Lipmann in 1941. He proposed a model based on an electric motor, in which phosphocreatine served as a "“P-accumulator”, ATP as the "wiring system”, and the oxidation- reduction system as the “metabolic dynamo" (90). This model was only used, however, as a schematic for illustrating "the machine-like functioning of the revolving sequence of reactions", and not as a tool for predicting the behavior of the system” Nearly half a century later, the qualitative and quantitative behavior of some aspects of muscle oxidative metabolism have now been successfully modeled by a simple electrical circuit analog (104). In this model, the creatine kinase system functions as a chemical capacitance (Pig. 1). PCr is represented by the charge accumulated on the capacitor, with capacitance (C) proportional to the total creatine level (Tc). The resistance component Rn depends on density and functional properties of the mitochondria and varies inversely with the oxidative capacity of the muscle fiber. The cytosolic free energy of hydrolysis of ATP is modeled as V0, and the mitochondrial value as the battery Vb. Current flow through circuit models the rate of hydrolysis/resynthesis of ATP or the flow of energy in the form of 'high-energy' phosphate (‘P). The ability of the CK system to temporally buffer ATP levels means that a work-to-rest transition, modeled as a step change in cytosolic ATPase rate, results in a new 51 Rm VW. i0 .1, avg Vb CY PICURE 1. Electrical circuit mdel. Key: V0 = cytoplasmic phosphorylation potential V}, = mitochondrial potential C = capacitance due to CK system R. = mitochondrial resistance term Icy = cytoplasmic ATPase activity (flow of “P) In. = mitochondrial ATP production rate (Prom Meyer (104)) steady state level of cytosolic free energy of hydrolysis not far from the original value of vs. The tenoral changes in PCr as modeled by this system will be monoexponential, with time constant for the RC circuit given by t=R.*C. Thus the model predicts that the time constant for PCr changes during transitions from one steady state (e.g. rest) to another (e.g. some submaximal contractile rate) will be proportional to the total creatine level for a given resistance value. According to this model, decreases or increases in total creatine content should result in respectively shorter or longer time constants for PCr changes in response to initiation or termination of a bout of moderate exercise, provided there is no change in the mitochondrial resistance value. The circuit-analog prediction that PCr time constants ought to vary linearly with total creatine content was tested recently in a study 52 utilizing the creatine analog B-CPA to induce Cr/PCr depletion in mscle cells (101). Since B-CPA-P is hydrolyzed only very slowly during submaximal mscle work, the replacement of Cr/PCr by analog results in reduction of the effective total creatine (Tc) pool. Utilizing feeding periods of 2, 4, 6, and 8 weeks, Tc was reduced in rat gastrocnemius mscle by amounts ranging from 40 to 901. 31P-NMR was used to follow PCr changes during stimulation at three submaximal (aerobic) rates and during subsequent recovery. The results show that PCr time constants did indeed scale linearly with Tc. This finding provides evidence against the creatine shuttle hypothesis, since a loss of Cr/PCr would result in larger spatial gradients of ATP and ADP and a substantial drOp in free energy of ATP if normal creatine content was a requirement for maintenance of normal ~P flux. This decreased deltaG(ATP) would be modeled as an increase in R. in the circuit model, thus countering the effect of a decreased Tc on PCr time constants. This RC circuit model can also be used to estimate the rate of energy use at the onset of stimulation. Utilizing the capacitative circuit analogy, the level of phosphocreatine remaining at time t after the onset of stimulation is given by For“) = Par“ 4- (Po-O - ”ssh-Vt where Pcr.. = steady state PCr concentration, PCro = initial PCr level, and time constant r = Rqu as noted earlier. The time rate of change of PCr is then given by g = gum. - Farah-Vt 53 The energy use in the first seconds of contraction will be reflected almost entirely by PCr hydrolysis, since glycolytic and oxidative mechanisms do not immediately contribute significantly to energy production (155). Therefore the value of the function dPCr/dt at t=0 should provide a good estimate of the initial energy cost of contraction. This application is used in the interpretation of experimental results in the fourth chapter of this paper. EXPERIMENTAL MANIPULATION 0P ADENINE NUCLEOTIDES One approach to resolving the issue of kinetic ([ADP]) vs. thermodynamic ([ATP]/([ADP]*[Pi])) control of oxidative phosphorylation rate is to study the effects of controlled alterations of these parameters in vivo. Muscle ATP can be depleted by ischemia (20, 27, 48) or by iodoacetate inhibition of glycolysis (25, 47), but these manipulations produce, respectively, structural damage or irreversible alteration of metabolic function and hence are unsuitable methods for respiratory control studies. Very intense work is also known to deplete ATP, but this effect is relatively short-lived (143). ATP stores depleted by heavy work can be prevented from recovering to normal levels, however, by blocking the purine nucleotide cycle (PRC). In rapidly contracting mscle there will be significant net hydrolysis of ATP to ADP, which in turn is converted to AMP via myokinase. AMP then enters the following series of reactions which constitute the purine nucleotide cycle (reviewed by Lowenstein (95)): 54 (A). MP + H20 ==== II'P + m3 (APP demuinase) (8). Ir? + asp + GTP ====> a»? + GDP + P1 (3»? synthetese) (c). M ====> MP + fuumrate (3MP lyase) Experiments by Meyer and Terjung (113, 108) used the drug hadacidin (N-formyl-hydroxyamdnoacetic acid: biochemistry reviewed by Shigeura (135)) to inhibit the PNC at step B. hadacidin has been shown by others (87) to produce very specific inhibition of sAMP synthetase. Infusion of the drug followed by intense stimulation was found to cause prolonged depletion of ATP levels by up to 502 in rat gastrocnemius (fast) muscle. PNC inhibition had no adverse effects on muscle perfommance at submaximal workloads, although the effect of the hadacidin-induced ATP depletion on subsequent work performance was not examined in these studies. In contrast to these results, recent reports of studies using the sAMP lyase inhibitor AICAr (5-amino-4-imidazolecarboxamide riboside: pharmacology reviewed by Eano (55)) indicated an apparent disruption of aerobic muscle function attributed by the authors to non-replenishment of Kreb's cycle intemmediates due to blockage of fumarate production at PNC step C (43). However, earlier reports of AICAr interaction with myriad other enzyme systems (e.g. 6, 55, 79) weakened the assumption of PNC specificity upon which this conclusion was based. Another method that has been used to deplete ATP in cardiac mscle is 2-deoxyglucose (2-DG) infusion. 2-DG is a glucose analog that, like glucose, is transported into cells and is phosphorylated by hexokinase. unlike glucose-6-P, 2-DG-P cannot be metabolized further, nor can it exit the cell once phosphorylated. It has been reported that this 55 results in the breakdown of ATP to AMP to adenosine and finally to inosine, which is released from the cell (78). The validity of this proposed mechanism for the total adenine nucleotide (TAN) depletion may be called into question, though, for several reasons. First, the dephosphorylation of AMP to adenosine by 5'-nucleotidase apparently does not occur to a large extent in non-ischemic conditions (48). Secondly the decline in ATP observed in the 31P-NMR spectra acquired in this study could be accounted for by a corresponding increase in IMP, but this change would have been masked by the large 2-DG-P peak which appears in the ammo spectral region as IMP. Finally, it is not clear that the chemical assay procedures used in this study were capable of discriminating between inosine and its corresponding nucleotide. In any case, a subsequent study applying this technique to a perfused rat heart preparation established that contractile function was not impaired despite 60-65! depletion of TAN (77). ATP depletion has also been observed in connection with creatine depletion induced by long-term feeding of creatine analogs to test animals (42, 137, 102). Although these studies uniformly reported unimpaired mscle performance at submaximal workloads, the biochemical and structural adaptations (136, 118, 134) that occur in response to this chronic treatment complicate any interpretations of these results regarding respiratory control. EXPERIMENTAL DESIGN AND RATIONALE The experimental portion of the present work consists of two separate studies. The first, as noted in chapter one, was undertaken to 56 investigate claims of adverse consequences of PNC inhibition on aerobic metabolism in muscle (43). This conclusion was judged suspect for three reasons. First, the drug AICAr infused in that study is known to interact with numerous other enzyme systems (e.g. 6, 55, 79) in addition to its action as an inhibitor of the final reaction in the purine nucleotide cycle. Secondly, the dose administered was extremely large (2.25 unol AICAr/100g body weight in a volume of 9 ml/100g, ip). This resulted in an intramuscular AICAr concentration of more than 1 IN (43, 46), far in excess of the amount required for maximal PNC inhibition (129). Finally, previous mscle studies using hadacidin, a drug recognized for the specificity of its action towards PRC inhibition (87), had demonstrated marked PNC blockage with no decrement in either aerobic or anaerobic performance (113, 108). To examine this question, three separate series of experiments were planned. In the first series, the experiment of Flanagan, et a1 (43), was replicated with the addition of procedures for monitoring systemic blood pressure, employed on the suspicion of possible systemic effects of the large doses of AICAr. The second phase consisted of another replication of the original experiment, conducted in this case inside an NMR spectrometer in order to monitor the levels of phosphorus metabolites and p11 during both stimulation and recovery. The final experimental series utilized isolated cat macles, perfused with control and AICAr solutions and studied via me during stimlation and recovery. The rationale for the use of isolated uncles was that, if the AICAr- associated decline in aerobic performance was indeed due to systemic effects of the drug, then the isolation of the Imuscle from the remainder 57 of the system should eliminate this effect on performance. The cat biceps and soleus mscles were chosen for study because of the suitability of their size for the saddle coil probe arrangement and because of the near homogeneity of fiber type, fast and slow respectively, in each mscle. The second of the two studies in this research plan called for the development of a method to induce and maintain significant ATP depletion in muscles, then for testing of the metabolic effects of this depletion on submaximal twitch and supramaximal tetanic stimlations. This study also consisted of three phases. In the first phase, anesthetized rats were infused with hadacidin and the right gastrocnemius suscle placed over a surface coil in an m probe. The distal end of the limb was then secured to a force transducer to measure isometric force production. The mscle was then electrically stimulated for varying periods of time while phosphorus metabolites were monitored via 31P-NHR to arrive at a protocol which would consistently produce and sustain a maximal depletion of ATP. The intense stimulation and hadacidn-induced PRC inhibition method for ATP depletion was selected because of the absence of confounding effects as noted in the preceding review. The in situ rat gastrocnemius model was chosen because of the existence of a large body of physical, chemical, and NMR data on that model, as well as the suitability of the animal size and mscle size and location for surface coil NHR studies in the available research magnet. The NH! method was incorporated into these studies for the reasons cited earlier, such as the noninvasive nature of the technique and the superior time resolution 31PM offers for both phosphorus metabolite levels and pH. Traditional chemical 58 methods were also employed in order to provide an ATP concentration reference for conversion of NMR peak areas into concentration units. The assay results also permitted calculation of total creatine content (PCr + Cr) upon which corrections for contraction-induced muscle swelling could be based. The second and third experimental series used the protocol developed in phase one to induce depletion of ATP, followed by an interval to allow recovery of other metabolites. In the second series, ATP-depleted muscles were subjected to low frequency twitch stimulations of sufficient duration to produce a steady state of force and metabolite levels. In the last series, the final stimulation bout consisted of trains of tetanic contractions of the same intensity used to induce the initial ATP depletion. The details of the experimental aims and.mmthods for each of these two major study areas are provided in the two following chapters. Additional background material is also introduced in the interpretations of the experimental results. III. UTILITY OP AICAR FOR METABOLIC STUDIES IS DIHIIISHED EY SYSTEMIC EFFECTS IN SITU INTRODUCTION The purine nucleotide cycle (PRC) can be viewed as a two-component process in which adenosine monophosphate (AMP) is first deaminated to inosine monophosphate (IMP), followed by reamination of IMP to AMP to complete the cycle. As described in the previous chapter, AMP deaminase (AMPDA) catalyzes the single reaction of the first component: APP + H20 ===> m: + mg. The subsequent regeneration of AMP requires two reactions catalyzed in turn by adenylosuccinate (sAMP) synthetase and sAMP lyase: er+asprtate+GrP===>sNP+®P+P1 sN‘P ===> NIP + funerate. Inquiries into the physiological role of the P80 in skeletal miscle energy metabolism have focused primarily on three possible functions reviewed by Lowenstein (95): l). removal of AMP to maintain the ratio of ATP to ADP and AMP: 2). production of a-onia to accelerate glycolysis by activation of phosphofructokinase: and 3). deamination of aspartate, producing fumarate to replenish the supply of Krebs cycle intermediates, thereby enhancing aerobic energy production. Operation of the first arm of the cycle alone is sufficient to carry out the first two functions. Production of fumarate, however, requires a coqlete turn of the cycle and coincident regeneration of AMP. Experiments by Tornheim and Lowenstein in 1972 on rat mscle 59 60 extracts demonstrated that the generation of ammonia by deamination of AMP occurred during ATP consumption, while reamination of IMP to AMP occurred when conditions favored ATP resynthesis. This prompted the authors to divide the PRC reactions into phases corresponding to 'energy drain' and 'energy excess'(l45). A 1977 study by Goodman and Lowenstein using both in situ and perfused rat hindlimb preparations showed that relatively intense stimulation protocols were necessary to cause a net reduction in muscle ATP levels and trigger the production of IMP. Little evidence was found for the cycling of purine nucleotides during work (52). Clinical interest in the PRC was sparked a year later by Pishbein's report on the discovery of a muscle disorder in humans characterized by the absence of AMPDA, the first enzyme in the cycle. Some victims of myoadenylate deaminase deficiency (MDD) exhibited slight muscle dysfunction during exercise of sufficient intensity to cause lactate accumulation, although others who were identified as MDD by genetic screening had shown no symptoms at all (36). The author speculated that the basis for this mildly impaired function was the inability of MDD muscle to utilize the PNC to maintain a high ATP to ADP ratio by removal of AMP. A.more recent clinical finding links reduced sAMP lyase activity and resultant accumulation of sAICAr with infantile autism syndrome (84). Several recent studies have utilized blockers of the second component reactions to assess the physiological importance of the anaplerotic role of the PRC in muscle. Studies using hadacidin (N- formyl-hydroxyaminoacetic acid), which inhibits the binding of aspartate to sAMP synthetase, indicated that IMP formation occurred only during 61 relatively intense stimulation, while reamination to AMP was largely delayed until the recovery phase after a burst of contractions (113). Furthermore, production of IMP was shown to occur primarily in fast- twitch glycolytic muscle fibers rather than in fibers with high oxidative potential (108, 116). These studies suggested a serial mode of operation of the two-component cycle, consistent with proposed PNC functions 1 and 2. In contrast, evidence in support of parallel operation of the PNC components was obtained using the compound 5-amino- A-imidazolecarboxamdde riboside (AICAr), which is phosphorylated in vivo by adenosine kinase to AICA ribotide (AICAR), an inhibitor of the sAMP lyase reaction (129). In these studies, intraperitoneal administration of AICAr was associated with a marked impairment of the capability of rat gastrocnemius muscle to maintain twitch force during in situ stimulation at 0.75 Hz (43), a rate known to be within the steady-state aerobic capacity of normal rat hindlimb muscle (68). Assuming that the effects of AICAr are confined to sAMP lyase inhibition in muscle, this result suggests that the second arm of the PRO is required for normal aerobic energy production, i.e., that fumarate production is an important physiological role of the PNC in skeletal muscle. The present study was designed to clarify the effects of AICAr infusion on rat skeletal muscle. The results suggest that the previously reported effects of AICAr on muscle force were secondary to effects on systemic arterial pressure and that AICAr has no direct effect on muscle energy production during mild stimulation. 62 METHODS Three separate series of experiments were performed. In the first series, adult male Sprague-Dawley rats weighing 270 t 10 g (SE, n=l6) were anesthetized with pentobarbital sodium (50 mg/kg, ip) and prepared for in situ stimulation of the gastrocnemius muscle essentially as described by Flanagan, et al (43). Three exceptions were made to their reported procedure: a catheter (PB-50) was inserted into the carotid artery for measurement of arterial blood pressure, muscle temperature was maintained at 37°C with a heat lamp throughout the experiment, and stimulation was begun immediately following the 28 minute ip infusion period to ensure a consistent infusion volume (9 ml/100 g, the average infusion volume per animal in the Flanagan report) of either isotonic saline or 250mM AICAr (Sigma Chemical: total dose, 2.25 mmol/100g). Reviewing the procedure in brief, an ip catheter (PB-50) was inserted for delivery of AICAr or saline, followed by insertion of the carotid catheter. Prior to infusion the right gastrocnemius muscle was exposed and prepared for stimulation via the tibial nerve. During infusion, the distal end of the mscle was connected to a force transducer, and the length of the muscle was adjusted to give a maximal isometric twitch in response to a supramaximal pulse (8 V, 5 ms). AICAr was infused into 10 rats, two of which died during infusion or stimulation and were therefore excluded from the data analysis. A control group of eight rats received an infusion of isotonic saline. Immediately following the infusion period, the muscle was stimulated for 10 minutes at 0.75 Hz. The gastrocnemius muscles of both the stimulated and nonstimulated legs were then immediately clamp-frozen using metal tongs precooled in liquid nitrogen. The frozen samples were stored at 63 -80° C until extraction. The presence of AICAr and AICAR in the muscles of the AICAr- infused animals was verified by high resolution 1H-NMR spectroscopy of extracts of the frozen muscles. The frozen samples were pulverized in a mortar under liquid nitrogen, then extracted in cold alcoholic perchloric acid (96). These extracts were diluted 20:1 with deionized water and treated 4 times each with 5g Chelex-IOO (BioRad Laboratories) to remove metal ions (82). The chelated product was brought to a pH of 7.0 t 0.1 using dilute HCl and NaOH solutions, then lyophilized to dryness. The resulting precipitate was redissolved in 1 ml of deuterium oxide (1)20 or 2H20) and transferred to a 5m NMR tube. The use of 020 as a solvent eliminates the extremely large 1H peak due to water (1H20) that tends to obscure other proton peaks in normal aqueous solutions. Standard solutions were prepared by dissolving AICAr and AICAR (Sigma Chemical) in a volume of water equal to the diluted muscle extracts, then chelating, titrating, lyophilizing and redissolving in 020 as per the muscle extracts to give a final concentration of 100mM for each compound. Proton NMR spectra of extracts of 6 nonstimulated muscles (3 each, AICAr- and saline-infused), 4 stimulated muscles (2 each group), and the 2 standard solutions were acquired at 400 Hz in a Bruker AM 400 spectrometer while spinning the samples at 25 Hz. Chemical shifts are referenced to the deuterium.hydroxide (0H0) peak, assigned a value of 4.80 ppm. Following data acquisition on muscle extracts from the experimental animals, a small amount of the AICAR standard solution was added and the spiked samples reanalyzed. This procedure was then repeated using the AICAr standard. 64 The second phase of the study utilized a protocol identical to the first, except that the rats were mounted head-down in a customrmade NMR probe as described previously (102), and muscles were stimulated for only 5-6 min. In brief, catheters (PB-50) for AICAr/saline infusion and for supplemental anesthesia (4:1 saline:pentobarbital sodium (50 mg/ml)) were inserted into the peritoneal cavity of the anesthetized rat, and an intraarterial catheter placed as described above. The right sciatic nerve was exposed via a small incision on the lateral aspect of the hip. The nerve was then ligated and cut and the distal and placed in a bipolar platinumnelectrode. The nerve and electrode were then reinserted into the tissue pocket, using a small strip of Parafilm laboratory film for insulation from surrounding tissues. Cyanoacrylic glue was used to close the incision and secure the position of the electrode. The rat was then positioned in the NMR probe with the knee secured to a mount via a length of copper wire threaded beneath the patellar ligament. The achilles tendon was tied with copper wire to an isometric force transducer located at the top of the probe and mounted on an adjustable frame. Adjustment of the muscle length to optimize isometric twitch tension placed the belly of the gastrocnemius muscle directly over a 1 cm diameter surface coil. Phosphorus NMR spectra were acquired at 162 MHz in a Bruker AM400 7.3 cm bore magnet during infusion and during and after stimulation. A tube delivering 100! oxygen gas was placed near the animal's head to ensure sufficient oxygen delivery within the enclosed probe while inserted in the magnet bore. Eight rats weighing 262 t 23 g (88) were tested, four each receiving AICAr and saline infusions as described above. Carotid pressure was monitored in three of the four animals in each treatment group. 65 In the third experiment, 31P-NMR spectra and twitch force were recorded from isolated, arterially-perfused cat muscles before and after addition of AICAr to the perfusate. Adult cats (3.5-4.0 kg) were anesthetized with ketamine (33 mg/kg, ip) followed by pentobarbital sodium (30 mg/kg, ip). The biceps brachii (a representative mixed fast- twitch muscle) and soleus (slow-twitch) muscles were isolated with their associated vasculature and the muscles were perfused with a suspension of sheep red blood cells in Krebs-Hanseleit solution at 30°C as described previously (107). The isolated muscle with attached platinum electrodes was mounted on a force transducer and its length adjusted to give a maximal isometric twitch in response to a supramaximal pulse (15 V, 1 ms). The suspended muscle was inserted into a 20 mm diameter Helmholtz (saddle) coil in a specially designed NMR probe for acquisition of 31P-NMR spectra at 162 MHz. Spectra were obtained before, during and after 6 minutes of stimulation at 0.2, 0.5 or 1 Hz during infusion of control perfusate. AICAr was then added to the perfusate (2.5 mM net AICAr concentration), and after 30 minutes the same stimulation was repeated. One soleus and one biceps muscle were studied, each from a separate animal. It is important to note that each muscle serves as its own control in these experiments. Statistical comparisons between groups were by Student's t-test at the p<0.05 level. Results are expressed as means 1 38, unless noted otherwise in the text. RESULTS Wm Excluding the two AICAr-infused rats that died before the experiment could be completed, there was no significant difference in Force (fraction of mum!) 66 1.2‘[ 1'2'T 1‘ l lofll. :‘3 ”FRI FL \ g I \§\\I ° ”I I #5:: '6 “f {\s I c: : l \ t\r 0.8 9 0 6+ ‘0‘ Cf I ‘5 I L ‘ e a a 0-4' , O—O Control 3 04+ 0—0 Control A O- - O AICAr u a O- - I AICAr 0.2 g 0.2} a. 0.01 + *fi t ; t t t : f 4 0.0 L : t 4 L : : - %fi‘ 0 1 2 3 4 5 8 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time (min) Time (min) FIG!!! 2. Peak twitch force in rat gastrocnuins muscle in situ. A: 1st experimental series, animals horizontal, n = 8 per group. Initial force was 1280 t 95 and 1040 t 160 g in saline- and AICAr-infused, respectively. 8: 2nd series, animals head-down in NMR probe, n = 4 per group. Initial force was 550 t 55 and 525 f 55 g, respectively. A 120 Intusxon ' sum. T: 140 T Inlumn : sum]. t . h . ' v \ ‘ I f V [ . ' ; . o 2 100 \ 0 0‘0 a 120 ! i O/ . a \ L 1 a I I /u a x r 1\ : 0“”0—0—0—‘3 90 I : T~ .- 2 “°t.\ L l ' I ' I I o. ‘ , t ' I L - 80 \I r ’3’ I a. 100 I i : O—DControl E .‘ ' ‘.' ‘ L . i a 3 70 1 * ' O—OConu-ol '3 90 + . , t e- - encu- z A . E I .‘~_ ”T‘s I I T s 60 . .“'.MCM a no 3 r ‘r.’_-‘. § 5° L ‘ f 2 = = 5 7o 4 a ‘. ‘ 10 20 so 40 0 1b so do 40 Time (min) Time (mm) FIGIRE 3. Mean arterial pressure in rats during infusion and stimulation. A: 1st experimental series, animals horizontal, n = 8 per group. 8: 2nd series, animals head-down in NMR probe, n = 3 per group. 67 fraction of initial twitch force at any time during stimulation in AICAr vs. saline-infused animals (Figure 2A). However, a marked difference between groups was noted in arterial pressure patterns (Figure 3A). Mean arterial pressure in the AICAr-infused rats averaged nearly 20 Torr below those of the control group by 10 minutes of infusion and remained significantly different from control throughout the remainder of the infusion period. Again excepting the animals who died during the experiment, this difference in pressure tended to resolve towards the end of the infusion period, and was totally resolved by the end of stimulation. In the AICAr animal that died (i.e. zero arterial pulse pressure) near the onset of stimulation, twitch force declined to 132 of initial by 5 minutes of stimulation, and to 22 by 10 min. Proton spectra (Figure 4) of muscle extracts of the AICAr-infused animals showed two series of peaks (7.57,7.54,7.52 ppm, and 5.74,5.72,5.70 ppm) closely corresponding to a single peak and a doublet, respectively, in the AICAR (7.58, 5.71/5.70) and AICAr (7.53, 5.71/5.70) standards. These peaks were present in three of three nonstimulated and two of two stimulated muscles of AICAr-infused animals and were not evident in any of the extracts from control animals. Identification of these peaks as AICAR and AICAr was confirmed by spiking the extracts with standard compounds. In the downfield series, AICAR addition caused a selective increase in the amplitude of the 7.57 ppm peak, whereas AICAr added selectively to the 7.54 and 7.52 ppm peaks, but not to the 7.57 ppm peak. Addition of either compound caused an increase in amplitude of all 3 peaks in the upfield series. The splitting of AICAr and AICAR resonances when added to the muscle 7 extracts presumably reflects conformational changes in these molecules 68 Y I F 0.3 no 7.: m 0.3 0:0 :15 M. Tweak—Zn I r *1 T f 1 T 8.3 0.0 7.5 7.0 ..S ..O 3.3 FIGURE 4. ln-m spectra from extracts of rat mscles. Spectra are from perchlorate extracts of nonstimulated gastrocnemius muscles from saline infused (A) and AICAr-infused (B) rats. Peak assignments: a, b, and f, adenine nucleotides: c, AICAR: d and e, AICAr: g, h. and i, both AICAR and AICAr. Inset: Change in expanded regions of spectra before (bottom) and after sequential addition of AICAR (middle) and AICAr (top). (4000 scans, 1 second interval, sweep width 4 kHz, 16k data.) 69 due to interactions with other substances in the extracts. series. The single peaks at 8.59 and 8.31 ppm and the doublet centered at 6.20 ppm have been identified as corresponding to proton resonances from adenine nucleotides (34). If an average total adenine nucleotide content of 8 m is assumed (107), comparison of the integrals of the adenine nucleotide peaks with those of the AICAR peak at 7.57 ppm yielded an estimated AICAR concentration of 0.5 t 0.2 mM (n=3) in the nonstimulated muscles of AICAr-infused animals. This estimate is in good agreement with the value of 0.554 1 0.058 mM measured by high performance liquid chromatography (HPLC) in the Flanagan study (43). The concentration of AICAr averaged roughly twice that of AICAR, as indicated by the greater size of the 7.54 ppmupeak (Figure 4). Resonances from lactic acid (1.36 ppm) and creatine-phosphocreatine (3.08 ppm) were also easily resolved in proton spectra of the same extracts. Assuming a total creatine content of 41 mM (83), lactate concentration in the nonstimulated muscles of saline-infused animals was 4.5 t 0.90 mM (n=3), vs. 9.3 f 2.01 mM (n=3) for the AICAr group. mm In AICAr-infused rats mounted head-down in the NMR probe, mean carotid pressure declined from 10717 to 83:5 Torr (n=3) in AICAr infused rats after 28 minutes of infusion, but was stable (1181:15, n=3) in saline-infused animals (Figure 3B). In contrast to the first experiment, this pressure difference persisted during the stimulation. Furthermore, by the end of the infusion period but before stimulation, 31P-NMR spectra (Figure 5) showed a significant increase in the ratio Pi:PCr in muscles of AICAr-treated (0.1910.04, n=4) animals compared with controls (0.11t0.03). Despite these differences, there was no ACONTROL m 70 ATP gamma alpha nets 3. AICAR ~s “WM now It I «10.0 FIGURE 5. 31P~NMR spectra from rat gastrocnemius muscle in situ. Spectra acquired before (bottom spectrum), during (next 5 spectra) and after 0.75 Hz stimulation of a saline infused (A) and an AICAr-infused (8) animal. Each spectrum is average over one minute (20 scans, 3 second interval, sweep width 7 kHz, 2k data). 71 significant difference in twitch force between the two groups after 5 minutes of stimulation at 0.75 Hz (Figure 28). Phosphocreatine resynthesis after stimulation was dramatically impaired in the muscles of the AICAr-infused rats compared to controls (Figure 5). However, upon removal from the NMR probe, the hindlimbs of the AICAr-treated animals were clearly cyanotic compared to saline-infused controls, suggesting that the failure of recovery was due to reduced hindlimb blood flow in the AICAr-treated animals. Wheel”; Perfusion with AICAr had no effect on the pattern of stimulation- induced PCr changes in either the isolated biceps brachii (Figure 6) or soleus muscle (Figure 7). Muscle function, as indicated by twitch force, was likewise unaffected by AICAr perfusion (Figure 8). ACONTROL EAICAR ‘II' 7 We In“, I t T 9.0 0.. -IU.U .26.. '0. 0;? .‘0'.. I -260. "I "I FIGURE 6. 31m spectra from isolated cat biceps miscle. Spectra acquired before (bottom spectrum), during (next 3 spectra) and after stimulation at 0.2 Hz. (A) is before and (B) is 30 minutes after the addition of 2.5 mM AICAr to the perfusate. Bach spectrum is an average over two minutes (40 scans, 3 second interval, sweep width 7 kHz, 2k data). 72 acowma. 8. MAR a", It II! I.“ III I.» I.- I .é‘ " a; Q I o V V V I ‘s. .0. ".e. ."e. "I ”a "is. m "a FIGURE 7 . 312-“ spectra from isolated cat soleus miscle. Spectra acquired before (bottom spectrum), during (next 3 spectra) and after stimulation at 1 Hz. (A) is before and (8) is 30 minutes after the addition of 2.5 mM AICAr to the perfusate. Each spectrum is an average over two minutes (40 scans, 3 second interval, sweep width 7 kHz, 2k data). 1.5- l.4<[ AICAr ,.m--m--m 1.3 fl , zl' Biceps. 0.2 Hz .’ u_——o———o———a l..2 I I I O 0 T\ ,,,,,, 0 g, V ‘ 0 4b E o I- “ 20 4+ < 2'0" 0 e : t t : t 0.0 r : c : t 0 30 60 90 120 150 180 0.0 0.5 1.0 1.5 2 0 2.5 30 TIME (seconds) ““6 (min) | 35.0 1 C 3 D 7.21 f? 0.01 0—0 Control .5 1 ,_ 7.0 a _‘ 25.01 e ----- e ATP-depleted a ‘ o 2000‘ 6.8%- > D“ E 15.0 0 6.6 4» m 100 o 6‘ ‘- 8 5. . 6.24 o r < ------- sTm ----------- > 0.0 c 5.0 : : ; t : t A. : 0.0 05 10 1.5 20 25 .3 0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 5.0 3.5 4.0 TIME (min) TIME (min) um 15. Peak ismtric tetanic force, ATP, phosphocreatine, and pH in control and ATP-depleted muscles during and after final tetanic Iti-Illt i“ e A: Peek isometric tetanic force. (Percent of initial force at the start of the first series of tetani applied at the beginning of the depletion protocol (see Pigure 11A)). 8: ATP. (Calculated from [OR spectra as described in text.) C: Phosphocreatine. (PCr levels are calculated from 1.! spectra as described in text.) D: Intracellular pH. (Calculated from ch-ical shift of Pi (120)). Points are means : SB, n = 5 muscles per group. 104 (C) acid load (ml/9) = pH * B + [PCr] '1' a (102) The coefficient a represents the molar proportion of hydrogen ions consumed during net PCr hydrolysis and is calculated from the pK.'s of Pi and PCr (6.8 and 4.6, respectively) to be 0.69 at pH 6.50 (final pH of stimulated control muscles) and 0.54 at pH 6.79 (ATP-depleted muscles). The result of this calculation suggests that the control muscles produced more than twice as much acid (presumably lactic acid) as the ATP-depleted muscles (32 vs. 15 umol/g) during tetanic stimulation. Applying the same formula to data from the twitch IIE. experiments produced a similar relative difference, although the acid load during this moderate stimulation was much lower (2.3 umol/g in controls, 1.2 in ATP-depleted muscles) compared to during tetanic stimulation. DISCUSSION mm 15.. specific hypothesis which inspired this study was that, if muscle respiration is feedback-regulated primarily by cytoplasmic ADP, then acute depletion of ATP and total adenine nucleotide ought to result in decreased PCr at any stimulation rate. This prediction followed directly from the asst-ed equilibrium of the creatine kinase reaction (equation A), which is commonly used to calculate free ADP in muscle (103, 146). This specific hypothesis was apparently negated, even considering only the results of muscles at rest, before the final 105 stimulations (Table 3). PCr was not lower in the ATP-depleted muscles: in fact it was slightly higher than in controls. Likewise, pH in the resting, recovered muscles did not increase, but rather showed a slight decrease. Thus, the calculated ADP content in ATP-depleted muscles at rest was less than half that in the control muscles. Although we have not measured oxygen consumption in this study, it does not seem plausible that the resting oxygen consumption of the ATP-depleted muscle could be less than half that in the controls, considering that PCr levels were stable in both groups, and that intracellular pH was only slightly decreased by ATP depletion. 0n the other hand, the calculated phosphorylation potential was essentially identical in the two groups. Thus, the straightforward interpretation of the results in resting muscles is that phosphorylation potential rather than ADP per se is the regulated metabolic variable (104, 112, 80). The results during twitch stimulation also clearly favor phosphorylation potential rather than ADP as the parameter controlling respiration. The twitch rate used was previously shown to be predominantly supported by aerobic metabolism in normal rat fast twitch muscle (68), a conclusion confirmed by the low acid accumulation observed in both groups in this study. During this stimulation, in which both groups developed the same force, calculated ADP in the ATP-depleted muscles barely exceeded ADP in the control muscles at rest. In contrast, changes in phosphorylation potential were similar, although not identical, in the two groups. Considering this result, together with other recent observations (100) and model calculations (125), the hypothesis that skeletal muscle respiration is controlled in a simple Michaelis-Menten fashion by cytoplasmic ADP seems clearly untenable. 106 However, there is an unanticipated and remarkable feature of our results which complicates their application to the simple hypothesis discussed above. The results strongly suggest that the energy cost of isometric force development was decreased by the ATP-depletion protocol. mm During the twitch experiment, the estimated initial rate of PCr hydrolysis in the ATP-depleted muscles was roughly half that in the F‘— controls, despite the fact that twitch force was identical in the two h groups. It is unlikely that anaerobic glycolysis made a significant contribution to ATP supply in either group, considering that pH became initially alkaline, and that even after 8 minutes only a small acid load had accuumulated. Therefore, the initial rate of PCr hydrolysis provides a good estimate of the ATPase rate associated with the twitch contractions. In the control muscles, the initial rate was 6.95 umol/g/min, which (for 0.75 Hz or 45 twitches/min) corresponds to 0.15 umol PCr hydrolyzed/g/twitch. This compares reasonably well with the ATP cost per twitch estimated from steady-state oxygen consumption measurements in rat hindlimb muscle by Hood, et a1 ((68), 0.22 umol/g/twitch, assuming P:O ratio = 3). In contrast, the initial rate of PCr hydrolysis in the ATP-depleted muscles was 3.19 umol/g/min, corresponding to an ATP cost of only 0.07 umol/g/twitch. This remarkable result is confirmed by the dramatic difference in acid accumulation during tetanic stimulation. Although final force deveIOpment in the ATP-depleted muscles was slightly higher, these muscles accuumulated less than half as much acid as controls during this supramsximal stimulation. Furthermore, the initial rate of PCr . 107 hydrolysis was less, and there was no further loss of ATP, in the ATP- depleted muscles. The most straightforward interpretation is that the energy cost of tetanic contractions was substantially decreased in the ATP-depleted muscles compared to controls. A potential problem with this interpretation is the fact that the m spectra are recorded from only an ill-defined superficial portion of the muscle (83), whereas force is recorded from the entire gastrocnemius-plantaris group in our preparation. For example, it might be argued that the superficial fibers were for some reason less activated after the stimulation/recovery protocol, and that this problem is more serious in the ATP-depleted group, thereby attenuating the measured metabolic changes. However, three points argue against this possibility. First, and most obviously, forces were not significantly different in the two grouups. Second, the metabolic results in control muscles during the final stimulation are very similar to many previous results obtained in the same rat preparation without any prior stimulation protocol (114, 113, 68, 103). Finally, the PCr time constant data is not consistent with this possiblity. PCr time constant measurements naturally reflect only active fibers, wherein PCr is changing. If the superficial, predominantly fast twitch glycolytic fibers were not contracting, then the observed time constants would necessarily reflect a greater contribuution from the deeper, more oxidative fibers. Thus, the time constant, which is inversely related to oxidative capacity (104), should have decreased had the superficial fibers been preferentially inactivated. It should also be emhasized that the apparent change in muscle energy cost could not be due to the administration of hadacidin per se. 1r 108 Hadacidin was previously shown to have no effect on PCr hydrolysis or lactic acid accumulation during twitch or tetanic stimulation of rat muscles which were not previously stimulated to deplete ATP (113). This result was confirmed by our pilot experiments. Depletion of the phosphocreatine content in rat muscle by chronic feeding of the creatine analog B-guanidinopropionate (BGPA) is also accompanied by decreased ATP and total adenine nucleotide contents (137, 42). The chronic ATP depletion observed during BGPA feeding is similar in extent to that induced acutely in these experiments. Surprisingly, supramaximal (5H2) twitch stimulation of creatine-depleted muscles had effects remarkably similar to those observed during tetanic stimulation of ATP-depleted muscles in this study, i.e., higher steady- state force development, decreased net phosphagen and ATP utilization, and dramatically decreased acid accumulation, compared to control muscles (102). These changes accompanying creatine depletion have been attributed to increased mitochondrial content (53) and/or to altered myosin expression (118). Of course neither of these adaptations could have occurred during our acute ATP-depletion protocol. It is possible that the chronic ATP depletion which occurs during BGPA feeding is at least partially responsible for the observed metabolic changes. Unfortunately, we know of no mechanism which can account for this effect of ATP depletion on ATP utilization. Lowered ATP use has been observed under other conditions, for example, during single prolonged isometric tetani in mouse muscles (19) and during contractions at low temperatures (126). In addition, ATPase activity has been correlated with the intrinsic speed of shortening in comparative studies of muscles from vertebrate and invertebrate animals (7). However, to our 1F 109 knowledge, there is no evidence that variations in ATP content in the 4- 8 mM range influence contraction kinetics in skinned fibers, or ATPase rates in isolated myosin. Another logical possibility is that the increased IMP in the ATP-depleted muscles somehow modulates cross-bridge activity. Finally, it is conceivable (70, 71), although unlikely (112), that the ATP remaining after the ATP-depletion protocol is not primarily free in the cytosol, and hence that ATP is kinetically limiting for myosin ATPase. Previous studies have demonstrated a correlation between ATP content and the energy cost of maintaining isometric tension in different fiber types in the hamster (51). This suggests an energy cost reduction mechanismlbased on an increased efficiency (energy cost for maintenance of tension) rather than altered economy (energy cost for development of tension) as the terms are commonly defined (81, 67, 21). In any case, whatever the mechanism, our results suggest that high nucleotide content is an important factor supporting the high ATPase rate of fast twitch muscle. A prediction emerges from this conclusion and remains to be examined: the maximum velocity of shortening ought to be reduced in ATP-depleted muscles. .9that_Bf£a£§s_2£_filf_nenlasienu ATP depletion was associated with some other interesting and unanticipated effects. First, the time constants for PCr changes during and after twitch stimulation were about 50! longer than in the control muscles. According to an electrical analog model of muscle respiration (104), PCr time constants during submaximal stimulation are inversely dependent on muscle aerobic capacity. Thus, our results suggest that aerobic capacity was significantly decreased by ATP depletion. This 110 could be viewed as a result of inhibition of IMP reamination, and hence of fumarate production (95), rather than of ATP depletion per se. However, this inferred decrease in aerobic capacity was not associated with greater fatigue during stimulation in the ATP-depleted muscles, apparently because the contracting ATP-depleted muscles used less ATP than did controls contracting at the same rate and with the same peak force. In this context, the smaller change in phosphorylation potential in ATP-depleted muscles during twitch stimulation is still consistent i with the proposal that phosphorylation potential is the key regulator of respiration. On balance, less respiratory drive was required to maintain the lower ATP turnover in ATP-depleted muscles. Another surprising effect of the ATP-depletion protocol was the staircase effect observed during repetitive tetanic stimulation. This effect occurred in both groups during the second of the two initial tetanic stimulation bouts, but only in the ATP-depleted group after the intervening recovery period. Therefore, the staircase cannot be a direct effect of hadacidin treatment per se. A staircase of peak force is commonly associated with repetitive low frequency twitch stimulation in fast twitch muscles, as illustrated in Figure 13A of the present chapter and in Figure 2 of chapter three. However, this pattern of potentiated force has to our knowledge never been observed to occur in a series of tetanic trains as reported here. The mechanism for this unique observation is obscure, although recent studies of twitch potentiation in mouse skeletal muscle have uncovered a possible link between this staircase effect and the level of phosphorylation of myosin light chains (124). Along this line, it is interesting to note that in rat gastrocnemius muscles chronically depleted of PCr, this twitch lll potentiation disappeared (102). Inasmuch as this treatment also produced an ATP depletion similar in magnitude to that produced acutely in the present experiments, it would be of interest to determine whether this chronic ATP depletion is associated with the tetanic potentiation phenomenon reported in our acute experiments. If so, the mechanism of the two potentiation phenomena would likely be of different origin. Finally, there was a slight but significant decrease in r“ intracellular pH associated with ATP depletion. This could arise by many mechanisms. One possibility is that the high IMP content in ATP-depleted muscles results in higher basal rates of glycogenolysis and lactic acid production by activating glycogen phosphorylase (18). On the other hand, it is apparent that IMP is not the sole, or even a major determinant of lactic acid production during tetanic stimulation, inasmuch as high IMP was already present at the onset of stimulation in the ATP-depleted muscles, and yet these accumulated much less acid than control muscles. In sumnary, acute depletion of ATP from rat fast twitch muscle results in metabolic changes consistent with decreased ATP turnover during isometric contractions. Despite this surprising complication, the results are consistent with the proposal that phosphorylation potential, rather than ADP alone, is the cytoplasmic factor which regulates respiration in muscle. ”Research is not a systematic occupation but an intuitive artistic vocation." A. Szent-Cydrgyi, 1963 (142) The first portion of this research addressed the problem of conflicting claims regarding the importance of the purine nucleotide cycle in aerobic energy metabolism in skeletal muscle. The experiments reported in chapter three using the drug AICAr resolved this question satisfactorily. The aerobic performance decrement attributed to PNC inhibition in previous studies (43) was demonstrated to be more likely E caused by systemic effects of the particular drug AICAr being used as a PHC inhibitor in those experiments. The significance of this finding is twofold. First, future investigations of PNC function in vivo should not rely on the drug AICAr as a PRC blocker because of these confounding effects. Secondly, the use of verifiably selective PHC inhibitors to study muscle energy metabolism now cannot be questioned on the grounds of interference with aerobic pathways on the basis of the previous AICAr report (43). This conclusion directly impacted the design of the principal research plan for this thesis, relying as it did on the use of the selective PNC inhibitor hadacidin to produce an in vivo model of ATP depletion in muscle. The primary question being addressed in this dissertation was: Is kinetic control (by amount of the phosphate acceptor ADP) of the rate of oxidative phosphorylation sufficient to explain the regulation of mitochondrial respiratory rate in contracting skeletal muscle? 112 113 The results reported in the previous chapter clearly indicate that control by ADP alone is unlikely, and that the theory of thermodynamic control via the regulatory parameter [ATP]/([ADP]*[Pi]) is more consistent with these observations. In addition to this main outcome, some other rather surprising and unexpected observations arose from these studies. First, depletion of ATP was associated with an apparent reduction in energy cost of muscular contractions. If the association is confirmed as a true cause-effect relationship, this result may be of value in interpreting the findings of previous studies in which ATP depletion was but one of several variables associated with an experimental manipulation (e.g. 102, 53, 118). Testing of the prediction of reduced maximum shortening velocity in ATP-depleted muscles should help resolve this question. Further studies directly measuring oxygen consumption in ATP-depleted muscles are also suggested by these results. The apparent reduction in oxidative capacity, as inferred (104) from the lengthened time constants of PCr changes in ATP-depleted muscle, is another interesting but as yet unexplained finding. The reduction in acid accumulation in ATP-depleted muscle, suggesting reduced glycolytic/glycogenolytic activity, is also consistent with reduced energy cost of contraction. Both of these issues should be examined to determine the mechanism by which ATP depletion produces these results. For example, examination of mitochondria from acutely ATP-depleted muscle may reveal if oxidative capacity is in fact reduced, thereby providing another test of the predictive capacity of the circuit model ( 104). Studies of glycogen content in ATP-depleted muscle would help determine if the decreased glycolytic flux is due to substrate 114 limitation or is more likely caused by enzyme inhibition. Finally, the staircase or potentiation of tetanic force observed in these studies is a phenomenon that has not been previously reported in the muscle literature. The discovery of the potentiation phenomenon in the second of two tetanic trains separated by only a short rest interval was a serendipitous result of trying a varierty of methods to achieve the goal of maximum ATP depletion. 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