Role of Calcium Signaling in the Activation of Mitochondrial Nitric Oxide Synthase and Citric Acid Cycle

Home , Calcium cycle

Biochimica et Biophysica Acta 1658 (2004) 64–71 www.bba-direct.com Review Role of calcium signaling in the activation of mitochondrial nitric oxide synthase and citric acid cycle

Nathaniel Traaseth, Sarah Elfering, Joseph Solien, Virginia Haynes, Cecilia Giulivi*

Departments of Chemistry and Biochemistry and Molecular Biology, University of Minnesota, 10 University Drive, Duluth, MN 55812, USA Received 18 February 2004; received in revised form 21 April 2004; accepted 26 April 2004 Available online 15 June 2004

Abstract

An apparent discrepancy arises about the role of calcium on the rates of oxygen consumption by mitochondria: mitochondrial calcium increases the rate of oxygen consumption because of the activation of calcium-activated dehydrogenases, and by activating mitochondrial nitric oxide synthase (mtNOS), decreases the rates of oxygen consumption because nitric oxide is a competitive inhibitor of cytochrome oxidase. To this end, the rates of oxygen consumption and nitric oxide production were followed in isolated rat liver mitochondria in the G presence of either L-Arg (to sustain a mtNOS activity) or N -monomethyl-L-Arg (NMMA, a competitive inhibitor of mtNOS) under State 3 conditions. In the presence of NMMA, the rates of State 3 oxygen consumption exhibited a K0.5 of 0.16 AM intramitochondrial free calcium, agreeing with those required for the activation of the Krebs cycle. By plotting the difference between the rates of oxygen consumption in State 3 with L-Arg and with NMMA at various calcium concentrations, a K0.5 of 1.2 AM intramitochondrial free calcium was obtained, similar to the K0.5 (0.9 AM) of the dependence of the rate of nitric oxide production on calcium concentrations. The activation of dehydrogenases, followed by the activation of mtNOS, would lead to the modulation of the Krebs cycle activity by the modulation of nitric oxide on the respiratory rates. This would ensue in changes in the NADH/NAD and ATP/ADP ratios, which would influence the rate of the cycle and the oxygen diffusion. D 2004 Elsevier B.V. All rights reserved.

Keywords: Mitochondria; Nitric oxide; Calcium; Krebs cycle; Dehydrogenase; Cytochrome oxidase; Oxygen; Nitric oxide synthase

1. Introduction Calcium also plays an important role in the regulation of the citric acid cycle by activating pyruvate dehydrogenase, Cells need mechanisms that ensure that the rate of NAD+-dependent isocitrate dehydrogenase, and 2-oxoglu- formation of ATP matches tissue requirements for this tarate dehydrogenase [1–5], thus allowing the same signal molecule. In most mammalian cells, ATP is mainly formed that stimulates muscle contraction to activate the production through mitochondrial oxidative phosphorylation. Changes of ATP to sustain it. Hence, and owed to many feedback in ATP synthesis can be completed through alterations in the mechanisms, oxygen consumption, NADH reoxidation, and activities of the mitochondrial dehydrogenases, which gen- ATP production are tightly coupled to coordinate its NADH erate NADH and FADH for the respiratory chain. Three of production with energy expenditure. these dehydrogenases are of particular importance (i.e., Nitric oxide has been proposed as a modulator of the pyruvate dehydrogenase, NAD+-dependent isocitrate dehy- oxygen consumption, in a role beyond that mediated through drogenase, and 2-oxoglutarate dehydrogenase) because they the activation of soluble guanylate cyclase. The modulation can be activated by increases in calcium, [NAD+]/[NADH] of oxygen consumption is attained by the competitive or [ADP]/[ATP] ratios. Calcium, among its many biological inhibition of cytochrome oxidase by nitric oxide [6–8]. This functions, is an essential metabolic regulator. It stimulates can be efficiently achieved by stimulating mitochondria to glycogen breakdown, triggers muscle contraction, and produce nitric oxide for they are endowed with a mitochon- mediates many hormonal signals as a second messenger. drial nitric oxide synthase (mtNOS; see Refs. [9–15]). Experimental evidence for nitric oxide production by mito- * Corresponding author. Tel.: +1-218-726-7590; fax: +1-218-726-7394. chondria has been provided by our laboratory (using purified E-mail address: [email protected] (C. Giulivi). rat liver mitochondria [12,13]) and others [9–11,14,16,17]

0005-2728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2004.04.015 N. Traaseth et al. / Biochimica et Biophysica Acta 1658 (2004) 64–71 65 using a variety of approaches and biological systems. MO), except for sucrose and magnesium chloride (Mal- mtNOS has been identified as the nNOS isoform alpha, linckrodt), fatty acids free bovine serum albumin (ICN), precluding a novel alternative spliced product [18].Itis EGTA (Acros), and fura-2 (Molecular Probes). likely coded by the same gene as nNOS since nNOS K.O. mice have no mtNOS [16]. In contrast to nNOS, mtNOS has 2.2. Isolation of rat liver mitochondria been reported to have two distinct posttranslational modifications: acylation different from that found in eNOS, and Liver mitochondria were isolated from adult Wistar rats phosphorylation at the C-terminus [18]. Clearly, the modu- (180–200 g) by differential centrifugation [12]. The medi- lation of O2 consumption by endogenous nitric oxide is um used to isolate mitochondria contained 220 mM manni- transient and reversible as long as nitric oxide is generated in tol, 70 mM sucrose, 2 mM HEPES (pH 7.4), 0.1% defatted small quantities. Effects attributed to higher concentrations albumin, and 0.5 mM ethylene glycol bis(6-aminoethyl achieved during a sustained production of nitric oxide may ether)-N,N,NV,NV-tetraacetic acid (EGTA). With this tech- include the inhibition of the respiratory chain at different nique, it is possible to arrest calcium redistribution, which is levels [19], increased rate of oxygen free radical production necessary when studying the effects of hormone pretreat- [19–21], cytochrome c release [22], and nitration of proteins ment [31,32] (see also below). Experiments with 45Ca2+ [23,24], among others. tracer have established that EGTA prevents calcium uptake Considering that the constitutive forms of nitric oxide and that at 0–4 jC there is very little calcium efflux from synthase, i.e., nNOS and eNOS, account for the rapid, mitochondria, even in the presence of Na+. Purified mito- transient, calcium-dependent production of nitric oxide chondria were obtained by Percoll gradient centrifugation [25–27], thus, it would be expected that increases in [12], subsequently washed with 150 mM KCl to dissociate mitochondrial calcium are required to activate mtNOS. In arginase nonspecifically bound proteins. The Percoll purifi- line with this assumption, stimulation of nitric oxide pro- cation step greatly reduces the contamination by endoplas- duction by mtNOS was observed by bolus additions of mic reticulum and plasma membranes. The respiratory calcium to mitochondria [14]. The authors proposed that control ratio (RCR) was 5.5 F 0.5 (n = 12) with succinate. uptake of calcium by respiring mitochondria may lead to Protein was determined by the Lowry assay using bovine increased peroxynitrite formation in mitochondria, which in serum albumin as a standard [33]. turn causes calcium release [28] via the pyridine nucleotide- dependent pathway [29] followed by mtNOS deactivation. 2.3. Measurement of nitric oxide production These observations have been interpreted as part of a feedback loop that prevents calcium overloading and allows mtNOS activity was evaluated by absorption spectropho- its release preserving membrane potential [14]. Considering tometry by following the oxidation of oxymyoglobin. This the above situations, an apparent discrepancy arises in terms assay is based on the oxidation of oxy- to metmyoglobin by . G of the role of calcium on the rate of oxygen consumption: NO [12], sensitive to N -monomethyl-L-Arg (NMMA) mitochondrial calcium increases the rate of oxygen con- inhibition. This oxidation was followed at 630 nm in a sumption as a result of the activation of calcium-activated spectrophotometer Cary 1E or in a dual-wavelength, double- dehydrogenases, and by activating mtNOS, decreases the beam DW-2C at 22 jC in the presence of 0.2 mM oxy- oxygen consumption due to the nitric oxide–cytochrome myoglobin using the following buffer: 125 mM KCl, 5 mM oxidase interplay. Albeit many studies have been published potassium phosphate, 1 mM HEDTA, 0.2 mM EGTA, 50 on the regulation of citric acid cycle by calcium, compar- mM MOPS, pH 7.4, maintaining MgCl2 at a constant isons between the effects of calcium under conditions of concentration of 1 mM. Oxymyoglobin was prepared from mtNOS inhibition or stimulation have not been reported a 1 mM solution of commercially available horse heart earlier. Because mitochondria are an important cellular myoglobin in 0.1 M HEPES buffer, pH 7.4, and desalted by calcium buffer, and the mechanisms controlling this cation gel filtration. concentration are relevant to mitochondrial metabolism and to cell proliferation, apoptosis, and necrosis [30], we studied 2.4. Determination of respiratory rates the regulation of these processes, i.e., Krebs cycle vs. mtNOS activities, by changing mitochondrial free calcium Rates of oxygen consumption were measured polaro- concentrations. graphically at 25 jC with a Clark oxygen electrode (Hansatech Instruments Limited Kings Lynn, Norfolk, UK). Typical incubations are carried out with 0.5–1 mg 2. Materials and methods mitochondrial protein in 1 ml volume of incubation medi- um (125 mM KCl, 5 mM potassium phosphate, 1 mM 2.1. Chemicals and biochemicals HEDTA, 0.2 mM EGTA, 50 mM MOPS, pH 7.4, maintaining MgCl2 at a constant concentration of 1 mM) at All reagents used were of analytical grade. All chemicals 25 jC for 5 min to achieve steady state conditions, and biochemicals were purchased from Sigma (St. Louis, followed by a suitable respiratory substrate (e.g., 10 mM 66 N. Traaseth et al. / Biochimica et Biophysica Acta 1658 (2004) 64–71 a-ketoglutarate), and the additions of calcium and/or 0.3 2.2 F 0.3 nmol nitric oxide/min mg protein. The inhibition mM ADP as indicated. of the mitochondrial oxygen consumption was ascribed to the production of nitric oxide by mtNOS for it was abro- 2.5. Calcium concentration evaluation gated upon the addition of 2 mM oxymyoglobin (which converts nitric oxide to nitrite with the formation of met- Mitochondria (10 mg protein/ml) were loaded with fura- myoglobin), NMMA (a competitive inhibitor of NOS), and 2 2 by incubating with 10 AM of the dye for 20 min [34,35]. D-Arg , and it was found resistant to the inhibition by The excess of dye was removed by several washes per- NMMA. The inhibition of cytochrome oxidase activity formed by centrifugation, and the RCR and P/O numbers has been interpreted as the competition between nitric oxide were checked after this treatment to assure that mitochon- and oxygen for the binding to cytochrome oxidase [8]. This dria were still coupled and functional as the original inhibition is reversible, with no changes in the Vmax and preparations. The fluorescence of the mitochondrial suspen- increases in the apparent Km for oxygen in the presence of sion was measured at kexc 350 (calcium-sensitive k) and 365 nitric oxide [8]. nm (calcium-insensitive k) and kem 510 nm, and rationing This rate of nitric oxide production was obtained without the emission at the calcium-sensitive with the calcium- the exogenous supplementation of calcium to mitochondria insensitive wavelength. The calculations regarding calcium as it was observed by our lab previously [8,12]. Thus, it was concentration required the measurement of the minimum assumed that the concentration of this cation in our mito- (with EGTA and Triton X-100) and maximum (plus 10 mM chondrial preparations was sufficient to sustain a mtNOS CaCl2) fluorescence intensities [36]. activity. This assumption was confirmed by evaluating the calcium content of these mitochondria which resulted to be 2.6. Statistical methods (0.9 F 0.1) AM. Given that the reported K0.5 of purified nNOS from various species is between 0.2 and 0.4 AM The results represent the mean F S.E. of three replicates [27,37,38], and assuming a Michaelis–Menten behavior for from one experiment. Each experiment was carried out a mtNOS (and that all substrates and cofactors are not limiting minimum of three times, and the results showed represent for its activity), then the production of nitric oxide evaluated all results obtained. The effect of treatment was compared under these conditions (without exogenous supplementation with control values by one-way analysis of variance of calcium) would represent a 75% of the Vmax. (ANOVA). Tests were carried out using StatSimple version 2.0.5 from Nidus Technologies (Toronto, Canada). 3.2. Effect of calcium on respiratory rates of NMMA-treated liver mitochondria

3. Results and discussion To investigate the effect of calcium concentrations on Krebs cycle activity alone, we evaluated the rates of oxygen 3.1. Production of nitric oxide with endogenous mitochon- consumption in the presence of NMMA under States 4 and drial calcium 3 (substrate-supplemented mitochondria and phosphorylat- ing mitochondria, respectively). To evaluate the effect of Purified and coupled rat liver mitochondria supple- calcium on the oxygen consumption when mtNOS is mented with 10 mM of either succinate or a-ketoglurate activated, we followed the rates of oxygen consumption consumed oxygen at a rate of 10 F 1 and 11 F 1 nmol and nitric oxide production in the presence of L-Arg under oxygen/min mg protein, respectively. Upon the addition of State 3 (Fig. 1) and State 4 (not shown) conditions. 0.3 mM ADP, the oxygen consumption increased to 52 F 4 Given that mitochondrial dehydrogenases are sensitive and 56 F 1 nmol oxygen/min mg protein with succinate and to changes in the 0.1–10 AM calcium concentration range, a-ketoglurate as respiratory substrates, respectively. When these preparations were supplemented with L-Arg (3 Amol/ mg mitochondrial protein), these rates were decreased (in 1 average) by 43 F 3% at 220 AM oxygen . Under these 2 In previous experiments from our lab [12], 10 min of preincubation conditions, the rate of nitric oxide production was with D-Arg or direct addition of it to mitochondria, followed by L-Arg addition, to evaluate nitric oxide production, did not prevent this generation. We indicated that in the presence of both L- and D-Arg, resulted in the preferential—if not exclusive—transport of the L-isomer to 1 As the inhibition of cytochrome oxidase is dependent on the [nitric mitochondria, thus allowing mtNOS to sustain its activity. The conditions oxide]/[oxygen] ratio [8], to avoid the situation of a nonstationary function used in this experiment that allowed us to observe the inhibition of nitric as pO2 falls during the experiment, the rates of oxygen consumption oxide production were performed with toluene-permeabilized mitochondria reported in this study were evaluated between 150 and 220 AM for (obtained as described before [12]), to overcome the specificity of the cytochrome oxidase activity and the inhibition of the oxygen consumption transporter and to concentrate on the direct effect of D-Arg on mtNOS by nitric oxide was not significantly affected within this range of oxygen activity. In addition, these experiments were performed in the presence of a concentration [8]. Current experiments in our lab are studying these cocktail of proteolytic inhibitors to suppress the eventual proteolytic processes at more physiological oxygen concentrations. activity that may serve as a source of endogenous L-Arg. N. Traaseth et al. / Biochimica et Biophysica Acta 1658 (2004) 64–71 67

transport. To evaluate the possibility of calcium-dependent uncoupling, we evaluated the P/O values in the presence of succinate as a function of calcium concentration. The P/O ratios were constant up to 5 AM extramitochondrial free calcium or 9 AM intramitochondrial free calcium, suggesting that uncoupling was not significant at the calcium concentrations used in this study (1.51 F 0.02 and 1.55 F 0.03 at 0 and 6 AM intramitochondrial free calcium, respectively). Thus, the increase in oxygen consumption upon addition of calcium was attributed mainly to the activation of matrix dehydrogenases. In this regard, our experimental K0.5 is in accordance with those reported for a-ketoglutarate dehydrogenase (0.12–0.8 AM [43]; see Ref. [64]). Previous reports indicated that increases in matrix free calcium elevated the activities of three dehydrogenases of the citric acid cycle, namely, pyruvate, oxoglutarate, and isocitrate dehydrogenases [1–5], and some studies indicated Fig. 1. Effect of intramitochondrial free calcium concentration on the rate of oxygen consumption by rat liver mitochondria. Rat liver mitochondria that the most significant effects of calcium are exerted via (0.4–0.5 mg protein) were incubated in the reaction buffer with HEDTA/ stimulation of the a-ketoglutarate dehydrogenase flux (Ref. EGTA/MOPS, 10 mM a-ketoglutarate, 0.3 mM ADP, and calcium. The [43] and references therein). If this is the case, under our oxygen consumption was evaluated under State 3 with L-Arg (triangles) or experimental conditions, an extramitochondrial free calcium NMMA (open circles) using an oxygen electrode. The intramitochondrial concentration of 0.5 AM, which results in a matrix free free calcium concentrations were evaluated by using fura-2. The kinetics for A 2+ the rate of oxygen consumption in the presence of L-Arg only (closed calcium concentration of 1.5 M (at 1 mM Mg and 0.2 + circles) were obtained assuming that the observed rate of oxygen mM Na ; experimentally evaluated using fura-2 and sup- consumption with L-Arg was equal to the following equation: ported by the values obtained in Ref. [43]), would result in a decrease in the apparent Km for a-ketoglutarate from 2.5 to 2þ 2þ vobs ¼ vKrebsð1Þ þ vLÀArgð1Þ ¼½Ca 1Vm=ðEC50ð1Þ þ½Ca 1Þ 0.6 mM [43]. The higher affinity of a-ketoglutarate dehy- 2þ 2þ drogenase for a-ketoglutarate would lead to a 1.3-fold þ½Ca Vm=ðEC þ½Ca Þ 2 50ð2Þ 2 increase in the oxygen consumption at 10 mM a-ketoglutarate. In line with this, the increase in oxygen consumption observed under our experimental conditions was consistent it was essential to control the concentration accurately with that expected for such a change in the apparent Km using appropriate buffers. Because EGTA is suitable at (Fig. 1). pH V 7.2, to cover a wider range that included pH 7.4, we used a combination of EGTA, N-(2-hydroxyethyl) 3.3. Effect of calcium on respiratory rates in L-Arg- ethylenediamine triacetate (HEDTA) and N-(morpholino) supplemented liver mitochondria propanesulfonic acid (MOPS; see Refs. [39–41]). Mitochon- dria (isolated as described under Materials and methods) were To investigate the effect of calcium on oxygen con- incubated for 10 min in this buffer, and the concentrations of sumption by nitric-oxide-producing mitochondria, experi- extramitochondrial free calcium in the reaction mixture were ments were performed with mitochondria incubated with calculated utilizing published equilibrium constants, at a L-Arg. The rates of oxygen consumption in State 3 with fixed Mg concentration, with or without the addition of L-Arg increased with extramitochondrial free calcium ADP [4,42]. Using fura-2 as an indicator for matrix free concentrations, although at a lower extent than those in calcium, it was possible to assess the effects of intramito- thepresenceofNMMA(Fig. 1). This effect was chondrial free calcium on mitochondrial metabolism. expected because of the competitive inhibition of cyto- In the presence of NMMA, the rates of State 3 oxygen chrome oxidase by nitric oxide [8]. Because the ob- consumption of liver mitochondria increased with extra- served rate of oxygen consumption in the presence of L- mitochondrial free calcium concentrations. The effect of Arg is the result of two opposite effects, i.e., (1) the calcium on oxygen consumption is clearly shown in a inhibition of cytochrome oxidase by nitric oxide species Lineweaver–Burk plot (Fig. 1), which indicated that the produced by calcium-dependent mtNOS activity, and (2) K0.5 was 0.16 AM intramitochondrial free calcium con- the calcium-dependent activation of the Krebs cycle, then centration (equivalent to 0.07 AM for extramitochondrial by subtracting from the NMMA-supplemented rates free calcium). The increase in oxygen consumption can be of oxygen consumption (solely Krebs activation), the explained in terms of calcium activation of dehydro- L-Arg-supplemented rates (both effects), the resulting genases and/or mitochondrial uncoupling by calcium rates are those representing solely the effect of calcium 68 N. Traaseth et al. / Biochimica et Biophysica Acta 1658 (2004) 64–71 on mtNOS activity (Fig. 1). The kinetics of oxygen consumption showed a K0.5 of 0.64 and 2 AM extra- and intramitochondrial free calcium concentrations, respectively. It can be concluded that the activation of the Krebs cycle required a K0.5 of 0.60 AM of intramitochondrial free calcium (from NMMA data, see above), whereas the effect of nitric oxide on the respiratory rates exhibited a K0.5 of 2.0 AM. Thus, an extramitochondrial free calcium concentration (or as an extension, a cytosolic one) of 0.2 AM (concentration approximately equivalent to 0.6 AM intramitochondrial free calcium concentrations at 1 mM Mg2+ and 0.15 mM Na+ evaluated by fura-2 and confirmed by others under similar conditions [43]) will result in 50% of the Krebs cycle activation and 23% of the Vmax for nitric oxide production. In other words, even at this low, physiological cytosolic (or extramitochondrial) calcium concentration the amount of nitric oxide produced under these conditions would be sufficient to decrease the oxygen consumption in State 3. Fig. 2. Effect of intramitochodrial free calcium concentrations on the rate of nitric oxide production by intact mitochondria. Rat liver mitochondria 3.4. Effect of calcium on rate of nitric oxide production by (0.3–0.4 mg protein) were incubated with 1 mM oxymyoglobin and either liver mitochondria L-Arg (closed circles) or NMMA (open circles) in reaction buffer. The rate of oxymyoglobin formation was followed spectrophotometrically as When the rates of nitric oxide production by mitochon- indicated in Materials and methods. dria were independently evaluated varying the concentration of calcium, a sigmoidal curve was obtained with a K0.5 of 0.9 AM of intramitochondrial free calcium (Fig. 2; upon stimulation with hormones or other agents [47–50], equivalent to 0.26 AM extramitochondrial free calcium). the concentrations found in this study are within this No significant rate of nitric oxide production was obtained physiological range, and these two pathways could be when mitochondria were incubated in the presence of either activated simultaneously or sequentially, depending on the NMMA (regardless of the calcium concentration), indicat- calcium concentration. ing that the generation of nitric oxide was sustained by the The sigmoidal kinetics can be explained by either the mtNOS (Fig. 2),or20AM ruthenium red, an inhibitor of cooperative binding of calcium to calmodulin, which in the calcium uniporter (not shown). The K0.5 value was turn, activates NOS activity, or by considering the kinetics similar to that calculated from the difference in respiratory of the calcium uniporter, which is second-order in calcium rates with L-Arg and NMMA (K0.5 =1.2 AM; Fig. 1), concentrations. However, the large apparent Km for the indicating that both processes are biochemically linked, uniporter (10–20 AM; see Refs. [44,45]) makes it unlikely. and is low enough that the influx of calcium would not A Hill plot of these rates vs. calcium concentrations compromise the electrical potential difference across the resultedinanapparentnH of 2.98 as expected for a inner membrane. Of note, our experimental K0.5 was three positive cooperativity. Two macroscopic association con- 5 5 times higher than those reported for purified nNOS (K0.5 of stants were calculated of 6.7 10 and 0.67 10 , com- 0.2–0.4 AM [28,38,39]) or for less purified preparations parable to those obtained for calmodulin in the presence of from brain and N1E-115 neuroblastoma cells [39,51]. Mg2+ (3.0 105 and 0.62 105;seeRef. [46]). The Although mtNOS is the nNOS-1 alpha isoform, the appar- sigmoidal response (as opposite to a hyperbolic one) acts ent discrepancy can be attributed to the posttranslational as an ‘‘off–on switch’’ because a change in calcium modifications found in mtNOS that had not been reported concentration from 0.125 to 0.25 AM would result in a for nNOS [18]. 4-fold increase in the rate. To obtain the same increase in Our data demonstrated that the Krebs cycle dehydro- velocity, a hyperbolic curve (with the same K0.5) would genases (among them probably the a-ketoglutarate dehy- require a 5.4-fold increase in calcium concentration. Al- drogenase) are more sensitive to lower levels of calcium though other reports indicated the calcium dependency of a than mtNOS (compare 0.16 with 0.9 AM, respectively). mtNOS activity, the sigmoidal kinetics was not observed However, because cytosolic calcium of heart cells oscillates and comparisons between K0.5 are not possible for the from below 0.1 to over 1 AM from beat to beat (although experimental conditions (e.g., amount of mitochondria, matrix free calcium is difficult to predict), and 1 AM preparation procedure, and methodology) are not compara- cytoplasmic calcium concentrations have been reported ble [23]. N. Traaseth et al. / Biochimica et Biophysica Acta 1658 (2004) 64–71 69

4. Concluding remarks carrier is driven by the mitochondrial proton motive force [60]. It should be noted that the increased DW should have In this study, we investigated the effect of calcium on the important effects on mitochondrial calcium uptake as the activation of the Krebs cycle and mtNOS. Because it has initial rate of calcium uptake in liver mitochondria is pro- been indicated that the basal level of calcium in mitochon- portional to the magnitude of DW [61]. This suggests that not dria is about 0.2 AM and approaches 1 AM on treatment of only cytosolic calcium may regulate mitochondrial function, the preparations within hormones or other agents that but, once mtNOS is activated, subsequent incoming calcium increase cytoplasmic calcium concentration [47–50], the could be further modulated, e.g., by decreasing the available calcium concentrations used in this study were within this cytosolic calcium to nearby mitochondria. With higher con- physiological range. In this regard, the levels of calcium did centrations of calcium, it is also possible to activate the other not uncouple mitochondria (as it was observed by evaluat- two dehydrogenases (namely pyruvate and isocitrate), which ing P/O values with calcium concentrations) or were beyond have lower affinity for calcium activation3 [4] and the any reported physiological limit. increases in State 3 respiratory rates might result in signif- We have confirmed previous reports that low AM levels icant decreases in the oxygen concentration around these of calcium were required for the activation of dehydro- mitochondria within milliseconds. If oxygen supply is lim- genases [1–5]. In contrast to previous studies, our results iting, nitric oxide, by slowing down the respiratory rates of precluded the putative activation of mtNOS by either those mitochondria in which mtNOS has been activated by calcium or L-Arg, for NMMA, a competitive inhibitor of calcium, would allow a smoother gradient of oxygen to the mtNOS, had been present in those preparations in which the rest of the cell(s) [62], thus modulating the oxygen avail- sole effect of calcium on the Krebs cycle had to be ability and as a consequence, the activities of the dehydro- evaluated. genases as it has been observed in our study. This effect Using this approach, at extramitochondrial free calcium might explain the relatively slow reoxidation of NAD(P)H, concentrations (equivalent to cytosolic ones) as low as 70 compared to the return of mitochondrial calcium to basal nM were sufficient to exert an increase in the oxygen levels when hepatocytes are stimulated with vasopressin, consumption, resulting primarily from the increased Krebs aside from the increases in both DW and DpH [63]. cycle activity. It has been reported that a calcium-mediated A key objective for the near future will be to try to obtain activation of a-ketoglutarate dehydrogenase and the conse- more direct evidence for the control that matrix calcium quent decrease in a-ketoglutarate concentration in the ma- exerts on the Krebs cycle and mtNOS (among other effects) trix, results in the release of the inhibition of aspartate in intact cells or organs under conditions considered as aminotransferase by its competitive inhibitor a-ketoglurate ‘‘normoxic’’ (oxygen concentrations of 20 AM or below) for [52]. Aspartate aminotransferase is an enzyme required for the cells and tissues. the operation of the malate/aspartate shuttle [53], and its ensuing activation results in the increased aspartate efflux from mitochondria and glutamate influx in mitochondria Acknowledgements [54], mediated by the reported calcium-stimulated aspartate/glutamate carrier [55]. This activation would eventually The authors thank the excellent technical assistance of decrease the cytosolic [lactate]/[pyruvate] ratio, promoting Ms. Laura Yager. These studies had been supported by pyruvate and glucose oxidation. Combining these two effects grants from the National Institutes of Health GM66768 and initiated by calcium, an increase in matrix NADH would ES011407), Cottrell Research Corporation (CC5675), and result, as a consequence of the activation of a-ketoglurate American Chemical Society-Petroleum Research Fund dehydrogenase, and transport of reducing equivalents from (37470-B4). the cytosol via the malate/aspartate cycle would ensue. At higher calcium concentrations, the effect of nitric oxide on the respiratory rates would be evident, especially considering References the sigmoidal shape of the kinetics of nitric oxide production. 2+ A [1] R.M. Denton, J.G. McCormack, Ca transport by mammalian mito- The K0.5 for this production was 0.9 M intramitochondrial chondria and its role in hormone action, Am. J. Physiol. 249 (1985) free calcium concentration, which is within the physiological E543–E554. range and below (200 times in average) the concentration [2] J.G. McCormack, A.P. Halestrap, R.M. Denton, Role of calcium ions required to release cytochrome c to initiate apoptosis [58] or in regulation of mammalian intramitochondrial metabolism, Physiol. uncoupling (10 times; this study). Rev. 70 (1990) 391–425. The interaction of nitric oxide with cytochrome oxidase decreased the respiratory rates as observed in this and other 3 studies [8,14]. As a consequence, it has been reported that An alternative mechanism can be proposed considering that during a transient rise of cytosolic calcium, the mitochondrial calcium increases at a the mitochondrial membrane potential (DW) increases [14], slower rate, but stays elevated longer that the cytosolic one [56,57], thus an effect that has been proposed to activate the malate/ allowing the activation of dehydrogenases other than a-ketoglurate aspartate shuttle [59], given that the aspartate/glutamate dehydrogenase. 70 N. Traaseth et al. / Biochimica et Biophysica Acta 1658 (2004) 64–71

[3] R.M. Denton, J.G. McCormack, Ca2+ as a second messenger within D.J. Stuehr, Proteomic method identifies proteins nitrated in vivo mitochondria of the heart and other tissues, Annu. Rev. Physiol. 52 during inflammatory challenge, Proc. Natl. Acad. Sci. U. S. A. 98 (1990) 451–466. (2001) 12056–12061. [4] R.M. Denton, J.G. McCormack, Calcium and the regulation of [24] S.L. Elfering, V. Haynes, N. Traaseth, C. Giulivi, Aspects, mecha- intramitochondrial dehydrogenases, Methods Toxicol. 2 (1993) nism, and biological relevance of mitochondrial protein nitration sus- 390–403. tained by mitochondrial nitric oxide synthase, Am. J. Physiol. 286 [5] R.G. Hansford, Relation between mitochondrial calcium transport and (2004) H22–H29. control of energy metabolism, Rev. Physiol., Biochem. Pharmacol. [25] B. Mayer, K. Schmidt, P. Humbert, E. Bohme, Biosynthesis of endo- 102 (1985) 1–72. thelium derived relaxing factor: a cytosolic enzyme in porcine aortic 2+ [6] G.C. Brown, C.E. Cooper, Nanomolar concentrations of nitric oxide endothelial cells Ca dependently converts L-arginine into an acti- reversibly inhibit synaptosomal respiration by competing with oxygen vator of soluble guanylyl cyclase, Biochem. Biophys. Res. Commun. at cytochrome oxidase, FEBS Lett. 356 (1994) 295–298. 164 (1989) 678–685. [7] M.W.J. Cleeter, J.M. Cooper, V.M. Darley-Usmar, S. Moncada, [26] D.S. Bredt, S.H. Snyder, Isolation of nitric oxide synthase, a cal- A.H.V. Schapira, Reversible inhibition of cytochrome c oxidase, the modulin-requiring enzyme, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) terminal enzyme of the mitochondrial respiratory chain, by nitric 682–685. oxide. Implications for neurodegenerative diseases, FEBS Lett. 345 [27] B. Mayer, M. John, E. Bohme, Purification of Ca2+ calmodulin-de- (1994) 50–54. pendent nitric oxide synthase from porcine cerebellum. Cofactor— [8] C. Giulivi, Functional implications of nitric oxide produced by mi- role of tetrahydrobiopterin, FEBS Lett. 277 (1990) 215–219. tochondria in mitochondrial metabolism, Biochem. J. 332 (1998) [28] M. Schweizer, C. Richter, Peroxynitrite stimulates the pyridine nucle- 673–679. otide linked Ca2+release from intact rat liver mitochondria, Biochem- [9] T.E. Bates, A. Loesch, G. Burnstock, J.B. Clark, Immunocytochem- istry 35 (1996) 4524–4528. ical evidence for a mitochondrially located nitric oxide synthase [29] H.R. Lo¨tscher, K.H. Winterhalter, E. Carafoli, C. Richter, Hydroper- in brain and liver, Biochem. Biophys. Res. Commun. 213 (1995) oxides can modulate the redox state of pyridine nucleotides and 896–898. the calcium balance in rat liver mitochondria, Proc. Natl. Acad. Sci. [10] T.E. Bates, A. Loesch, G. Burnstock, J.B. Clark, Mitochondrial nitric U. S. A. 76 (1979) 4340–4344. oxide synthase: a ubiquitous regulator of oxidative phosphorylation, [30] C. Richter, Reactive oxygen and nitrogen species regulate mito- Biochem. Biophys. Res. Commun. 218 (1996) 40–44. chondrial Ca2+ homeostasis and respiration, Biosci. Rep. 17 [11] L. Kobzik, B. Stringer, J.-L. Ballingand, M.B. Reid, J.S. Stamler, (1997) 53–65. Endothelial type nitric-oxide synthase in skeletal muscle fibers: mito- [31] J.G. McCormack, R.M. Denton, Role of Ca2+ ions in the regulation chondrial relationships, Biochem. Biophys. Res. Commun. 211 of intramitochondrial metabolism in rat heart. Evidence from studies (1995) 375–381. with isolated mitochondria that adrenaline activates the pyruvate de- [12] C. Giulivi, J.J. Poderoso, A. Boveris, Production of nitric oxide by hydrogenase and 2-oxoglutarate dehydrogenase complexes by in- mitochondria, J. Biol. Chem. 273 (1998) 11038–11043. creasing the intramitochondrial concentration of Ca2+, Biochem. J. [13] A. Tatoyan, C. Giulivi, Purification and characterization of a nitric- 218 (1984) 235. oxide synthase from rat liver mitochondria, J. Biol. Chem. 273 (1998) [32] J.G. McCormack, Studies on the activation of rat liver pyruvate de- 11044–11048. hydrogenase and 2-oxoglutarate dehydrogenase by adrenaline and [14] P. Ghafourifar, C. Richter, Nitric oxide synthase activity in mitochon- glucagons. Role of increase in intramitochondrial Ca2+concentration, dria, FEBS Lett. 4 18 (1997) 291–296. Biochem. J. 231 (1985) 597–608. [15] S. French, C. Giulivi, R.S. Balaban, Nitric oxide synthase in porcine [33] O.H. Lowry, N.H. Rosenbrough, A.L. Farr, J.R. Randall, Protein heart mitochondria: evidence for low physiological activity, J. Phys- measurement with the Folin phenol reagent, J. Biol. Chem. 193 iol. Heart Circ. Physiol. 280 (2001) H2863–H2867. (1951) 265–275. [16] A.J. Kanai, L.L. Pearce, P.R. Clemens, L.A. Birder, M.M. Van Bibber, [34] G.L. Lukacs, A. Kapus, Measurement of the matrix free Ca2+ con- S.Y. Choi, W.C. de Groat, J. Peterson, Identification of a neuronal centration in heart mitochondria by entrapped fura-2 and quin2, Bio- nitric oxide synthase in isolated cardiac mitochondria using elec- chem. J. 248 (1987) 609–613. trochemical detection, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) [35] J.G. McCormack, H.M. Browne, Studies on mitochondrial Ca2+ 14126–14131. transport and matrix Ca2+using fura-2-loaded rat heart mitochondria, [17] M.O. Lo´pez-Figueroa, C. Caamano, M.I. Morano, L.C. Ronn, H. Biochim. Biophys. Acta 973 (1989) 420–427. Akil, S.J. Watson, Direct evidence of nitric oxide presence within mi- [36] P.T. Jain, S.H. Change, P.P. Gutry, I.K. Berezesky, B.F. Trump, The tochondria, Biochem. Biophys. Res. Commun. 272 (2000) 129–133. relationship between [Ca2+] and cell death using in vivo model: a [18] S.L. Elfering, T.M. Sarkela, C. Giulivi, Biochemistry of mitochondrial study using the ced-1 mutant strain of C. elegans, Toxicol. Pathol. 21 nitric-oxide synthase, J. Biol. Chem. 277 (2002) 38079–38086. (1993) 572–583. [19] J.J. Poderoso, M.C. Carreras, C. Lisdero, N. Riobo´, F. Scho¨pfer, A. [37] D.S. Bredt, S.H. Snyder, Nitric oxide, a novel neuronal messenger, Boveris, Nitric oxide inhibits electron transfer and increases superox- Neuron 8 (1992) 3–11. ide radical production in rat heart mitochondria and submitochondrial [38] H.H. Schmidt, J.S. Pollock, M. Nakane, L.D. Gorsky, U. Fo¨rstermann, particles, Arch. Biochem. Biophys. 328 (1996) 85–92. F. Murad, Purification of a soluble isoform of guanylyl cyclase-acti- [20] T.M. Sarkela, J. Berthiaume, S.L. Elfering, A.A. Gybina, C. Giulivi, vating-factor synthase, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) The modulation of oxygen radical production by nitric oxide in 365–369. mitochondria, J. Biol. Chem. 276 (2001) 6945–6949. [39] G.A. Rutter, R.M. Denton, Regulation of NAD+-linked isocitrate de- [21] J.J. Poderoso, M.C. Carreras, F. Scho¨pfer, C.L. Lisdero, N.A. Riobo, hydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within C. Giulivi, A.D. Boveris, A. Boveris, E. Cadenas, The reaction of toluene-permeabilized rat heart mitochondria. Interactions with regu- nitric oxide with ubiquinol: kinetic properties and biological signifi- lation by adenine nucleotides and NADH/NAD+ ratios, Biochem. J. cance, Free Radic. Biol. Med. 26 (1999) 925–935. 252 (1988) 181–189 (Erratum in Biochem. J. 253 (1988) 935). [22] P. Ghafourifar, U. Schenk, S.D. Klein, C. Richter, Mitochondrial [40] A.P. Thomas, R.M. Denton, Use of toluene-permeabilized mitochon- nitric-oxide synthase stimulation causes cytochrome c release from dria to study the regulation of adipose tissue pyruvate dehydrogenase isolated mitochondria. Evidence for intramitochondrial peroxynitrite in situ. Further evidence that insulin acts through stimulation of py- formation, J. Biol. Chem. 274 (1999) 31185–31188. ruvate dehydrogenase phosphatase, Biochem. J. 238 (1986) 93–101. [23] K.S. Aulak, M. Miyagi, L. Yan, K.A. West, D. Massillon, J.W. Crabb, [41] P.J. Midgley, G.A. Rutter, A.P. Thomas, R.M. Denton, Effects of N. Traaseth et al. / Biochimica et Biophysica Acta 1658 (2004) 64–71 71

Ca2+and Mg2+ on the activity of pyruvate dehydrogenase phosphate of glucagon and other Ca2+ mobilizing hormones on the malate as- phosphatase within toluene-permeabilized mitochondria, Biochem. J. partate cycle, Arch. Biochem. Biophys 264 (1998) 310–320. 241 (1987) 371–377. [54] T. Sugano, K. Nishimura, N. Sogabe, M. Shiota, N. Oyama, S. Noda, [42] D.D. Perrin, Stability Constants of Metal Ion Complexes, Parts A and M. Ohta, Calcium-dependent activation of the malate–aspartate shut- B, Pergamon, London, 1979. tle by norepinephrine and vasopressin in perfused rat liver, Arch. [43] B. Wan, K.F. LaNoue, J.Y. Cheung, R.C. Scaduto, Regulation of citric Biochem. Biophys. 264 (1988) 144–154. acid cycle by calcium, J. Biol. Chem. 264 (1989) 13430–13439. [55] F.M. Lasorsa, P. Pinton, L. Palmieri, G. Fiermonte, R. Rizzuto, F. [44] E. Carafoli, The calcium cycle of mitochondria, FEBS Lett. 104 Palmieri, Recombinant expression of the calcium-sensitive aspartate/ (1979) 1–5. glutamate carrier increases mitochondrial ATP production in ago- [45] T.E. Gunter, D.R. Pfeiffer, Mechanisms by which mitochondria trans- nist-stimulated Chinese hamster ovary cells, J. Biol. Chem. 278 port calcium, Am. J. Physiol. 258 (1990) C755–C786. (2003) 38686–38692. [46] T.H. Crouch, C.B. Klee, Positive cooperative binding of calcium to [56] M.J. Jou, T.I. Peng, S.S. Sheu, Histamine induces oscillations of bovine brain calmodulin, Biochemistry 19 (1980) 3692–3698. mitochondrial free Ca2+ concentration in single cultured rat brain [47] H. Miyata, H.S. Silverman, J. Sollot, E.G. Lakatta, M.D. Stern, R.G. astrocytes, J. Physiol. 497 (1996) 299–308. Hansford, Measurement of mitochondrial free Ca2+ concentration in [57] D.F. Babcock, B. Hille, Mitochondrial oversight of cellular Ca2+ living single rat cardiac myocytes, Am. J. Physiol. 261 (1992) signaling, Curr. Opin. Neurobiol. 8 (1998) 398–404. H1123–H1134. [58] A. Andreyev, G. Fiskum, Calcium induced release of mitochondrial [48] S.P. Allen, D. Stone, J.G. McCormack, The loading of fura-2 into cytochrome c by different mechanisms selective for brain vs. liver, mitochondria in the intact perfused rat heart and its use to estimate Cell Death Differ. 6 (1999) 825–832. matrix Ca2+ under various conditions, J. Mol. Cell. Cardiol. 24 [59] X.M. Leverve, A.J. Verhoeven, A.K. Groen, A.J. Meijer, J.M. Tager, (1992) 765–773. The malate/aspartate shuttle and pyruvate kinase as targets involved in [49] S.P. Allen, V.M. Darley-Usmar, J.G. McCormack, D. Stone, Changes the stimulation of gluconeogenesis by phenylephrine, Eur. J. Bio- in mitochondrial matrix free calcium in perfused rat hearts subjected chem. 155 (1986) 551–556. to hypoxia reoxygenation, J. Mol. Cell. Cardiol. 25 (1993) 949–958. [60] K.F. LaNoue, A.J. Meijer, A. Brouwer, Evidence for electrogenic [50] R. Rizzuto, A.W. Simpson, M. Brini, T. Pozzan, Rapid changes of aspartate transport in rat liver mitochondria, Arch. Biochem. Biophys. mitochondrial Ca2+ revealed by specifically targeted recombinant 161 (1974) 544–550. aequorin, Nature (Lond.) 358 (1992) 325–327 (Erratum in: Nature [61] T.E. Gunter, K.K. Gunter, S.S. Sheu, C.E. Gavin, Mitochondrial cal- 360 (1992) 768). cium transport: physiological and pathological relevance, Am. J. [51] U. Fo¨rstermann,L.D.Gorsky,J.S.Pollock,K.Ishii,H.H.H.W. Physiol. 267 (1994) C313–C339. Schmidt, M. Heller, F. Murad, Hormone-induced biosynthesis of en- [62] C. Giulivi, Characterization and function of mitochondrial nitric- dothelium-derived relaxing factor/nitric oxide-like material in N1E- oxide synthase, Free Radic. Biol. Med. 34 (2003) 397–408. 115 neuroblastoma cells requires calcium and calmodulin, Mol. Phar- [63] L. Robb-Gaspers, P. Burnett, G.A. Rutter, R.M. Denton, R. Rizzuto, macol. 38 (1990) 1–7. A.P. Thomas, Integrating cytosolic calcium signals into mitochondrial [52] R.C. Scaduto Jr., Calcium and 2-oxoglutarate-mediated control of metabolic responses, EMBO J. 17 (1998) 4987–5000. aspartate formation by rat heart mitochondria, Eur. J. Biochem. 223 [64] M. Reers, R.A. Kelly, T.W. Smith, Calcium and proton activities in rat (1994) 751–781. cardiac mitochondria. Effect of matrix environment on behavior of [53] T. Strzelecki, D. Strzelecka, C.D. Koch, K.F. LaNoue, Sites of action fluorescent probes, Biochem. J. 257 (1989) 131–142.