JOURNAL OF EXPERIMENTAL ZOOLOGY 283:501–509 (1999) O2 Consumption and Metabolic Activities in Resting Cardiac Myocytes From Rainbow Trout B. MORTENSEN AND H. GESSER* Institute of Biological Sciences, Department of Zoophysiology, University of Aarhus DK-8000, Aarhus C, Denmark ABSTRACT In mammalian cardiac tissue, the O 2 consumption during mechanical rest is strikingly high. As this may relate to endothermy, the O2 consumption of resting atrial myocytes from an ectotherm, the rainbow trout, was examined. It was 1.46 ± 0.13, 1.96 ± 0.33, 4.34 ± 0.47, and 6.37 ± 0.79 nmol min–1 mg protein–1 at 5, 10, 15, and 20°C respectively. These values are about one fourth of the maximal O2 consumption recorded with 30 µM of the uncoupler dinitrophenol. Inhibition of mitochondrial ATP synthetase with 5 µg ml–1 oligomycin reduced resting O2 consumption by 33 ± 9, 33 ± 1, and 24 ± 7% at 10, 15, and 20°C, respectively. Thus, only about one third of resting O2 consumption supports ATP synthesis, whereas the rest probably represents a futile proton cycle across the mitochondrial membrane. Thus, oligomycin-insensitive O2 consumption was not diminished by 30 µM ruthenium red, which inhibits mitochondrial Ca2+ uptake. About one half of the 30% of the O2 consumption coupled to ATP synthesis supports Na-K ATPase, inasmuch as 2 mM ouabain in the absence of extracellular Ca2+ reduced O2 consumption by about 15% at 10 and 15°C and somewhat less at 20°C. With 1.26 mM Ca2+, the reduction disappeared, probably due to the Na-Ca exchange. As examined at 15°C, changes in extracellular Ca2+, i.e., Ca2+ load, from 0 to 1.26 and 7 mM did not affect O2 consumption. Despite an apparent respiratory “overcapacity,” lactate measurements indicate that between 10 and 20% of the ATP synthesis in the resting cells is anaerobic. Inhibition by ouabain suggests that about 30% of the lactate production supports Na+-K+-ATPase. J. Exp. Zool. 283:501509, 1999. © 1999 Wiley-Liss, Inc. In mammals, the fraction of cellular respiration remaining after cessation of contractile activity is higher in cardiac muscle than in other muscles. This resting respiration supports ATP requiring processes including transport ATPases involved in the cellular regulation of ions such as Na+, K+, and Ca2+ (Gibbs, ’78; Chapman et al., ’82). The main part of the resting respiration, however, does not seem to be coupled to ATP synthesis (Challoner, ’68; Haworth et al., ’83; MorenoSanchez and Hansford, ’88). Instead, it may directly support mitochondrial transport of ions such as Ca2+ (McCormack et al., ’90). In addition, a futile proton leak across the inner mitochondrial membrane seems to be generally present in tissues of mammals (Brand et al., ’94) and have also been recorded in one ectothermic species, a reptile, at 37°C (Brand et al., ’91). The futile proton leak appears to participate in temperature regulation of endothermic vertebrates. For instance, oligomycin-insensitive respiration of mitochondria from heart and skeletal muscle of the ground squirrel is increased during winter (Brustovetsky et al., ’92). Furthermore, proton leak has been suggested to fulfill three other © 1999 WILEY-LISS, INC. functions. First, it enables aerobic energy liberation to follow rapid changes in energy demand. Secondly, it may counteract formation of harmful free radicals. Thirdly, it may regulate different carbon fluxes (Rolfe and Brand, ’97). The first function should be important in cardiac muscle with respect to its rapid and large variations in contractile activity and its almost exclusive dependence on aerobic energy liberation. In contrast, activity changes in skeletal muscle typically rely heavily on anaerobic glycolysis. If the high cellular respiration recorded for resting cardiac muscle from mammals represents a means for rapid changes between different activity levels and also for removal of free radicals and for regulation of carbon fluxes, it should not only occur in endothermic species but also in ectothermic species at their different in vivo temperatures. The present study of rainbow trout concerns the components of resting myocardial respiration at Grant sponsor: Danish Natural Science Research Council; ‘‘Center for Respiratory Adaptation.’’ *Correspondence to: H. Gesser, Institute of Biological Sciences, Department of Zoophysiology, University of Aarhus DK-8000, Aarhus C, Denmark. E-mail: firstname.lastname@example.org Received 15 April 1998; Accepted 11 August 1998. 502 B. MORTENSEN AND H. GESSER different temperatures within the in vivo range. In addition to components not associated with ATP synthesis, the interest was directed to the ionic regulation depending directly or indirectly on NaK-ATPase, which presumably constitutes a major part of the energy demand in resting muscle. The contribution of these different processes to cellular respiration was estimated with various agonists and antagonists. In order to reduce diffusion limitations, the experiments were performed on suspensions of cells isolated from atrial tissue of rainbow trout. Calorimetric studies show that suspensions of cardiac myocytes from mammals are well suited for metabolic studies like the present (Ponce-Hornos et al., ’90). This seems to be true for trout cardiac myocytes as well, according to, among other things, their high ability to maintain cellular energy state (Milligan, ’91). MATERIAL AND METHODS Rainbow trout (Oncorynchus mykiss) weighing 250–300 g were kept in fresh water tanks at 15 ± 2°C and fed regularly. The solutions used in the different procedures were based on solution A containing (mM): 100 NaCl, 2.5 KCl, 1.8 MgCl2, 0.63 NaH2P04, 10 HEPES and as substrates 5 of each glucose, Na-glutamate, Na-fumarate, and Na-pyruvate. Unless otherwise stated, solution A also contained 10 mM 2-3-butanedione monoxime (BDM) in an attempt to obtain a well-defined resting state. BDM inhibits the interaction of actin and myosin and the action potential dependent influx of Ca2+ into the cell (Backx et al., ’94, Ikenouchi et al., ’94). Furthermore, BDM has been shown to counteract dissection injury (Mulieri et al., ’89). At both 10 and 20°C, 10 mM BDM inhibits about 60% of the twitch force developed by isolated ventricular strips of trout. The concentration providing full inhibition amounted to more than 30 mM (unpublished observations) and was not chosen because of a concern about possible side effects. Immediately before use and after its pH had been brought to 7.4 (20°C) with imidazol, solution A was equilibrated with pure O2 followed by the addition of 1 mg/ml bovine serum albumin. Atrial cells were isolated enzymatically on the basis of methods previously used (e.g., Mitra and Morad, ’85). The thin walled atrium was used to favor a homogenous enzyme treatment. The fish was decapitated and the heart excised and transferred to an ice-cold solution A with 250 IU/ml heparin to inhibit blood coagulation. A plastic tube was inserted in bulbus and 5 ml of solution A with heparin were injected to remove blood from the ventricular and atrial chambers. To isolate atrial cells, the heart was perfused at a rate of 4 ml sec–1 with 50 ml of solution A and then by 20 ml of solution A to which had been added 1 mg/ml collagenase (0.15 IU/ml) (Boehringer Mannheim) and 0.5 mg/ml (about 5,000 BEE units/ml) trypsin (Sigma, St. Louis, MO). The latter solution was recirculated for 30 min. Care was taken to remove air bubbles and to ensure that the atrium wall was extended to secure access of the solution to the cells. After the perfusion, which was performed at 20°C, the heart was placed on an ice-cold petri dish and the atrium was cut free. Atrial cells were suspended in 3–4 ml of solution A with 1.25 mM CaCl2. After being spun down for 1 min at 100g, cells were resuspended in 3–4 ml of A with 1.25 mM CaCl2 and stored at 4°C until being used for recordings of O2consumption or lactate production. The cells were used the day of preparation in some introductory experiments to establish suitable protocols. For the rest they were stored overnight. The quality of the cells was evaluated immediately before and after recording of respiration or lactate production. The number of intact and defective cells was determined at a 10-times magnification in a hematocytometer by counting cells in four squares containing 8 · 10–9 liters. The cells should be long (about 150 µm) and thin (about 10 µm) and have a smooth surface. The preparations contained two types of defective cells of which one was round. According to literature, these cells are muscle cells in a supercontracted state although the possibility that also non-muscle cells are included in this group cannot be excluded. The metabolic activity of supercontracted muscle cells is unclear. According to measurements of heat development in suspensions of cardiac myocytes from rat, guinea pig, and rabbit, supercontracted cells do not seem to contribute significantly to metabolic activity (Ponce-Hornos et al., ’90), whereas their activity in terms of oxygen consumption was somewhat above normal for turtle cardiac myocytes (Watson et al., ’94). The other type of defective cells was long and slender but with blebs on the cell membrane, probably indicating a disturbed ion regulation (Watson et al., ’94, Uchida and Doi, ’94). The preparations of the present study contained up to 15% abnormal cells, of which maximally 5% were supercontracted and the rest were long and slender but with blebs. The dye tryptan blue, which was used to test the barrier function of the cell membrane (e.g., Watson et al., ’94), entered supercontracted cells but was excluded from all the slender cells irrespective of O2 CONSUMPTION OF TROUT MYOCYTES the presence of blebs. The quality of the preparations was also examined at room temperature (about 20°C) by incubating about 20 cells in 250 µl solution with or without BDM. Irrespective of the presence of BDM, all of the slender cells without blebs were quiescent, whereas the majority of those with blebs displayed spontaneous contractions. This spontaneous activity was not assessed quantitatively, because the activity varied between cells as well as for the individual cell. Thus, cascades of spontaneous contractions of a frequency up to about 1 Hz alternated with periods of rest. In the absence of BDM, all of the slender cells could be stimulated to contraction by electrical square pulses of a duration of 5 msec delivered by a stimulator (Grass SD 9, Quincy, MA) through two platinum electrodes. Cell quality was influenced by the experimental temperature. Thus most of the cells became blebbed and spontaneously active at 20°C, whereas the temperatures below 20°C did not affect cell quality. • Cellular O2 consumption (MO2), referring either to mg cellular protein or to ml cell suspension, was recorded polarographically by an electrode (Radiometer E5046) in a thermostated 1.4 ml glass vessel filled with suspension, which was stirred by a magnetic stirrer. With intervals of five seconds, O2 tension was recorded and stored on computer. The initial O2 tension was 250–300 mmHg and the final one• not below 150 mmHg. In this tension range, MO2 was not limited by oxygen availability. This was demonstrated in an experiment run at 15°C in • which MO2 was stable, although oxygen tension fell from 150 to 120 mmHg and respiration was stimulated by 30 µM of the uncoupler dinitrophenol (Fig. 1). Lactate was measured spectrophotometrically as NADH produced by the LDH reaction coupled to the glutamate-pyruvate transaminase reaction (Boehringer, Mannheim). Values of O2-consumption and lactate production were related to tissue protein determined as described by Peterson (’77). The cell sample had been incubated at the experimental temperature for at least 10 min before recordings of respiration or lactate production were started. Recordings were then performed for 15–20 min before any experimental intervention was applied. Values are presented as mean ± SEM with the number of measurements in parenthesis. Significance was assessed with ANOVA and Student test for paired and unpaired data. Arcsin transformation was performed for normalized data (Sokal and 503 Fig. 1. Decrease in pO2 with time at 15°C in a suspension • of myocytes; MO2 was stimulated with 30 µM of the uncoupler • dinitrophenol. MO2 = 7.8 nmol O2 min–1 ml suspension–1. Rohlf, ’81). Significance was considered present at P < 0.05. RESULTS • MO2 of resting myocytes increased with temperature, although the difference between 5 and 10°C. was not statistically significant (Table 1). Due to this fact, 5°C was not included in the further experiments. According to experiments per• formed at 15°C, the MO2 recorded is exclusively due to the mitochondrial respiratory chain being completely inhibited by either 2 mM NaCN (n = 2) or 12.5 µM rotenone (n = 2). • In order to estimate its maximum, MO2 was recorded in the presence of 30 µM of the uncoupler • dinitrophenol, DNP. MO2 increased several times by a percentage that did not differ significantly among the temperatures applied (Table 2A). Preliminary experiments at 10 and 20°C had shown that DNP elicited its maximal effect at 30 µM, as • MO2 did not change upon an elevation of DNP from 30 to 60 µM (data not shown). Oligomycin—a well-established inhibitor of mitochondrial ATP synthetase—was applied to • to ATP estimate the fraction of MO2 coupled • synthesis. With 5 µM oligomycin, MO2 was lowered by about one third and that, to an extent, did not vary significantly among the temperatures applied (Table 2B). The maximal effect of • oligomycin on MO2 was developed at 5 µmol liter–1 according •to experiments run at 10 and 20°C, in which MO2 was measured subsequently at 5 and 15 µmol liter–1 oligomycin. The reduc• tion in MO2 after the first and second addition 504 B. MORTENSEN AND H. GESSER • TABLE 1. MO2 (nmol min1 mg protein1) and temperature1 °C • M O2 5 10 15 20 1.46 ± 13 (9) 1.96 ± 0.33 (5) NS2 4.34 ± 0.47 (7) P < 0.01 6.37 ± 0.79 (7) P < 0.05 • 1 Mean values ± S.E.M. Number of samples within parentheses. The significance levels refer to the difference in MO2 between the temperature indicated and that which is 5°C lower. 2 NS = nonsignificant. of oligomycin respectively was 34 ± 4% and 33 ± 10% (n = 3) at 10°C and 29 ± 5 and 26 ± 7 (n = 3) at 20°C. Oligomycin was dissolved in • DMSO, which did not affect MO2 when applied in concentrations used in the present experiments, i.e., less than 2% of the total volume. Oligomycin should block mitochondrial ATP synthetase completely and should therefore remove the influence of an increased ATP demand • on MO2. This was tested with veratridine, which is known to stimulate energy degradation of myocardial cells by increasing the cytoplasmic Ca2+ activity and the ATP hydrolysis of the contractile system (Moreno-Sanchez and Hansford, ’88). In experiments performed at 10 and 15°C, • MO2 was measured in a solution, from which BDM and its inhibition of the ATP hydrolysis by the contractile system were excluded. Without oligomycin, 30 µM veratridine stimulated • M O 2 to an extent, which was significantly higher at 15 than 10°C (Table 2C1). When veratridine was added 20 min after oligomycin had TABLE 2. MO2 (nmol min1 ml suspension1) recorded just before the addition of the agent indicated, and change (%) in MO2 after stabilization in presence of the agent1 °C 10 A. Dinitrophenol (DNP), 30 µmol liter • MO2 1.00 DNP % 435 B. Oligomycin, 5 µg ml–1 • MO2 1.42 oligom % –32 C1. Veratridine, 30 µmol liter–1, no BDM • 1.23 MO2 veratridine % 59 15 20 –1 ± 0.12 (11) ± 57 P < 0.001 1.97 ± 0.30 (11) 385 ± 44 P < 0.001 2.58 ± 0.37 (11) 556 ± 62 P < 0.01 ± 0.48 (11) ± 10 P < 0.025 2.52 ± 0.30 (11) –33 ± 1 P < 0.001 4.55 ± 0.79 (11) –24 ± 7 P < 0.025 5.34 ± 0.73 (4) 200 ± 39 P < 0.025 P < 0.025 (10 vs. 15°C) C2. Veratridine, 30 µmol liter–1 in the presence of oligomycin, 5 µg ml–1, no BDM • 1.51 ± 0.46 (7) 2.80 ± 0.52 (8) MO2, oligom veratridine % 4 ± 7 NS 1 ± 3 NS D1. Cyanide (CN), 0.25 mmol liter–1 in the presence of iodoacetate (IAA), 1 mmol liter–1 • MO2, IAA 2.14 ± 0.70 (6) CN % –38 ± 7 P < 0.01 D2. Oligomycin, 5 µg ml–1 in the presence of iodoacetate (IAA) and cyanide (CN) • 1.43 ± 0.51 MO2, IAA, CN oligom % –32 ± 2 P < 0.001 E1. Ouabain, 2 mmol liter–1, with Ca2+, 1.26 mmol liter–1 • MO2 1.41 ± 0.07 (12) 2.98 ± 0.62 (9) ouabain % 8 ± 8 NS 2 ± 8 NS E2. Ouabain, 2 mmol liter–1, without Ca2+ • MO2, 0 CA2+ 2.07 ± 0.33 (12) 3.01 ± 0.59 (9) ouabain % –16 ± 4 P < 0.01 –14 ± 4 P < 0.01 F. Extracellular Ca2+ mmol liter–1 • MO2, 0 Ca2+ 2.08 ± 0.96 (10) –1 ± 8 NS 1.26 Ca2+ % • 2.01 ± 0.86 (10) MO2, 1.26 Ca2+ 4 ± 8 NS 7 Ca2+ % G. Ruthenium red, 30 µmol liter–1 with oligomycin, 5 µg ml–1 • MO2, oligom 1.79 ± 0.40 (5) Ruth. red % 5 ± 6 NS 1 ± 0.34 (4) ± 18 P < 0.05 4.02 ± 0.83 (13) 20 ± 8 P < 0.05 3.49 ± 0.75 (10) –7 ± 6 NS The experiments were run at the temperatures given at the top of each column. Unless otherwise stated, significance levels refer to the effect of the agent. cf. Table 1 footnotes. O2 CONSUMPTION OF TROUT MYOCYTES been applied, the stimulation of respiration by veratridine was removed at both temperatures (Table 2C2). The fraction of respiration sensitive to oligomycin has been shown to increase with the rate of ATP hydrolysis, i.e,. when the energy demand is increased relative to the energy liberating capacity (Haworth et al., ’83). Therefore, the effect of oligomycin was recorded following an attempt to increase energy demand relative to the energy liberating capacity by decreasing the latter with 0.25 mM NaCN and 1 mM iodoacetate. Cyanide in this concentration decreases both the electron flow in the respiratory chain and as a result the proton gradient across the mitochondrial inner membrane (Brand et al., ’94). Furthermore, a shift from aerobic to anaerobic ATP synthesis was prohibited by an inhibition of glycolysis with iodoacetate (Hartmund and Gesser, ’96). Iodoacetate was added first followed 10 min later by cyanide. During the 10 min following the addition of cyanide, • MO2 stabilized at a value lower than the initial one (Table 2D1). The subsequent addition of oli• gomycin, 5 µg/ml, reduced MO2 further by a percentage that did not differ significantly from that recorded in the absence of cyanide and iodoacetate (Table 2D2). In cardiac myocytes, a substantial part of the ATP hydrolysis supports ionic homeostasis, which to a major extent either directly or indirectly depends on Na+-K+-ATPase (Skou, ’92; Brand et al., ’94). A survey of the literature covering a wide range of cell types provides evidence that 2 mmol liter–1 should give a complete inhibiton of Na+-K+ATPase in trout heart cells. For instance, a K0.5 of 20 µmol liter–1 for ouabain was found with cultured chick cardiac myocytes (Stimers et al., ’90). –1 ouabain entailed a significant Two mmol liter • elevation of MO2 at 20°C and no significant change at either 10 or 15°C (Table 2E1). This heterogenous response may depend on the Na+-Ca2+ exchange across the cell membrane allowing Na+ and Ca2+ to accumulate together in the cell following an inhibition of Na+-K+-ATPase at a rate that increases with temperature. Therefore, experiments were performed in the nominal absence of extracellular•Ca2+. Here, a tendency of ouabain to decrease MO2 appeared, which was significant at all temperatures except 20°C. The effects at the three temperatures were not statistically different (Table 2E2). The different effects of ouabain recorded with and without extracellular Ca2+ are most probably 505 an expression of the function of Na-K-ATPase acting through the Na-Ca exchange in the maintenance of a low cytoplasmic Ca2+ activity. This • function entails the possibility that the MO2 associated with Na-K-ATPase is underestimated, • when the decrease in MO2 by ouabain is recorded in a Ca-free solution. This reasoning implies that extracellular Ca2+ should enhance the Na-K• ATPase activity and MO2. Furthermore, extracel• lular Ca2+ is of interest regarding the part of MO2, which is insensitive to oligomycin, as mitochondria are able to accumulate Ca2+ by a mechanism directly supported by respiration (McCormack et al., ’90). The effect of Ca2+ was examined at 15°C. In one series of experiments, cells were incubated in a solution of which Ca2+ was maintained in sequence at 0, 1.26, and 7 mmol liter–1 with 15 min at each concentration. Neither the first nor the • second elevation of Ca2+ changed MO2 significantly (Table 2F). The respiratory-supported mechanism by which mitochondria takes up Ca2+ is inhibited by ruthenium red (McCormack et al., ’90). Therefore, ruthenium red in a concentration of 30 µM, which is in the upper range of concentrations used on whole myocyte preparations (e.g., Siegmund et al., ’92), was applied in another series of experiments. These experiments were run in the presence of oligomycin to block the respiration supporting ATP synthesis and to accentuate the respiratory dependent part of cellular Ca2+ handling. No significant effect of ruthenium red on • MO2 was detected (Table 2G). Lactate production was assessed in experiments run at 15°C and revealed a significant anaerobic energy liberation (Table 3). Evidence exists that transport processes across the cell membrane preferentially utilize anaerobically produced energy (Campbell and Paul, ’92). Therefore, the effect of ouabain on lactate production was examined in a separate series of experiments performed in a Ca2+-free solution to avoid possible side effects of an increase in cellular Ca2+. The average amount of lactate released during 20 min fell significantly during the following 20 min with ouabain, whereas no significant change was recorded for the cells subjected to the same protocol but without ouabain (Table 3). According to the numbers in Table 3, the ouabain inhibited lactate production amounted to 28 ± 12% (P < 0.05). The value for the lactate production rate recorded in the presence of extracellular Ca2+ did not differ significantly from the two values obtained in the absence of extracellular Ca2+ (Table 3). 506 B. MORTENSEN AND H. GESSER TABLE 3. Lactate production rate (nmol min1 mg1 protein) at 15°C in 1.26 and 0 mmol liter1 Ca2+ and change (%) in lactate production in 0 Ca2+ after 20 min in presence of either 2 or 0 mmol liter1 ouabain1 Ca2+ mmol liter–1 126 Lactate, nmol min–1 mg–1 0 0.99 ± 0.16 (13) 20 min, % 1 0.78 ± 0.09 (8) +Oua –43 ± 5 P < 0.001 0 0.87 ± 0.10 (8) –Oua –15 ± 11 NS Significance levels refer to the changes in lactate production. cf. Table 1 footnotes. DISCUSSION The respiration recorded in the present study is exclusively mitochondrial in relation to its inhibition by either cyanide or rotenone. The temperature dependence of resting respiration was similar to that seen in other biochemical or physiological processes having a Q10 well above one. In a study similar technically to the present one, ventricular myocytes from turtle displayed an oxygen consumption of about 1 nmol mg protein–1 min–1 at about 20°C (Watson et al., ’94), i.e., about six times lower than that recorded for trout atrial cells at 20°C in the present study. This difference may relate to differences in cellular metabolic capacities between the two species (Christensen et al., ’94) and the finding that atrial tissue is likely to have a higher metabolic activity than ventricular tissue as judged from its higher activity of Ca2+ activated myosin ATPase (Degn and Gesser, ’97). In this respect, it also should be noted that the O2 consumption recorded for trout atrial tissue is well below maximal as it was enhanced 3.5–5.5 times by the uncoupler DNP. Resting respiration relative to the maximal respiratory capacity estimated by the O2 consumption in the presence of DNP was not significantly affected by temperature in the range of 10–20°C. It is doubtful, however, if a respiration of the magnitude released by the DNP ever occurs in vivo. Considering this, trout atrial cells seem to resemble the mammalian heart in which resting metabolism measured in terms of O2 consumption or heat production constitutes a substantial fraction of the metabolism during mechanical activity (Gibbs, ’78; Chapman et al., ’82). For technical reasons it should be noted that the cells at 20°C were of lower quality than those at the other temperatures; almost all had blebs. The fact that their behavior was reasonably within the range of the other cells seems to indicate that the presence of cells with blebs did not seriously disturb the results. Veratridine was used to examine the efficiency of the oligomycin inhibition. Evidence exists that it attaches itself to open sarcolemmal Na+ chan- nels and potentiates their opening (Horackova and Vassort, ’74; Honerjäger and Reiter, ’75) so that there is an intracellular increase in Na+ and subsequently in Ca2+, due to the Na-Ca exchange. The stimulating effect of veratridine on respiration fell with decreasing temperature, i.e., from 15 to 10°C. It is possible that the frequency of spontaneously opened channels to which veratridine is able to attach decreases with decreasing temperature. The removal of the stimulation of respiration by veratridine and dose-response experiments indicates that the dose of oligomycin used was sufficient to block ATP-synthetase completely. The effect of oligomycin suggests that about 70% of the resting O2-consumption of trout atrial myocytes is not coupled to ATP production. The fraction of respiration coupled to ATP synthetase was not significantly influenced by temperature within the range covering the in vivo temperatures of rainbow trout. In contrast, the fraction of respiration coupled to ATP synthetase decreased with lowering of temperature in mitochondria from rat liver in a way that seemed to depend on adenosine translocase (Quentin et al., ’94). Regarding the value of resting O2 consumption not coupled to ATP synthetase—about 70%—trout myocardium resembles the myocardium of mammalian, or endothermic, species (Challoner, ’68; Haworth et al., ’83; Moreno-Sanchez and Hansford, ’88). The absence of the effect of blocking mitochondrial Ca2+ uptake with ruthenium red in the presence of oligomycin and of varying the Ca2+ concentrations in the extracellular solution suggests that cellular Ca2+-handling in resting trout heart cells does not contribute substantially to cellular respiration. A similar conclusion was reached for guinea pig heart (Hanley et al., ’94). Although, other transports such as phosphate and adenosine (Quentin et al., ’94) were not examined, it seems probable that the predominant part of the nonphosphorylating respiration is associated with a futile proton cycle, i.e., a proton leak. The size of this respiration in cells from an ectothermic vertebrate and the fact that its fraction of resting respiration appears to be the same within O2 CONSUMPTION OF TROUT MYOCYTES the in vivo range of temperatures complies with the contention that the futile proton cycle represents a fundamental property of the living cell. Although its function is unclear, its presence in cells of ectothermic species suggests that it has not evolved with endothermy. A similar conclusion was reached in a comparison of lizard and rat tissues (Brand et al., ’91). The fraction of respiration not sensitive to oligomycin may represent an overestimation because an inhibition of ATP-synthetase blocks one pathway of proton flux across the inner mitochondrial membrane, whereby protons should tend to accumulate and increase proton motive force. As a result proton flux along the remaining pathways should be enhanced and respiration should decrease less than expected from a simple subtraction of the proton flux coupled to ATP-synthetase (Brand et al., ’94). However, as oligomycin reduced respiration only by about 30%, the increase in proton motive force and the overestimate of the nonphosphorylating part should be modest. The large fraction of oligomycininsensitive respiration may represent a capacity of the respiration supporting ATP synthetase to increase promptly upon an increase in cellular ATP demand (Rolfe and Brand, ’97). A study of heart cells from rat accords with this shift from a nonphosphorylating to a phosphorylating respiration in that an enhancement of ATP degradation increased the fraction of respiration inhibited by oligomycin (Haworth et al., ’83). On this basis it is not unexpected that the fraction of respiration that is insensitive to oligomycin in noncontracting heart cells would be considerably higher than in cells such as hepatocytes (Brand et al., ’94), which may be less removed from their ATP demand in vivo than noncontracting heart cells. The results of the present study deviate, however, from the outlined model inasmuch as the decrease in proton gradient presumably accompanying the approximate 40% inhibition of mitochondrial respiration with cyanide did not change the fraction of respiration insensitive to oligomycin. The expectation was that the oligomycin-sensitive respiration, i.e., that coupled to ATP synthetase, should increase and that associated with a proton leak should decrease. This is because a partial inhibition of mitochondrial respiration should lower proton motive force and total proton flow. Together with an iodoacetate inhibition of anaerobic ATP synthesis, this should tend to decrease the cellular energy state and thus increase the part of the remaining proton flow coupled to ATP synthetase. 507 A major portion of the phosphorylating respiration—about one third of resting respiration— inhibited by oligomycin seems to support the Na+-K+ pump. This portion was not significantly affected by temperature, such that the energy degraded by the Na-K pump changed in pace with total cell respiration. In the resting cell at steady state, the Na-K pump rate should be equal to the passive flux of Na+ and K+ across the membrane. Hence, this flux should have a Q10 of that typical for enzyme catalyzed processes suggesting that it occurs predominantly through specific channels. The contribution of the Na-K ATPase to the energy degradation is probably underestimated in the present experiments performed in a Ca2+-free solution. Here, the Na+ load is diminished as Ca2+ does not leak into the cell and consequently does not have to be removed by the sarcolemmal Na-Ca exchange at the cost of an influx of Na+. The importance of this exchanger was suggested by the increased O2 consumption and the contractures of the cells, when ouabain was applied in the presence of extracellular Ca 2+. It should also be restated, however, that without an increase in cellular Na+, changes in the cellular Ca2+ load in terms of extracellular Ca2+ had no substantial influence on O2 consumption. The fraction of respiration supporting Na-K ATPase might also be underestimated since an inhibition of this enzyme may tend to increase the proton motive force across the inner mitochondrial membrane and thus increase the resulting proton flux. This effect is likely to be small as the decrease in respiration due to ouabain only amounted to about 15%. Measurements at 15°C suggest that about 10– 20% of ATP is produced by anaerobic metabolism in assuming a P/O ratio of 3 of the respiration inhibited by oligomycin and 1–1.5 ATP per lactate. The lactate production of 0.98 nmol min–1 mg–1 protein should be compared to an O2 consumption of about 4.5 nmol min–1 mg–1 protein of which only about a third represents ATP production according to the oligomycin experiments. Apart from O2 consumption, ouabain also decreased lactate production by about one fourth, indicating that the Na+-K+ pump under aerobic conditions is supported by both aerobic and anaerobic metabolism. Roughly estimated, the anaerobic contribution seems to cover one tenth of the pump activity. The functional advantage of such an incomplete substrate utilization, i.e., from glucose and/or glycogen to lactate, is not clear. In 508 B. MORTENSEN AND H. GESSER the fully aerobic smooth muscle, lactate is produced at a rate that correlates with the Na+-K+ pump activity and it has been proposed that glycolysis offers an advantage due to its cytoplasmic localization close to the pump sites (Campbell and Paul, ’92). In conclusion, the respiration of resting trout atrial cells constitutes about one fourth of the respiratory capacity and only one third of it appears to support ATP synthesis, whereas the remaining two thirds predominantly cover a futile proton cycle across the inner mitochondrial membrane. Despite the apparent aerobic overcapacity, a small but significant fraction of the ATP production is supported by anaerobic glycolysis. A substantial part of the phosphorylating aerobic metabolism as well as of the anaerobic metabolism appears to support Na-K ATPase. The respiratory rate changes with a Q10 of about two or above in the temperature range of 5–20°C, whereas the relative contributions of the processes mentioned essentially stay the same. ACKNOWLEDGMENTS The development of the PC-based recording of data by engineer E. Larsen and the technical assistance of A. Wetter are deeply appreciated. LITERATURE CITED Backx PH, Gao WD, Azan-Backx MD, Marban E. 1994. 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