Effects Of Ca²⁺ On Oxidative Phosphorylation In Mitochondria From The Thermogenic Organ Of Marlin

Several large pelagic fishes of the suborder Scombroidei (mackerels, tunas and billfishes) have independently evolved a unique form of endothermy. The billfishes (family Istiophoridae), swordfish (family Xiphiidae) and the butterfly mackerel (family Scombridae) heat the brain and eyes using a specialized thermogenic organ (the heater organ) that has evolved from muscle (Carey and Teal, 1966; Carey, 1982; Block, 1987). Telemetry studies have shown that swordfish diving in cold water are able to maintain their cranial temperature up to 14 °C above that of the surrounding water (Carey, 1990). By generating metabolic heat, the heater organ dampens the effects of rapid temperature changes on the central nervous system.

Biochemical studies show that heater tissue has exceptional aerobic metabolic capacity (Tullis et al. 1991; Ballantyne et al. 1992), however, a mechanism by which oxidative activity is stimulated needs to be described. In mammalian brown fat, the only other tissue described that functions primarily to provide heat, a high rate of respiration is maintained by a unique uncoupling protein in the mitochondrial inner membrane. The uncoupling protein is a proton pore that collapses the mitochondrial proton gradient, thus driving respiration at a high rate and diverting respiratory energy from ADP phosphorylation. The proton pore is closed in the presence of guanine nucleotides, providing a mechanism for the cellular control of heat production (Nicholls and Locke, 1984). The mitochondria in heater tissue, unlike their counterparts in brown fat, lack uncoupling protein (Block, 1986) and must maintain high oxidative activity in another way. The presence of an extensive sarcoplasmic reticulum (SR) and T-tubule network in heater tissue indicates a Ca²⁺-based mechanism for stimulation (Block, 1986, 1987, 1991). Structural and biochemical evidence suggests that thermogenesis in heater cells is initiated by ‘excitation–thermogenic coupling,’ a Ca²⁺-activated thermogenic cycle under nervous control (Block, 1994). This cycle is based on the release of Ca²⁺ through Ca²⁺-release channels in the extensive SR network with a consequent increase in metabolic activity in the heater cell.

The interaction of Ca²⁺ and mitochondria during the thermogenic cycle is the focus of this study. Vertebrate mitochondria are known to possess energy-dependent Ca²⁺ uptake and efflux pathways. Ca²⁺ is taken up by an electrophoretic Ca²⁺ uniporter that responds to the mitochondrial membrane potential (Crompton, 1985). Ca²⁺ enters through the uniporter with two positive charges at the expense of the membrane electrical gradient. By collapsing the electrical gradient, this process stimulates respiration and competes with ATP synthesis. Ca²⁺ taken up through the uniporter is lost from mitochondria through a number of routes. The most active efflux pathway in mitochondria from excitable cells is an electrogenic nNa+/Ca²⁺ exchanger (Jung et al. 1995) which is, in turn, driven by an energy-consuming Na+/H+ antiporter (Carafoli, 1982; McCormack et al. 1990). The activities of the Ca²⁺ uptake and efflux systems normally maintain matrix Ca²⁺ levels below 100 nmol l⁻¹, but this can rise substantially under conditions involving increases in average cytosolic Ca²⁺ concentration such as hormonal activation, muscular activity and ischaemia (McCormack et al. 1990). Such changes in matrix free Ca²⁺ concentration have been identified as an important mediator in the regulation of mitochondrial oxidative phosphorylation in vertebrate tissues (McCormack et al. 1990; McCormack and Denton, 1993).

The very close apposition of heater organ mitochondria with the extensive SR, along with the small cytoplasmic space (Block and Franzini-Armstrong, 1988), raises the possibility that released Ca²⁺ may be taken up directly into the mitochondria during the Ca²⁺ release cycle. If the activity of the uptake uniporter and an efflux pathway were sufficiently high, respiration could then be uncoupled during transient high Ca²⁺ levels. This would result in enhanced respiration and heat production. Rapid uptake of extramitochondrial Ca²⁺ added at high concentration has been observed in isolated mitochondria from the heater organ of swordfish (Ballantyne et al. 1992), indicating that heater organ mitochondria have an active Ca²⁺ uptake mechanism. Thus, Ca²⁺ uncoupling could be a mechanism for activating thermogenesis in heater cells, but would result in a reduced output of ATP. On the other hand, if the mitochondria were relatively insensitive to physiological levels of Ca²⁺, stimulation of energy consumption and heat production would depend on other processes such as cellular consumption of ATP and the modulation of the supply of substrate and NADH.

In this study, we have examined energetic aspects of Ca²⁺ cycling in marlin heater organ mitochondria. Red locomotory muscle mitochondria were studied for comparison. We found that mitochondria from both tissues actively take up Ca²⁺ from the medium, but that significant uncoupling occurs in heater mitochondria only at free Ca²⁺ concentrations higher than those predicted to be found in the heater cell. These results support a model in which heat production is stimulated by ATP consumption in the heater cell. A futile Ca²⁺ cycle involving the SR Ca²⁺-release channel and the SR Ca²⁺-ATPase is discussed.

Heater organs from blue marlin (Makaira nigricans Lacépède) were dissected from recently caught specimens and chilled in ice-cold 0.9 % NaCl. Samples (3 g) of heater tissue were dissected from the center of the organ and trimmed free of attached rete and extraocular muscle. The tissue piece was minced in 10 volumes of ice-cold homogenizing buffer containing 0.25 mol l⁻¹ sucrose, 5 mmol l⁻¹ K-Hepes, pH 7.2, 2 mmol l⁻¹ EGTA, 1 % (w/v) serum albumin. The minced tissue was homogenized with three passes of a loose-fitting Potter-Elvhjem homogenizer and centrifuged for 5 min at 120 g in a Sorvall centrifuge. The supernatant was filtered through two layers of cheesecloth and centrifuged for 5 min at 9750 g. The resulting pellet was resuspended in 0.2 mol l⁻¹ sucrose, 50 mmol l⁻¹ KCl, 5 mmol l⁻¹ K-Hepes, pH 7.2, 2 mmol l⁻¹ EGTA and 1 % serum albumin (wash buffer) and centrifuged for 5 min at 9750 g. The wash step was repeated once and the final pellet resuspended in 0.5 ml of wash buffer per gram starting tissue. Mitochondrial protein content was assayed using the BCA (bicinchoninic acid) method (Pierce) with serum albumin as the protein standard. Mitochondrial preparations were kept on ice and maintained respiratory control for up to 8 h. However, most experiments were performed within 3 h of isolation.

Mitochondria from red swimming muscle were prepared in the same way. Approximately 5 g of red muscle was dissected from the base of the tail and trimmed free of white muscle and connective tissue. The homogenization procedure was sufficient to release a large number of mitochondria, however, it is likely that the majority of interfibrillar mitochondria remained with the myofibrils and were discarded. We chose to accept this bias in favor of subsarcolemmal mitochondria as the possible damage to the mitochondria caused by more vigorous homogenization methods would be detrimental to the study.

The time between capture of the fish by sportfishing boats and dissection of tissues varied and influenced the characteristics of the mitochondria. Experimental fish were selected using a variety of criteria including time of capture, evidence of muscle rigor and skin discoloration. Preparations were made from the freshest fish possible and were not used if the respiratory control ratio was below 2.5.

Respiration rates were measured using a Strathkelvin 1302 oxygen electrode and Strathkelvin 781 oxygen meter. The electrode was mounted in a glass 2 ml side-port water-jacketed chamber maintained at 25 °C using a circulating water bath. Additions were made through a groove in the Teflon chamber plug and the volume was adjusted to compensate for added liquid. Mitochondria were suspended at approximately 0.5 mg ml⁻¹ in an assay buffer containing 0.15 mol l⁻¹ KCl, 5 mmol l⁻¹ K-Hepes, 5 mmol l⁻¹ potassium phosphate, pH 7.2 at 25 °C. The assay mixture contained, as carryover from the wash medium, 10 mmol l⁻¹ sucrose, 100 µmol l⁻¹ EGTA and 0.05 % serum albumin. Substrates and inhibitors were added as indicated in the Figure legends. P/O ratios were calculated using the method of Estabrook (1967) from total oxygen consumed. Levels of oxygen and adenylates were assayed as described previously (O’Brien and Vetter, 1990).

For Ca²⁺ flux measurements, the chamber plug of the respiration chamber described above was modified to fit a Radiometer F2112Ca calcium electrode. The calomel reference electrode (Radiometer K401) was kept in a separate water-jacketed chamber filled with 0.15 mol l⁻¹ KCl and was connected to the assay chamber by means of an agar bridge made with 0.15 mol l⁻¹ KCl. The 95 % response time of the Ca²⁺ electrode to step addition of 10 µmol l⁻¹ Ca²⁺ in respiration buffer was 2.1 min, but increased to 5.5±0.8 min in the presence of mitochondria and EGTA. Somewhat faster responses were obtained at higher Ca²⁺ concentrations. Ca²⁺ fluxes and oxygen consumption were measured simultaneously for all experiments. The Ca²⁺ solutions used were calibrated using the Ca²⁺ electrode by comparison to standard curves made in 0.15 mol l⁻¹ KCl with a Radiometer Ca²⁺ standard. The solutions were checked daily by comparison of known additions of the Ca²⁺ solution to mitochondria assay buffer with comparable additions of the Ca²⁺ standard. Each individual assay performed was followed by an addition of 50 nmoles of the Ca²⁺ standard after addition of 2 µmol l⁻¹ Ruthenium Red to the assay mixture to inhibit mitochondrial Ca²⁺ uptake. This served as an internal control for endogenous buffering of added Ca²⁺.

Unless otherwise noted, the data are means ± S.E.M. of measurements from 4–8 preparations from different fish. Comparisons between treatments were made using a Kruskal-Wallis one-way analysis of variance (ANOVA). Differences at the P<0.05 level were considered significant.

Mitochondria isolated from both heater organs and red locomotory muscle were well coupled and respired on a variety of substrates. Isolated heater organ mitochondria typically displayed respiratory control ratios (RCRs) above 3.0 and red muscle mitochondria frequently showed higher values (Table 1). With heater organ mitochondria, it was necessary to omit Mg²⁺ from the assay medium as Mg²⁺-stimulated ATPase activity was present in the preparations and interfered with P/O ratio measurements. This activity was very low in red muscle mitochondrial preparations, but Mg²⁺ was omitted from all experiments in order to maintain similar conditions throughout. Preliminary experiments with red muscle mitochondria (results not shown) indicated that P/O ratios and RCRs were not different in media containing Mg²⁺.

The P/O ratios measured using 10 mmol l⁻¹ glutamate plus 5 mmol l⁻¹ malate as the substrate were within the range reported for mammalian mitochondria (Table 1). The P/O ratio measured for red muscle mitochondria was significantly higher than that measured for heater organ mitochondria, but state 4 and state 3 respiration rates and respiratory control ratios were not significantly different.

Free Ca²⁺, when added to heater organ mitochondria at micromolar concentrations, was taken up in an energy-dependent fashion. In Fig. 1, the respiration-dependent Ca²⁺ uptake pathway and Na⁺-induced efflux pathway in heater organ mitochondria are followed using a Ca²⁺ electrode. Ca²⁺ added to non-energized mitochondria was not taken up until a respiratory substrate was provided. Upon addition of 5 mmol l⁻¹ malate plus 10 mmol l⁻¹ glutamate, oxygen consumption (results not shown) and Ca²⁺ uptake were stimulated. The respiration-dependent Ca²⁺ uptake was completely abolished by 1 µmol l⁻¹ Ruthenium Red. Ca²⁺ efflux from the Ruthenium-Red-inhibited respiring mitochondria could not be measured until 5 mmol l⁻¹ NaCl was added to the medium. After Na⁺ addition, Ca²⁺ was released at a steady rate.

To study the effects of Ca²⁺ on respiratory coupling, we measured the P/O ratios and respiratory rates of mitochondria over a range of concentrations of added Ca²⁺. Fig. 2 shows the effects of 11.1 µmol l⁻¹ added free Ca²⁺ on respiration and oxidative phosphorylation by red muscle mitochondria. Ca²⁺ was added in these experiments to mitochondria respiring in state 4 in the presence of excess substrate (10 mmol l⁻¹ glutamate plus 5 mmol l⁻¹ malate). Following stabilization of state 4 respiration, ADP was added to stimulate state 3 respiration. In Fig. 2A, most of the added Ca²⁺ was rapidly taken up (Ca²⁺ electrode trace) with a concomitant increase in state 4 respiration. The mitochondria were still able to phosphorylate ADP in the presence of free Ca²⁺, but the P/O ratio measured (1.99) was well below the control P/O ratio of 2.88 measured in the absence of Ca²⁺. The addition of 2 µmol l⁻¹ Ruthenium Red (Fig. 2B) prevented Ca²⁺ uptake and stimulation of state 4 respiration. The P/O ratio measured under these conditions was 2.82, showing that the reduction in the P/O ratio caused by added Ca²⁺ is due to uptake through the Ruthenium-Red-sensitive Ca²⁺ uniporter.

Heater organ mitochondria showed similar effects of added Ca²⁺ on the P/O ratio to those found in red muscle mitochondria. Fig. 3 shows the concentration-dependence of these effects. As levels of free Ca²⁺ and Ca²⁺ loaded into the mitochondria increased, the P/O ratio was progressively reduced (significant reductions are indicated by an asterisk at each Ca²⁺ concentration) and the mitochondrial preparations tended to lose respiratory control. The addition of 2 µmol l⁻¹ Ruthenium Red before Ca²⁺ addition prevented this effect. The effects of Ca²⁺ levels on eight heater organ (Fig. 3A) and six red muscle (Fig. 3B) mitochondrial preparations are shown. However, a number of red muscle preparations lost respiratory control at higher Ca²⁺ concentrations and could not be included in the analysis. The P/O ratios plotted represent only those from preparations that had measurable respiratory control at the designated Ca²⁺ concentration. The reductions in P/O ratio compared to the Ruthenium-Red-treated controls were statistically significant above 10 µmol l⁻¹ added Ca²⁺ for both types of mitochondria, while at 1.1 µmol l⁻¹ free Ca²⁺, only red muscle mitochondria showed a significantly reduced P/O ratio. The Ca²⁺-dependent reductions in the P/O ratio described above were associated with a loss of respiratory control caused, in part, by a decrease in the state 3 respiratory rate. Fig. 4 shows the reduction in state 3 respiratory rates compared to Ruthenium-Red-treated controls over the range of Ca²⁺ concentrations tested. Due to the variation between preparations in absolute respiratory rate, the state 3 respiratory rate of each preparation was normalized to the state 3 rate of the same preparation in the absence of added Ca²⁺ for statistical comparisons. Both heater organ (Fig. 4A) and red muscle (Fig. 4B) mitochondria showed declines in state 3 respiration rate relative to Ruthenium-Red-treated controls. In red muscle, a significant reduction in state 3 respiratory rate occurred at 1 µmol l⁻¹ free Ca²⁺ as well as at each higher Ca²⁺ concentration. Heater organ mitochondria showed a significant reduction in state 3 respiration rate compared to the Ruthenium-Red-treated control at free Ca²⁺ levels above 10 µmol l⁻¹.

In heater organ mitochondria, the respiration rates in Ruthenium-Red-treated controls were significantly higher in the presence of added Ca²⁺ than in its absence (Fig. 4A). This was not the case for red muscle mitochondria (Fig. 4B). This effect counteracts the inhibition of state 3 respiration by external Ca²⁺ in heater organ mitochondria. In fact, the state 3 respiration rate was significantly different from that of control preparations without Ca²⁺ only at 80 µmol l⁻¹ free Ca²⁺, which is well above the expected physiological range. These observations indicate that two processes occurred together in the heater organ mitochondria experiments: (1) a Ruthenium-Red-sensitive inhibition of respiration caused by added Ca²⁺, as is the case with red muscle mitochondria, and (2) a Ruthenium-Red-resistant effect that stimulates respiration in the presence of externally added Ca²⁺ in the micromolar range.

We examined the influence of low concentrations of Ca²⁺ on the activity of 2-oxoglutarate dehydrogenase, an important tricarboxylic acid (TCA) cycle enzyme that is directly modulated by Ca²⁺ (McCormack and Denton, 1990). In this experiment, state 3 respiration of heater organ mitochondria on 2-oxoglutarate was measured under conditions in which the supply of substrate was limiting. A concentration of 200 µmol l⁻¹ 2-oxoglutarate was found empirically to give somewhat less than half-maximal state 3 respiration rates in the presence of 75 µmol l⁻¹ ADP. Added Ca²⁺ had complex effects on the respiration rate under these conditions. Fig. 5 shows the effect of Ca²⁺ on respiration rate normalized to the control (no Ca²⁺ addition) state 3 respiration rate for each preparation. The mean ± S.E.M. state 3 respiration rate was 33.0±7.8 nanoatoms oxygen min⁻¹ mg protein⁻¹. Free Ca²⁺ levels below 1 µmol l⁻¹ increased the state 3 respiration rate by up to 15 % above the control value. However, at 1 µmol l⁻¹ and above, steady-state respiration was inhibited, as was seen in experiments involving saturating concentrations of glutamate. In the experiments involving Ca²⁺ concentrations of 1 µmol l⁻¹ and above, the addition of Ca²⁺ induced a transient increase in the respiration rate that preceded the inhibited steady state. The transient respiration rates are shown in Fig. 5 (open symbols). The transient increases in respiration were not linearly related to Ca²⁺ concentration, but were consistently of the same size as the increased state 3 rate observed at 0.2 µmol l⁻¹ Ca²⁺. The kinetics of respiration rate changes suggest the presence of two processes: (1) a relatively rapid activation by submicromolar Ca²⁺ levels and (2) a slower inhibition by micromolar Ca²⁺ levels similar to that observed with excess glutamate as the substrate.

Regulation of metabolism by Ca²⁺ is important in many cell types, and in excitable cells the roles of Ca²⁺ are complex. Both the basal cytoplasmic level of free Ca²⁺ and transient Ca²⁺ concentration peaks are of physiological importance, and may serve quite different roles. In muscle cells, the contractile apparatus is triggered by large-amplitude Ca²⁺ release events while the basal, or perhaps time-averaged, myoplasmic Ca²⁺ concentration regulates factors such as the activity of mitochondrial enzymes that affect energy supply (McCormack and Denton, 1990). The heater organ of billfishes is an example of a tissue in which the normal muscle Ca²⁺ release cycle has been modified and harnessed to generate heat (Block, 1987). Heater cells lack the contractile elements of muscle, showing almost no detectable myosin ATPase activity (Block, 1986; Tullis, 1994). However, they retain an extensive network of sarcoplasmic reticulum and T-tubules with a configuration unique among muscle tissues. An average of 63 % of the heater cell volume is occupied by mitochondria, and a large fraction of the remaining cell volume is taken up by SR and T-tubule membrane systems with stacks of sarcoplasmic reticulum forming a network surrounding each mitochondrion (Block, 1987; Block and Franzini-Armstrong, 1988). In addition, the heater cell plasma membrane is endowed with a rich supply of acetylcholine receptors (Block, 1994). These histological characteristics suggest that heater cells function by a depolarization-induced Ca²⁺ release process, as in normal muscle.

A number of biochemical studies indicate that the heater organ may have Ca²⁺ release and uptake properties similar to fast-twitch oxidative muscle. The heater organ expresses only the α (RyR1) isoform of the SR Ca²⁺-release channel (Block et al. 1994), unlike the majority of other skeletal muscles in non-mammalian vertebrates, which express two different isoforms (α, and β or RyR3) together (Airey et al. 1990; Olivares et al. 1991; O’Brien et al. 1993). We have shown previously that expression of the α isoform of the Ca²⁺-release channel alone is associated with muscles specialized for high-frequency contraction (O’Brien et al. 1993). In addition, the heater organ expresses the SERCA1 or fast isoform of the Ca²⁺-ATPase (Tullis, 1994). The presence of the fast Ca²⁺ release and uptake proteins suggests that heater organs are capable of performing a rapid cycle of Ca²⁺ release and reuptake.

Knowledge of the interaction between Ca²⁺ and mitochondria in heater organs is also important for understanding the thermogenic cycle. The high density of mitochondria in heater organs and the close proximity of the mitochondria to the SR suggest that Ca²⁺ released from the SR may be taken up directly by the mitochondria. This process could make an important contribution to the mechanism of thermogenesis. In Fig. 6, two models depicting different potential Ca²⁺ cycles in heater cells are shown. Fig. 6A shows a model in which direct Ca²⁺ uptake by mitochondria through the Ca²⁺ uniporter makes a major contribution to the thermogenic cycle. In this model, Ca²⁺ uptake and subsequent Na⁺-dependent efflux stimulates respiration by consuming the energy in the electrical and proton gradients. A portion of the respiratory energy may be diverted away from oxidative phosphorylation to support this process and this would lead to a reduction in ATP output. Following a transient increase in Ca²⁺ level, oxidative phosphorylation would return to normal as the myoplasmic Ca²⁺ concentration is reduced by the action of Ca²⁺-ATPase. An elegant demonstration of this sort of effect has been made in cultured neuroblastoma cells (Loew et al. 1994). Optical measurements of mitochondrial membrane potential and cytoplasmic Ca²⁺ concentration revealed mitochondrial depolarizations in response to transient increases in cytoplasmic Ca²⁺ levels induced by high extracellular K⁺ levels or hormones. These results suggest that the mitochondrial protonmotive force is reduced during a transient increase in Ca²⁺ level and thus oxidative phosphorylation may be reduced.

An alternative model, shown in Fig. 6B, is one in which the activities of the mitochondrial Ca²⁺ uptake and efflux pathways are relatively low and ATP output is maintained during a transient increase in Ca²⁺ level in the cell. In order to stimulate respiration and thermogenesis, one or several ATP-consuming processes in the cell must operate at a high level. This model would not exclude uptake of a small amount of Ca²⁺ into the mitochondrial matrix, which could activate several matrix dehydrogenases and thus enhance respiration (McCormack et al. 1990; McCormack and Denton, 1993). The model in Fig. 6B depicts Ca²⁺ pumping by the SR Ca²⁺-ATPase as the major ATP-consuming process, although some contribution by the several other ATPases present would be expected. Sustained or frequent Ca²⁺ release events would be needed to maintain the activity of the ATPase and support thermogenesis. The main differences between these models lie in the properties of the mitochondrial Ca²⁺ uptake and release pathways, and characterization of the mitochondrial response to Ca²⁺ will help to choose between the models.

Mitochondria from both the heater organ and red muscle were able to take up Ca²⁺ from the medium, and Ca²⁺ uptake had a negative effect on respiratory coupling. At concentrations above 10 µmol l⁻¹ free Ca²⁺, the mitochondria showed significantly reduced P/O ratios and hence a reduced efficiency of coupling. At 1 µmol l⁻¹ free Ca²⁺, near the probable upper limit of physiological Ca²⁺ transients in muscle cells, the P/O ratio of heater organ mitochondria was not significantly reduced. Interestingly, a significant reduction in the P/O ratio was observed in red muscle mitochondria at 1 µmol l⁻¹ free Ca²⁺. Two factors make it impossible to evaluate this difference directly. Heater organ mitochondria differed significantly from red muscle mitochondria in having both a reduced respiratory control ratio and a lower P/O ratio in control experiments. Thus, a significant reduction in coupling (and, by inference, in protonmotive force) caused by Ca²⁺ uptake may put red muscle mitochondria in a condition similar to that of heater organ mitochondria assayed under control conditions. Nonetheless, it is evident that the heater organ mitochondrial preparations used were capable of maintaining control levels of respiratory coupling and P/O ratio at physiological levels of Ca²⁺.

One of the primary factors affecting respiratory control was the change in state 3 respiration rate in the presence of added Ca²⁺. Compared to control preparations treated with 2 µmol l⁻¹ Ruthenium Red, both heater organ and red muscle mitochondria showed reductions in the state 3 respiration rate in the presence of added Ca²⁺. These reductions were significant at free Ca²⁺ concentrations of 10 µmol l⁻¹ and above, and also at 1 µmol l⁻¹ for red muscle mitochondria. The heater organ mitochondria showed a second effect that was not shown by red muscle mitochondria. In heater organ mitochondria treated with Ruthenium Red, added Ca²⁺ significantly stimulated state 3 respiration. This effect may also occur in the absence of Ruthenium Red. This suggests that a stimulatory effect of Ca²⁺ on state 3 respiration in heater organ mitochondria may counteract the inhibitory effect of Ca²⁺ also observed. The basis for this stimulatory effect is not known. It is possible that the Ruthenium-Red-sensitive Ca²⁺ uniporter was not entirely blocked in the heater organ mitochondrial experiments and that some Ca²⁺ was taken up into the mitochondrial matrix. This could have the effect of activating matrix dehydrogenases (McCormack et al. 1990) and stimulating respiration, as will be discussed further below. However, no uptake of added Ca²⁺ was detectable with the Ca²⁺ electrode under these conditions, so any leak past the Ruthenium Red block must have been small relative to the normal rate of uptake.

The regulatory effects of Ca²⁺ on mitochondrial matrix dehydrogenases have been widely studied (see McCormack et al. 1990 for a review). Such effects may be important in the maintenance of elevated metabolism in the heater organ during calcium release events. In the present study, we examined the effects of Ca²⁺ on 2-oxoglutarate dehydrogenase activity in heater organ mitochondria. This experiment showed that Ca²⁺ uptake caused a small but consistent increase in 2-oxoglutarate dehydrogenase activity at concentrations above 0.2 µmol l⁻¹ free Ca²⁺. This activation was only transiently evident at concentrations above 1 µmol l⁻¹ free Ca²⁺, as the inhibition of respiration noted with saturating amounts of glutamate as the substrate was also observed under the conditions of this experiment. However, 0.2–1 µmol l⁻¹ free Ca²⁺ produced sustained activation of 2-oxoglutarate-supported respiration, indicating that probable physiological concentrations of Ca²⁺ can stimulate respiration. The degree of 2-oxoglutarate dehydrogenase activation observed, approximately 15 %, is smaller than many values reported for mammalian mitochondria, which can be as high as 200 % (McCormack et al. 1990). However, it is possible that the mitochondria used in this study contained elevated matrix concentrations of Ca²⁺ that were not depleted during the isolation procedure. In this case, true basal 2-oxoglutarate dehydrogenase activity may not have been measured and the degree of activation underestimated. In any case, it is evident that sub-micromolar Ca²⁺ levels can stimulate heater organ 2-oxoglutarate dehydrogenase and, by inference, respiratory activity. Thus, it is possible that during thermogenesis, the uptake of a small amount of Ca²⁺ may have an activating effect on mitochondrial oxidative phosphorylation and enhance ATP output. However, the present experiments suggest that this effect is marginal.

In summary, this study indicates that heater organ mitochondria are not significantly uncoupled under conditions likely to occur in the heater cell during a cycle of Ca²⁺ release. This property of heater organ mitochondria suggests that the model shown in Fig. 6B is a more accurate description of the mechanism of metabolic activation in the heater organ: heater organ mitochondria maintain ATP output during high Ca²⁺ transients. Respiration and ATP output may, in fact, be enhanced by the action of Ca²⁺ on mitochondrial matrix dehydrogenases. The biochemical properties of heater tissue indicate a relationship with fast-twitch oxidative muscle and suggest that the heater cell is designed for Ca²⁺ release events that are relatively brief and rapidly repeated. To date, no measurements of cytoplasmic Ca²⁺ transients have been made in heater cells, so the frequency of Ca²⁺ release, peak free Ca²⁺ concentration and average free Ca²⁺ concentration are not known. Nonetheless, the fast-twitch characteristics of the heater cell SR support the contention that released Ca²⁺ is probably quickly returned to the SR and that uptake of Ca²⁺ by the mitochondria is limited. These findings support the hypothesis that mitochondrial ATP output is maintained and that activity of an ATP-consuming process (e.g. Ca²⁺ uptake via the SR Ca²⁺-ATPase) is required to stimulate respiration and generate heat.

The authors are indebted to the Pacific Ocean Research Foundation for research and facilities support. We also gratefully acknowledge the assistance of the Hawaiian International Billfish Association in obtaining fresh specimens. This work was supported by NIH grant AR40246 and NSF grant IBN8958225 to B.A.B. J.O. was supported by postdoctoral fellowship AR08254 from NIH.