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ATP utilization associated in anaerobic frog muscle with recovery ROBERT R. DEFURIA AND MARTIN J. KUSHMERICK Department of Physiology, Harvard Medical School, Boston, anaerobic metabolism; muscle energetics; recovery energetics LABORATORY HAS FOUND an apparent P/O ratio Of 2 in frog sartorii, Rana pipiens, at 0°C during the metabolic restoration of ATP previously utilized during an isometric tetanus (34). This measured value was significantly lower than predicted from observations of optimally coupled mitochondria. Similar results were obtained for single sartorii isolated from Rana temporaria at 0°C and from Rana pipiens at 20°C (unpublished data). These data have two simple biochemical interpretations: a) there was significant suprabasal ATP utilization after mechanical relaxation, during metabolic recovery, possibly due to metabolic substrate cycling (3,8, 9, 32, 40); b) the low P/O ratio could be interpreted as partial uncoupling of oxidative phosphorylation in the muscle mitochondria (7, 13, 36). The experiments described in this paper were designed to find out if there was significant suprabasal ATP breakdown in frog sartorii during the metabolic restoration of ATP utilized during prior contraction. Our experiments rely on the fact that anaerobic glycolysis is the primary pathway for energy metabolism in our preparation; the experiments to be described suggest that this is true. Our strategy is based on the fact that the ratio of the suprabasal ATP synthesized by simple Embden-Meyerhof glycolysis and the suprabasal lactate produced during this recovery period is 1.5. That is, 3 net mol of ADP are phosphorylated when 2 mol of lactate are produced to give a P/lactate ratio of 3/2 (38, 39). We compared an experimentally determined P/lactate ratio with this known value of 1.5. ATP utilized during an isometric tetanus and suprabasal lactate proOUR c30 IWassachusetts 02115 duced by anaerobic sartorii during recovery from a tetanic stimulation were measured. Our experimental P/ lactate ratio was taken to be the quotient of these numbers. The total ATP turnover is thus equal to the sum of the ATP utilized during the tetanus and any suprabasal ATP utilization occurring during metabolic recovery. Since the muscles are frozen just after relaxation of isometric tension, the quick-freezing technique only measures ATP turnover during the tetanus. Thus, an experimental P/lactate ratio of less than 1.5 suggests that suprabasal ATP utilization occurred during the metabolic recovery period. Suprabasal ATP turnover associated with tetanic contractions after mechanical relaxation has not been found (11, 20, 26, 31). However, these studies were concerned with the possibility of ATP splitting associated with tetanic processes occurring during and shortly after mechanical relaxation and were not concerned with the broader issue of ATP breakdown above the basal rate associated with metabolic recovery. Estimates of P/lactate ratios have been previously made (1, 6, 29, 46). Interpretation of such data depend on the metabolic properties of the muscles used. In order to interpret our experimentally measured P/lactate ratio, we tested the following characteristics of our preparations: a) no significant ATP was resynthesized by glycolysis or oxidative phosphorylation during the tetanus; b) no significant ATP resynthesis by oxidative phosphorylation occurred during metabolic recovery; c) recovery did occur in anaerobic muscles, such that the muscle content of ATP, creatine phosphate, creatine, inorganic phosphate, and lactate was not altered by a stimulation-recovery cycle; d) lactate was the primary end product of this anaerobic recovery, and glycogen was the source of this lactate; e) total lactate collected in the bathing medium was an accurate measure of the amount of lactate produced by glycolysis during a recovery period. PROCEDURES, METHODS, AND EXPERIMENTAL DESIGN Rana pipiens were kept at 10°C in tap water for 2-10 wk. Pairs of sartorii were dissected the day before an experiment and left overnight at 0°C immersed in airequilibrated Ringer-phosphate buffer: 115 mM NaCl, 2.5 mM KCl, 3 mM Na,HP0,/NaH,P04, pH 7.1, 1.8 mM CaCl,, 1 mM MgSOd, and chloramphenicol 50 pglml. While at O”C, each muscle was subjected to two brief isometric tetani to test its stability. The first (2 s) was Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017 DEFURIA, ROBERT R., AND MARTIN J. KUSHMERICK. ATP utilization associated with recouery metabolism in anaerobic frog muscZe. Am. J. Physiol. 232(l): C30-C36, 1977 or Am. J. Physiol: Cell Physiol. l(1): C30-C36, 1977. -The ATP turnover during single isometric 0.5, 1-, and 3-s tetani in anaerobic frog sartorii was measured at 20°C. In parallel experiments, we measured the lactate production by sartorii during their metabolic recovery from such tetani. From these measurements, we obtained an experimental P/lactate ratio which was less than 1.5, the expected value for simple Embden-Meyerhof glycolysis when glycogen is metabolized to lactate. We interpreted these results as indicating a substantial amount of suprabasal ATP breakdown occurred during metabolic recovery. The operation of the fructose phosphate substrate cycle is suggested as a cause of this additional ATP breakdown. metabolism RECOVERY ATP c31 UTILIZATION Recovery of Added above baseline loo~5 Et =,;; 2 8o 6o+ ; Fa -z $ 40-J I3 ” t 5” ;i I2 l l a.mmm* Lactate l .memm* -@*mm 1 I IO ; .**m ** ****a-* 7 4 : IO added 20-A OO Observed l l gE” g= Fraction *mm*mam 1 l mm*l mmm.0 l mm** I I 20 FRACTION , 30 l *@ 1 Lactate Predicted 43.2 23.3 t2 5 6’ 3.6 1:: ? .mm*m@ L 40 1 I 50 NUMBER FIG. 1. A: base line of lactate production by an unstimulated muscle in a N,-bubbled bath at 20°C, Number of nanomoles of lactate in each serial lo-min fraction is plotted. Precision of lactate assay is equivalent to 1 nmol per fraction. Volume of muscle bath was 5 ml. Affluent and effluent flow rate was 0.307 ml/min. Dry weight of for these assays were calibrated with spectrophotometrically determined concentrations of NADH. Glycogen was assayed by the method of Handel (16). Our assay standards and lactate dehydrogenase (L/2500), malic dehydrogenase (4lO-l3), cu-glycerolphosphate dehydrogenase (G6751), were obtained from the Sigma Chemical Company; the identifying numbers in parentheses are catalog numbers. After most experiments, the dry weight of the muscle tissue was obtained by dessication for at least 24 h under vacuum over phosphorus pentoxide. All the data for tissue contents reported are normalized to the dry weight of the tissue, wet Sometimes only blotted weights or a total protein by the method of Lowry et al. (28) was obtained. The conversion factors were independently measured and were: 4,2 blotted wet wt/g dry wt and 764 mg protein/g dry wt. Our experiments were designed to measure the net suprabasal lactate produced by anaerobic muscles during metabolic recovery from a prior 0.5, 1-, or 3-s isometric tetanus, We also measured the ATP utilized by muscles during 0.5-, I-, and 3-s isometric tetani. The ratio of these two members is an experimental P/lactate ratio which is to be compared with 1.5, the known value of the P/lactate ratio of Embden-Meyerhof glycolysis when glycogen is metabolized to lactate. Recently, Scopes (38, 39) has assembled the glycolytic enzymes in vitro along with phosphorylase, adenylate kinase, creatine kinase, and AMP deaminase and found that 3 mol of ADP were phosphorylated when 2 mol of lactate were produced, verifying the P/lactate ratio of 1.5. Although there is the possibility of uncoupling carbon flux from ADP phosphorylation in glycolysis (for example, the hydrolysis of the acyl intermediate of glyceraldehyde dehydrogenase), which would lower the experimental PI lactate ratio, there is no evidence that these kinds of processes occur (38, 39). Another metabolic simplif”lcation of our preparation is that, although there are some Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017 soon after dissection; the second (1 s) on the morning of the experiment showed no deterioration. The muscle bath was brought to 20°C while the bathing solution was bubbled continuously with either O,-free N, or CO (99.99%). When required, the muscles were stimulated isometrically at 10 V and 40 Hz to evoke a fused tetanus (27). Muscle length on the apparatus was set approximately to the in vivo rest length by loading with 0.005 N tension. The ATP turnover during a tetanus was measured according to the protocol of Kushmerick and Paul (27) with the quick-freezing technique and assays previously described (24, 25-26). In this paper, we report measured APCrlg dry wt because no net breakdown of ATP was observed (see Table 4) and because there were no differences between APCr/g and A - P/g dry wt. The calculated values for A - P/g dry wt relevant to Table 4 are for series A: 12.0 t 0.9, 15.4 t 1.1, and 36.2 t 2.2 (for 0.5, 1.0, and 3.0 s tetani, respectively) and for series B: 14.1 t 0.9. These muscles were frozen 1 s after the isometric tension fell to the base line. Some muscles were allowed to recovery anaerobically. The lactate producti .on of’ those muscles was measured with a flowthrough apparatus where the effluent of the muscle bath was pumped to a fraction collector. The lactate content of each fraction was measured. The volume of the muscle bath was kept constant by maintaining an affluent flow rate from a Ringer reservoir equal to the effluent rate. Approximate values for the muscle bath volume were 4 ml and for the flow rates 0.3 mllmin. Pump stability was checked periodically by measuring the weight of individual fractions at constant bath volume; however, bath volume and flow rate varied slightly from one experiment to another. The average flow r&e of a particular experiment was determined from the weight of the reservoir before and after the experiment and the total duration of the experiment. Figure lB shows data from a control experiment done -periodically to test the flow stability and precision of the apparatus used to measure the lactate production of our isolated sartorius preparation. The suprabasal lactate content of each fraction after the injection can be predieted for a given muscle bath volume, amount of injetted lactate, flow rate, and fraction collection time. These values were compared with experimental values. These control experiments also test the validity of the base-line subtraction procedure which was used to obtain the suprabasal lactate content of each fraction. The base line content of each fraction collected during the injection recovery period was estimated by a linear extrapolation of the base line of fractions before and after the recovery period. The suprabasal content of each fraction was taken to be its total lactate content minus this base line estimate. Data in Table 3 and Fig. IA give further justification for this procedure. In order to measure the tissue content of glycogen and other relevant metabolites (Tables 1 and 2), muscles were quickly denatured and solubilized by immersion into 5 N NaOH at 100°C for 5 min; this solution was then chilled. Lactate, pyruvate, malate, a-glycerol phosphate, and alanine were assayed fluorometrically by standard enzymatic methods (18, 21, 37). The standards c32 R. R. DEFURIA conflicting views concerning the existence of the pentose phosphate pathway in muscle, this pathway would not contribute to the metabolism of any anaerobic tissue (15, 35, 40). TABLE 1 1 1 I I 1 20 30 40 50 60 70 FRACTION NUMBER 2. Basal lactate production of a muscle in a NP-bubbled bath at 20°C is interrupted by a l-s isometric tetanus at indicated points. Data are plotted as in Fig. 1. Total suprabasal lactate production after each stimulation is given above data points. Muscle bath volume was 3.8 ml. Flow rate was 0.295 mllmin. Fractions were collected every 8 min. Dry weight of muscle was 25.0 mg. FIG. l*mm*555 l nonomoles k l l *” 2nd L 20 30 CO from N2 bubbled change 10 siimulotion, 40 FRACTION NUMBER I 50 1 60 No. of animals 11 240 0.5% XL 1.2 5 2 0.7% 2 1.3 5 21 2.5% 4 5.0 5 Pyruvate 4 3.1% + 5.1 5 Alanine 6 0.6% k 7.0 5 Malate a-Glycerol phosphate units) Muscle content of glycogen, lactate, pyruvate, a-glycerol phosphate, malate, and alanine in aerobic sartorius muscles at 20°C after overnight storage (see METHODS). Mean values for all muscles are given in the first column, The second column is the average percentage differences between members of a pair. The last column gives the number of muscle pairs used. cogen depletions are summarized in Table 2. To test whether lactate accumulation accounted for the glycogen depletion when the muscles were stimulated, the experiment summarized in Fig. 4 was made. The data in Tables 1 and 2 and in Fig. 4 show that lactate production accounts for most of the glycogen metabolized in our preparations. The data in Table 3 show that the muscle content of ATP, creatine phosphate, creatine, inorganic phosphate, and lactate is not altered by a complete tetanusrecovery cycle, The measured ATP turnover during a 5-s tetanus at 20°C for aerobic sartorii was 72.0 k 4.8 (SEM; n = 10) pmol/g. In sartorii treated for 30 min with 0,5 mM iodoacetate and made anaerobic with NZ, the ATP utilization for an identical tetanus was very similar, 68.4 5 5.3 (SEM; n = 10) pmol/g. Table 4 shows the amount of ATP turnover during tetanic stimulation durations of 0.5, 1, and 3 s and the amount of suprabasal lactate produced during metabolic recovery from such tetani. Our experimental P/lactate ratios are determined from these two quantities and are given for each tetanus duration. All experimental P/ lactate ratios were less than 1.5. DISCUSSION -b-- t x 100 2 SE to co both 1 70 FIG. 3. Basal lactate production of a muscle in a N,-bubbled bath at 20°C was interrupted by a l-s isometric tetanus at point indicated. Experiment is repeated after changing to a CO-bubbled bath. Data are plotted as in Fig. 1. Suprabasal lactate production after each stimulation is given above data points. Muscle bath volume was 5 ml, flow rate was 0.380 ml/min with lo-min collection fractions. Dry weight of muscle was 47,O mg. If there was no suprabasal ATP utilization during the metabolic recovery, then the ATP utilized at the end of a tetanus by rapid-freezing techniques should have been equal to the high-energy phosphate resynthesized by glycolysis. The latter quantity is given by the suprabasal lacate production times 1.5, the known stoichiometric factor for the metabolism of glycogen coupled to ATP synthesis in the simple Embden-Meyerhof glycolytic pathway. This prediction is made because the metabolite contents were found to be unaltered by a stimulation recovery cycle (Table 3), and lactate is the only significant end product of the anaerobic recovery (Tables 1 and 2, Fig. 4). One way of comparing the tetanic ATP turnover with the amount of suprabasal ATP re- Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017 1 IO - Right/Left + 2.4 Glycogen (glucosyl -m&W- Left Mean muscles 0.6% Figure 2 shows data from a typical experiment designed to measure the suprabasal lactate produced by our preparations of isolated sartorii during metabolic recovery from a single isometric tetanus. Data for 0.5, l-, and 3-s tetani were obtained similarly in a series of muscles, and these data are summarized in Table 4. The data in Fig. 3 show that for a l-s tetanus the suprabasal lactate production is the same when the muscle is bubbled with 100% N, or 100% CO. The contents of relevant metabolites were measured in aerobic muscles stored overnight at 0°C (Table 1). Other paired muscles were dissected, treated similarly, and divided into experimental and control groups. The control muscles were treated exactly as those muscles summarized in Table 1. Experimental muscles were made anaerobic at 20°C and were incubated without stimulation in Ringer containing 1 mM NaCN for 25 h. The metabolite content of those muscles was then measured and compared with the initial content in a pairwise fashion. The accumulated metabolites and the gly- noncmoles J, KUSHMERICK 24 Lactate 269 M. uf unstimulated 1. Metabolite content Mean Content, prnol/g dry wt RESULTS 287nonunoles AND RECOVERY ATP c33 UTILIZAT1ON TABLE 2. OfglyC0gU-t Co??ZJIarisOn and metabolite depletion production Production Expt No. Lactate a-GIycerol phosphate Pyruvate pmollg 1 2 3 4 5 169 218 124 171 166 3 7 7 8 1 -15 -13 -9 -12 -13 Depletion Malate Glycogen glucosyl units Alanine dry wt -1 2 3 0 4 4 -1 1 0 -1 92.0 113.6 58.5 79.3 84.9 The total production of lactate, pyruvate, ar-glycerol phosphate, malate, and alanine is compared with the total glycogen depletion in five experiments. Paired muscles were used. The experimental member of each pair was incubated without stimulation at 20°C for 25 h in Ringer solution containing 1 mM NaCN. The control members of the pairs were treated as in Table 1. The data in this table are the differences in muscle content, experimental minus control. Total Lactate Production p moles Clycogen I Total in Glucosyl p moles lO* t l Om l l l -h l T-4 Depletion Units I 2nd stimulation 1st stimulotion V t IO begin N2 20 30 FRACTION 40 50 60 70 NUMBER FIG. 4. Total lactate produced by a muscle (right of a pair) at 20°C in a Ne-bubbled bath during two l-s tetanic stimulation and recovery periods is compared with its glycogen depletion during same total period. Data for lactate content in each fraction are plotted as in Fig. 1; sum of those quantities during whole anaerobic period is total lactate production. Glycogen content of control (Eeft) muscle at beginning of experiment was 233 pmol/g. Glycogen content of experimental (right) muscle was determined at downward arrow. Glycogen depleted during anaerobic incubation was difference between control and experimental muscle contents. Muscle bath volume was 3.3 ml and flow rate was 0.279 ml/min with 8-min collection fractions. Dry weight of muscle was 24.3 mg. at least 3 h prior to stimulation. Our results differ in that no significant resynthesis of ATP occurred during tetani .c contractions of up to 5 s durations at 20°C because ATP turnover in aerobic as well as i.odoacetatepoisoned, anaerobic preparations were the same. The data presented in Figs. l-4 indicate that the behavior of our anaerobic muscle preparation is predictable and reproducible. In Fig. 2, the suprabasal lactate production after a l-s tetanus was repeatable to within 6%. We found an average basal lactate production of 109 t 10 (SEM: n = 16) nmol/g per min, similar to those values reported by Karpatkin et al. (22). Since muscle lactate content was unaltered by a stimulation recovery cycle, Table 3, and the lactate content of fractions collected after recovery could be predicted from prestimulation collections, we conclude that a) there is an increase in the rate of lactate production by the muscle during the course of an experiment; b) this increase in the rate of lactate production did not influence our measurement of the amount of lactate produced. The data presented in Fig. 3 indicate that the suprabasal lactate production in N, and CO after a l-s tetanus is the same. It is known that CO is a potent and effective inhibitor of oxidative phosphorylation (41). Since we have found that the ATP breakdown in first and second tetani is the same (unpublished data) and because it is not expected that CO altered the extent of metabolic recovery (shown to be complete in our N,-bubbled preparation), these data suggest that there is no significant resynthesis of ATP by oxidative phosphorylation during metabolic recovery in our standard N,-bubbled preparation. Consistent evidence is that our average basal lactate production in an N,-bubbled bath was similar to that in 1 mM NaCN Ringer, 112.9 t 10 (SEM: n = 5) nmol/g per min; NaCN is known to block oxidative phosphorylation completely (41). The data shown in Table 3 indicate that a stimulation recovery cycle did not alter the cellular content of ATP, creatine phosphate, creatine, inorganic phosphate, and lactate. This suggests that metabolic recovery was complete and that bath lactate was a measure of the lactate produced during the recovery period. It has been reported that anaerobic frog gastrocnemii may undergo only partial metabolic recovery. This was presumably because the muscles were in a N, gas phase and intracellular lactate was forced to accumulate (1). The data presented in Table 1 indicate that at the beginning of an experiment, the content of glycogen, lactate, pyruvate, a-glycerol phosphate, malate, and alanine in both members of a pair of sartorii are the same. This fact allowed us to do the type of glycogen depletion experiments whose data are given in Table 2 which show a carbon balance, The lactate content determined in the controls for these experiments (Table 1) was substantially greater than those shown in Table 3, because of different experimental manipulations and analytical techniques. The result of this glycogen depletion experiment was that most of the glycogen metabolized can be accounted for by lactate production when the unstimulated sartorii were incubated in I mM NaCN Ringer. This conclusion is consistent with the data shown in Fig. 4 where the lactate production by our Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017 synthesized by glycolysis is to compare the experimental P/lactate ratio with the known stoichiometric factor 1.5. The experimental P/lactate ratios obtained for different tetanic durations in two series of experiments are lower than 1.5 (Table 4). Thus, there was suprabasal ATP utilized during the metabolic recovery after a tetanus. The amount of this additional ATP turnover can be appreciated if the data are further analyzed as in the bottom of Table 4. Here it is seen that the calculated additional ATP turnover during recovery is always significantly different from zero and is a significant fraction of the ATP turnover actually measured during the isometric tetanus. Moreover, the calculated additional ATP turnover during recovery is about one-quarter of the estimated base-line ATP utilization during the entire recovery period. Our measured ATP turnover at the end of an isometric tetanus in anaerobic frog sartorii is the same order of magnitude as that reported by Canfield, Lebacq, and Marechal (4). The experiments are not exactly comparable, however, because our preparation was anaerobic for c34 TABLE R. R. DEFURIA 3. Anaerobic recovery of initial AND Mean dry wt Fractional Differences 2 &q/n* 57.0 (51.0) 45.6 (50.5) 42.0 (44.5) 42.3 (40.7) 38.0 (35.8) -0.002 k 0.012 99.9 (96.4) 123.8 (122.1) 130.3 (129.6) 147,2 (147.2) 127.0 (118.0) 139.3 (138.4) -0.001 * 0.003 Creatine 73.3 (69.7) 94.2 (89.9) 65.1 (72.6) 54.6 (59.7) 57,l (59.7) 56.5 (58.2) 0,003 k 0.009 ATP 11.8 (11.1) 22.8 (19.6) 17.1 (21.5) 17.2 (18.7) 18.6 (18.6) 18.5 (18.3) 0.007 f 0.029 3.5 1.2 (1.4) 5.3 (5.3) Creatine phosphate phosphate Lactate 7.6 2.8 (6.9) (2.4) (3.2) 1.9 (2.1) The ATP, creatine phosphate, creatine, inorganic phosphate, and lactate contents of left and right sartorius from was subjected to a l-s isometric tetanus and allowed to recover for 4 h at 20°C in a N,-bubbled bath. Its contralateral but was otherwise treated identically. Chemical contents were obtained for each pair at the end of the recovery *Mean * SE. stimulated muscle of each pair are given in parentheses. 4. Experimental P/lactate ratios Series A Tetanus Duration, 0.5 I) Decrease in creatine phosphate or ATP turnover during tetanus, Fmollg dry wt* (n) in ATP content, j.mol/g dry 11.9 * 0.8 112> 0.9 -4 0.6 Series -. s 1.0 3.0 16.5 2 0.9 (13) 39.0 k 2.4 (11) -0.3 0.9 + 0.6 t 0.6 B 1.0 14.2 2 0.8 (9) 0.3 2 0.1 wt* 3) Suprabasal lactate pmollg dry wt” (n) production, 4) Experimental ratio P/lactate 14.2 2 0.6 (31 1.2 5) Tension-time integral N =s/cm2* ATP turnover experiments Lactate recovery experiments 6) Suprabasal ATP resynthesized glycolysis (data in row 2 x pmollg dry wt* 11.8 + 0.5 (9) .I7 % 4 109 * 3 by 1.5), 16.1 k 0.6 21.3 k 0.8 7) Difference between tetanic ATP I 4.2 t- 1.7 4.8 k 2.5 turnover and suprabasal ATP re- ’ i D < 0.0137 P < 0.025t synthesized (row 6 - row I), prnol/g dry wt* 8) Estimated basal ATP turnover during recovery (basal lactate produch tion x 1.51, pmollg dry wt 16 19 1 1.2 1.2 287 5 30 308 t 12 113 k 4 90 2 4 47.7 + 0.6 17.7 + 0.8 8.7 L 4.7 P < 0.05t 3.5 2 1.1 P < 0.005-t 25 21 The ATP utilized by anaerobic sartorius muscles after a complete 0.5, l-, and 3-s isometric tetanus at 20°C is given in row I. Suprabasal lactate produced during anaerobic recovery from similar tetani is given in row 3. These experiments3 were carried out as illustrated by Fig. 2, and the data were collected for the first tetanus only. The experimental P/lactate ratios obtained for each tetanus duration from the data given in rows I and 2 are given in row 4. The areas under the isometric myograms (tension-time integral) are given in row 5. Rows 2,6, 7, and 8 contain further analyses of the data. Experimental series A was made with a single batch of frogs obtained in September, 1975; series B was made with several batches of frog obtained during the winter months of 1974-75. *Mean * SE. t Student t test for independent and uncorrelated means. N,-bubbled preparation was found to be nearly stoichiometric with glycogen depletion over two stimulation and recovery cycles. We did not measure the muscle content of glycolytic intermediates in that experiment, but the return of the muscle to a predictable base line of lactate production suggests that a stimulation-recovery cycle did not significantly alter metabolite content. To the extent that other end products of glycolysis accumu- -0.017 2 0.081 six frogs. One of the pair mate was not stimulated period. The data for the lated [such as n-lactate (10) or succinate (l7)] or that oxidative phosphorylation contributed to the resynthesis of ATP in our N,-bubbled preparation, the value of our experimental P/lactate ratio is overestimated, and therefore our conclusion concerning the magnitude of ATP turnover during recovery is underestimated. There are several trivial explanations for the observed P/lactate ratio being lower than the known Embden-Meyerhof Pathway stoichiometry which can be excluded: a) systematic analytical errors which underestimate the ATP turnover or overestimate the lactate production were ruled out because the net amount of creatine phosphate breakdown was equal to the production of inorganic phosphate, and the latter was measured by an enzymatic assay calibrated in the same way as the lactate assay with solutions of known concentration so NADH (see METHODS). b) Bacterial metabolism of lactate was excluded by the data shown in Fig. 1. c) The total chemical energy utilized during the isometric tetani for the muscles used in ATP turnover measurements is not likely to have been different from those muscles used for recovery lactate measurements, since within each experimental series, A and& Table 4, frogs came from one batch, the stimulus conditions and temperature were the same, the shapes of the isometric myograms were not distinguishable, and because the areas under the tension record [the parameter called tension-time integral as used by Kushmerick and Paul (27)] were very similar as shown in Table 4. Our observed suprabasal ATP breakdown could be due to an, as yet, unidentified net exothermic process occurring during contraction but reversed after mechanical relaxation at the expense of ATP turnover. This possibility originates from a current problem in muscle energetics where more heat can be liberated during a contraction than is explained by the measured chemical changes (5, 12, 14, 19, 30, 44, 46). Our observed suprabasal ATP breakdown could be due to the restoration of electrochemical potentials to basal values by the sarcoplasmic reticulum, mitochondria, and the surface membrane. Any discussion of Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017 42.1 (39.8) Inorganic 21 Change J. KUSHMERICK metabolite composition pmollg TABLE M. RECOVERY ATP c35 UTILIZATION since the 1-malate-NADP oxidoreductase pathway is thought to be near equilibrium (40). Reaction A has not been observed, presumably because of the tight reciprocal covalent and allosteric control of glycogen phosphorylase and synthetase (43). The enzymes in reaction B are also thought to be under tight reciprocal control , but it does occur in liver (9) and in bee muscle (8). This fructose phosphate substrate cycle has not yet been studied in frog sartorii, but it can be measured by the method of Bloxham et al. (3). The following evidence indicates that this cycle can operate in frog sartorii; the enzymes are present (23) , can be active (2), and they may not always be under tight reciprocal control in the cell (42). The operation of these cycles are an energy cost associated with the structure of the metabolic pathways which must perform the task of net ATP restoration. If fructose phosphate cycling occurs in sartorii, it could explain an apparent low anaerobi c P/lactate and low aerobic P/O ratio, Cycling would not rule out the possibility of partially uncoupled mitochondria also contributing to a low P/O ratio. Received for publication 19 August 1976, REFERENCES 1. AMBROSOLI, G., AND P. CERRETELLI. The anaerobic recovery of frog muscle. Pfluegers Arch. 345: 131-143, 1973. 2. BENDALL, J. R., AND A. A. TAYLOR. The Meyerhof quotient and the synthesis of glycogen from lactate in frog and rabbit muscle. Biochem. J. 118: 887-893, 1970. 3. BLOXHAM, D. P., M. G, CLARK, P. C. HOLLAND, AND H. A. LARDY. A model substrate of the fructose diphosphatase-phosphofructokinase substrate cycle. Biochem. J. 134: 581-587, 1973. 4. CANFIELD, P., J. LEBACQ, AND G. MARECHAL. Energy balance in frog sartorius muscle during an isometric tetanus at 20°C. J. Physiol., London 232: 467-483, 1973. 5. CARLSON, F, D., D. J. HARDY, AND D, R. WILRIE. The relation between heat produced and phosphorylcreatine split during isometric contraction of frog’s muscle. J. PhysioZ., London 189: 209235, 1967. 6. CERRETELLI, P., P. DIPRAMPERO, AND G. AMBROSOLI. High energy phosphate resynthesis from anaerobic glycolysis in frog gastrocnemius muscle. Am. J. Physiol. 222: 1021-1026, 1972. 7. CHANCE, B. The energy-linked reaction of calcium with mitochondria, J. BioZ. Chem. 240: 2729-2748, 1965. 8. CLARK, M. G., D. P. BLOXHAM, P. C. HOLLAND, AND H. A. LARDY. Estimation of the fructose diphosphatase-phosphofructokinase substrate cycle in the flight muscle of Bombus affinis. Biochem. J. 134: 589-597, 1973. 9. CLARK, M. G., D. LEE, R. ROGNSTAD, AND J. KATZ. Futile cycles in isolated perfused rat liver and in isolated rat liver parenchyma1 cells. Biochem. Biophys. Res. Commun, 67: 212-219, 1975. 10. CORI, C. F., AND G. T. CORK Glycogen formation in the liver from d- and Z-lactic acid. J. BioZ. Chem. 81: 389-403, 1929. II. CURTXN, N., AND R. WOLEDGE. Energetics of relaxation in frog muscle. J. Physiol., London 238: 437-446, 1974, 12. DAVIES, R, E., M. J. KUSHMERICK, AND R, E. LARSON. ATP, activation, and the heat of shortening of muscle. 214: 148-151, 1967. 13, FLATMARK, T., AND J. I. PEDERSEN. Brown adipose tissue mitochondria. Biochim. Biophys. Acta 416: 53-103, 1975. 14. GILBERT, C., K. M. KRETZSCHMAR, D. R. WILKIE, AND R, C. WOLEDGE. Chemical change and energy output during muscular contraction. J. PhysioZ., London 218: 163-193, 1971. 15. GREEN, M., AND B. LANDAU. Contribution of the pentose cycle to glucose metabolism in muscle. Arch. Biochem. Biophys. 111: 569-575, 1965. 16. HANDEL, E. V. Estimation of glycogen in small amounts of tissue. Anal. Biochem. 11: 256-265, 1965. 17. HOCHACHKA, P, W., AND K. B, STOREY. Metabolic consequences of diving in animals and man. Science 187: 613-621, 1975. 18. HOHORST, H. Methods of Enzymatic Analysis, edited by Bergmeyer. New York: Academic, X963, p. 266-270. 19. HOMSHER, E., J. A. RALL, A. WALLNER, AND N. V. RICCHIUTI. Energy liberation and chemical change in frog skeletal muscle during single isometric tetanic contractions. J. Gen. Physiol. 65: l-21, 1975. 20. INFANTE, A. A., AND R. E. DAVIES. Adenosine-triphosphate breakdown during a single isotonic twitch of frog sarotius muscle. Biochem. Biophys. Res, Commun. 9: 410-415, 1962. 21. KARL, I,, A. PAGLIARA, AND D. M. KIPNIS. A microfluorometric enzymatic assay for the determination of alanine and pyruvate in plasma and tissues. J. Lab. CZin. Med. 80: 434-441, 1972. 22. KARPATKIN, S., E. HELMREICH, AND C. F. CORI. Regulation of glycolysis in muscle. II. Effect of stimulation and epinephrine in isolated frog sartorius muscle. J. BioZ. Chem, 239: 3139-3145, 1964. 23. KREBS, H. A., AND M. WOODFORD. Fructose 1,6-diphosphatase in striated muscle. Biochem. J. 94: 436-445, 1965, 24. KRETZSCHMAR, M., AND R. WILKIE. A new approach to freezing tissues rapidly. J. Physiol., London 202: 66-67P, 1969. 25. KUSHMERICK, M. J., AND R. E. DAVIES. The chemical energetics of muscle contraction. II. The chemistry, efficiency and power of maximally working sartorius muscles. Proc. Roy. Sot. London, Ser, B 174: 315-353, 1969. 26. KUSHMERICK, M. J*, R. E. LARSON, AND R. E. DAVIES. The chemical energetics of muscle contraction. I. Activation heat, heat of shortening and ATP utilization for activation-relaxation processes. Proc. Roy. Sot. London, Ser. B 174: 293-313, 1969. 27. KUSHMERICK, M. J., AND R. J. PAUL. Aerobic recovery metabolism following a single isometric tetanus in frog sartorius muscle at 0°C. J. PhysioZ., London 254: 693-709, 1976. 28. LOWRY, 0. H,, N. J. ROSEBRO~GH, A. L, FARR, AND R. J. RANDALL. Protein measurement with the folin phenol reagent, J. BioZ. Chem. 193: 265-275, 1951. 29. LUNDSGAARD, E. ober die energetik der anaeroben muskelkontraktion, Biochem. 2. 233: 322-343, 1931. 30. MEYERHOF, O,, AND W. SCHULZ ober die energieverhaltnisse bei der enzymatishcen milchsaurebildung und der synthese der phosphagene. Biochem. 2. 281: 292-305, 1935. 31. MOMMAERTS, W. F. H. M., AND A. WALLNER. The breakdown of adenosine triphosphate in the contraction cycle of the frog sartorius muscle. J. Physiol., London 193: 343-357, 1967. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017 these phenomena must be incomplete, since the stoichiometric coupling of ATP breakdown to ion movements and the magnitude and time course of the ion movements remain incompletely known. However, because of the biochemical evidence that substrate cycles can operate in muscle at the expense of a substantial amount of ATP turnover (ES), we consider such cycling a testable possibility to explain our results. Substrate cycling has not yet received much attention in connection with muscle energetics. When the enzymes which are specific for glycolysis and gluconeogenesis are present in the same cell, there is a possibility of cycling between those intermediates which are substrates and products of these enzymes. Cycling reactions which are not at equilibrium must expend net chemical energy during cycling (32). The following are examples of such substrate cycles: a) glycogen f-) Glu-1-P; b) F-6-P-1,6 FDP; c) phosphoenolpyruvatet, pyruvate; d) glucoses, Glu-6-P. These reactions will function as net ATPase’s if their reciprocal operation is not tightly controlled. Reactions A and B are the most likely prospects for substrate cycles in muscle, since pyruvate carboxylase and glucase-6-phosphatase are not present in muscle (33), and C36 AND M. J. KUSHMERICK system. The contractions. 40, 41, 42, 43. 44, 45. 46. anaerobic glycolytic response to simulated tetanic Biochem. J. 138: 119-123, 1974. SCRUTTON, M. C., AND M, F, UTTER. The regulation of glycolysis and gluconeogenesis in animal tissues. Ann. Rev. Biochem. 37: 249-302, 1968. SLATER, E. C. Application of inhibitors and uncouplers for a study of oxidative phosphorylation. Methods Enzymol. 10: 48-71, 1967. VAUGHAN, H., AND E. A. NEWSHOLME. The effects of calcium ions on the activities of hexokinase, phosphofructokinase and fructose 1,6-diphosphatase from vertebrate and insect muscles. Biochem. J. 114: 81-82P, 1969. WHELAN, W. J., AND M. P. CAMERON. Ciba Found. Symp. ControZ GZycogen Metab. 1964, p. 200-304. WXLKIE, D. R. Heat, work and phosphorylcreatine break-down in muscle. J. Physiol., London 195: 157-183, 1968. WOLEDGE, R. Heat production and chemical change in muscle, Progr. Biophys. MOL. BioZ. 22: 37-74, 1971. WOLEDGE, R. In vitro calorimetric studies relating to the interpretation of muscle heat experiments. Cold Spring Harbor Symp. Quant. BioZ. 37: 629-634, 1973. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 17, 2017 32. NEWSHOLME, E. A. Control of glycolysis and gluconeogenesis in liver and kidney cortex. Vitam. Harm. 25: l-52, 1967. 33, OPIE, L. H., AND E. A. NEWSHOLME. The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle, Biochem. J. 103: 391-399, 1967. 34. PAUL, R. J., AND M. J. KVSHMERICK. Apparent P/O ratio and chemical energy balance in frog sartorius muscle in vitro. Biochim. Biophys. 347: 483-490, 1974, 35. ROSSI, F., M. ZATTI, AND A. L, GREENBAWM. Evidence for the existence of the hexose monophosphate pathway for glucose metabolism in normal and denervated skeletal muscle of rats. Biochem.. J. 87: 43-48, 1963. 36. ROTTENBERG, H,, AND A. SCARPA. Calcium uptake and membrane potential in mitochondria, Biochemistry 13: 4811-4817, 1974. 37. SCOPES, R, K. Automated fluorometric analysis of biological compounds. AnaL. Biochem. 49: 73-87, 1972. 38. SCOPES, R. K. Studies with a reconstituted muscle glycolytic system. The rate and extent of creatine phosphorylation by anaerobic glycolysis. Biochem. J. 134: 197-208, 1973. 39. SCOPES, R. K. Studies with a reconstituted muscle glycolytic R. R, DEFURIA