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[20] R e g u l a t i o n o f M i t o c h o n d r i a l R e s p i r a t i o n b y Adenosine Diphosphate, Oxygen, and Nitric Oxide
By
E. COSTA, E N R I Q U E and JUAN J. PODEROSO
A L B E R T O BOVERIS, L I D I A
CADENAS,
Introduction Oxygen is required in adequate steady-state concentrations to sustain mitochondrial respiration and ATP production. The reaction of reduced cytochrome oxidase, the oxygen acceptor and terminal enzyme of the mitochondrial respiratory chain, with 02 is very fast (second-order reaction constant of about 107-108 M -1 S-1) l and the rate of electron transfer to cytochrome oxidase by the respiratory chain is the key factor to define the operational 02 concentration for half-maximal rate of 02 uptake ([02]o.5). The intracellular oxygen concentration in mammalian organs and tissues, in the 5-25/xM 02 range, is close and partially overlaps with the critical concentration, in the 2-6/xM 02 range, that limits the rate of mitochondrial respiration. 2,3 Nitric oxide, the product of the NO synthase of vascular endothelium, with estimated steady state concentrations in mammalian tissues in the 0.05-1/xM NO range, 4'5 has been recognized as a high affinity inhibitor of cytochrome oxidase activity and mitochondrial respiration in a competitive way with O2.6-I° Work on these topics has been performed with new and sensitive instruments: a two-channel respirometer (Oroboros Oxygraph, Oroboros, Schopfstrasse 18, A-6020 Innsbruck, Austria), developed for high-resolution respirometry, u and a specific NO-sensitive elec1 Q. Gibson and C. Greenwood, Biochem. J. 86, 541 (1963). 2 E. Gnaiger, R. Steinlechner-Maran, G. M6ndez, T. Eberl, and R. Margreiter, J. Bioenerg. Biomemb. 27, 583 (1995). 3 L. E. Costa, G. M6ndez, and A. Boveris, Am. J. Physiol. 273, C852 (1997). 4 R. G. Knowles, M. Merrett, M. Salter, and S. Moncada, Biochem. J. 270, 833 (1990). 5 j. j. Poderoso, J. G. Peralta, C. L. Lisdero, M. C. Carreras, M. Radisic, F. Schopfer, E. Cadenas, and A. Boveris, Am. J. Physiol. 274, Cl12 (1998). 6 M. W. J. Cleeter, J. M. Cooper, V. M. Darley-Usmar, S. Moncada, and A. H. V. Schapira, F E B S Lett. 345, 50 (1994). 7 G. C. Brown and C. E. Cooper, F E B S Lett. 356, 295 (1994). 8 y . Takehara, T. Kanno, T. Yoshioka, M. Inoue, and K. Utsumi, Arch. Biochem. Biophys.
323, 27 (1995). 9 j. j. Poderoso, M. C, Carreras, C. Lisdero, N. Riob6, F. Schopfer, and A. Boveris, Arch. Biochem. Biophys. 328~ 85 (1996). a0 A. Koivisto, A. Matthias, G. Bronnikov, and J. Nedergard, F E B S Letr 417, 75 (1997). u T. Hailer, M. Ortner, and E. Gnaiger, A n a l Biochem. 218, 338 (1994).
METHODS IN ENZYMOLOGY.VOL. 301
Copyright © 1999by AcademicPress All rightsof reproductionin any form reserved. 007645879/99 $30.00
[20]
REGULATION OF MITOCHONDRIAL RESPIRATION
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trode (ISO-NO, World Precision Instruments, 175 Sarasota Center Blvd, Sarasota, Florida 34240-9258), which are commercially available. High-Resolution Respirometry Classical respirometers with Clark-type 12 or vibrating 02 electrodes are useful for determining the rate of 02 uptake at 02 concentrations ranging from those of air-saturated reaction media to about 5/xM, but do not allow the precise measurement of 02 uptake at low 02 concentrations. Tightness of the 02 electrode chamber to prevent 02 diffusion from air, avoidance of materials with high Oe storage capacity, and a high sensitivity and stability of the polarographic Oe sensor are requisites for 02 measurements in the 0.1-5/xM Oe range. A high-resolution respirometer, designed to minimize Oe back diffusion, 11 is able to sense, with accuracy and reproducibility, changes in 02 concentration in the 0.1-1/xM range and in the rates of 02 uptake (dO2/dt) down to 1-10 nM/sec, z3,13,14 The properties that render this instrument particularly useful are (i) a design and a choice of inert materials, which do not absorb and release 02 (glass chambers, titanium stoppers, Viton O rings, etc.) to minimize 02 diffusion (back diffusion of 02 at low 02 concentrations is severalfold lower in this instrument than in previous and conventional reaction chambers); (ii) an appropriate 02 sensor which is a Clark-type gold cathode (Orbisphere Model 2120) with a relatively large area (2 mm diameter) to increase the sensitivity and stability of the signal and with an angular insertion into the glass chamber that improves signal stability by providing optimal stirring at the cathode surface; (iii) digital data acquisition (averaged from up to 30 data points each second); (iv) data output that provides readings of 02 concentration vs time and its first derivative every second and a continuous record, which is a high improvement over conventional line drawing on chart recorder traces. The respirometer software (Oroboros Dat Lab 2.1) permits automatic calibration and background and response time correction. Determination of Respiratory Rates and of Respiratory Control and ADP: O Ratios The classical parameters of mitochondrial respiration which are useful to describe mitochondrial function are (i) the rate of 02 consumption in le R. W. Estabrook, Methods" Enzymol. 10, 41 (1967). 13G. M6ndez and E. Gnaiger, in "Modern Trends in Biothermokinetics" (E. Gnaiger, F. N. Gellerich, and M. Wyss, Eds.), Vol. 31, p. 191. Innsbruck University Press, Innsbruck, 1994. 14R. Steinlechner-Maran, T. Eberl, M. Kunc, R. Margreiter, and E. Gnaiger, Am. J. Physiol. 271, C2053 (1996).
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metabolic state 315; the active respiration with maximal rates of 02 uptake and ATP synthesis in the presence of excess substrate and ADP; (ii) the rate of 02 consumption in state 4,15 the resting respiration, which depends on H + permeability and energy leaks of the mitochondrial inner membrane, in the presence of substrate and in absence of ADP, measured before ADP addition (state 4b) and after ADP has been phosphorylated (state 4a); (iii) the respiratory control (RC) or acceptor control ratio (ACR), the ratio of the rates of O2 consumption in state 3 and in state 4 (state 3 rate/state 4 rate), a sensitive parameter that depends on the close coupling between electron transfer and energy conservation, useful for assessing the integrity of the mitochondrial inner membrane; and (iv) total and net P/O, the ratio of moles of ADP added/total or extra moles of O consumed, respectively. A description of the determination of these respiratory parameters has been given early in this series. 12 Isolated mitochondria are usually suspended in a solution, 250 to 300 mOsm/liter, of a non-electrolyte (0.23 M mannitol and 0.07 M sucrose, or 0.25 M sucrose), added with a Ca 2+ chelator (0.5 to 1 mM EGTA or EDTA) and a buffer (Tris, HEPES or MOPS) taken to pH 7.3-7.4. Inorganic phosphate (5-10 mM) is included in the reaction medium to phosphorylate added ADP (0.2-0.5 raM). Some types of mitochondria (liver mitochondria) require the supplementation of the reaction medium with 3-5 mM Mg 2+, and in other cases (heart mitochondria), Mg 2+ is not required. Titration of the optimal Mg2+ concentration in the reaction medium is advised. The reaction media are usually equilibrated with 02 by aeration and intensive stirring; the solubility of oxygen at the reaction medium temperature is used to calibrate the electrode signal. The 02 concentration in air-saturated distilled water (given by Estabrook 12) and in electrolyte and nonelectrolyte solutions is a function of temperature and barometric pressure and is slightly lower in solutions than in pure water, depending on solute concentration and ionic strength. The 02 concentrations in two air-saturated solutions, which are widely used to suspend mitochondria for respiration studies (0.25 M sucrose and 0.15 M KC1) at 101.3 kPa (760 mm Hg) and in the temperature range 5-40°C, as determined by a precise kinetic method, 16 are shown in Fig. 1. The reagents used for the determination of respiratory parameters in isolated mitochondria must be of the highest purity commercially available; adenine nucleotides should be vanadium-free, and all solutions must be prepared in high quality, double-distilled and deionized water. The classical parameters of mitochondrial respiration, described before, and the ADP and Oz dependence of active respiration can be performed 15B. Chance and G. R. Williams,Nature (London) 1"/5,1120 (1955). amB. Reynafarje,L. E. Costa, and A. L. Lehninger,AnaL Biochem. 145, 406 (1985).
[20]
191
REGULATION OF MITOCHONDR1AL RESPIRATION
400 i
350
¢
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300
250
200
150
L
~
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10
15
20
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30
35
40
Temperature (°C)
FIG. 1. Oxygenconcentrationin air-saturated solutions at standard atmosphericpressure (101.3 kPa) and variabletemperature.©, 0.25 M sucrose;0, 0.15 M KC1.(FromB. Reynafarje, L. E. Costa, and A. L. Lehninger,AnaL Biochem. 145, 406 (1985).) in the high sensitivity respirometer with one mitochondrial sample in a simple assay consisting of two successive ADP pulses added to mitochondria in resting state 4 (Fig. 2). The first ADP pulse is given at a high 02 concentration, whereas the second ADP pulse is given at a relatively low O2 concentration. The respiratory rates in states 3 and 4 and the oxygen taken up under ADP stimulation, in the first ADP pulse, are used in the determination of the respiratory control and A D P / O ratios. 12
Determination of [ADP]o.5 and [Oe]o.5 for State 3 Active Respiration The rate of net state 3 O2 uptake (calculated as state 3 rate minus state 4 rate) follows a hyperbolic relationship with ADP concentration (Fig. 3). The ADP concentration required to reduce a half-maximal respiratory rate [ADP]0.5 (or KsoAop or, incorrectly, KmADp) was determined by hyperbolic fitting as 30/zM ADP for liver mitochondria and 52/zM ADP for heart mitochondria. 3 The state 3 rate of 02 uptake measured at the second ADP pulse (see Fig. 2) plotted as a function of 02 concentration follows a hyperbolic
192
BIOLOGICAL ACTIVITY
[201
200
7
,~ ~.~ 1oo
dO2/dt 460
650
860
1060
Tlme
1250
~4EO
[s]
FIG. 2. Oxygen concentration ( [ 0 2 ] ) and its first derivative (rate of 02 uptake; d[Oz]/dt) during the respiratory activity of rat liver mitochondria (0.35 mg protein/ml) suspended in 0.25 M sucrose, 0.5 mM EGTA, 5 mM MgCI2, 1.5% bovine serum albumin (BSA), 10 mM HEPES, and 6 rnM phosphate buffer (pH 7.35). ADP: 0.3 mM in both pulses. (From L. E. Costa, G. M6ndez, and A. Boveris, Am. J. Physiol. 273, C852 (1997).)
relationship in its dependence on 02 concentration (Fig. 4). State 3 respiratory rate, as Vmax, and [02]o.5 (also termed Ks0 02 and, incorrectly, Km 02) are calculated from these plots by hyperbolic fitting. The Vmax calculated in this way do not differ from the state 3 rate measured at high and saturating concentrations of O2 and ADP. Average Vmaxand [02]o.5 values are given in Table I.
•
•
...'"
..'''""""
•
t.5 O
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0
50
100
150 20D 2.50 300
350
400
450
[ADP] (pM)
FIG. 3. ADP dependence of the state 3 active respiration of rat liver mitochondria. The dotted line is the hyperbolic fitting of the net rates of 02 uptake. The calculated [ADP]05 is 27 p~M. (From L. E. Costa, G. M6ndez, and A. Boveris, Am. J. Physiol. 273, C852 (1997).)
[20]
REGULATION
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i
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193
RESPIRATION
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[o,11~) FIG. 4. Oxygen dependence of the state 3 active respiration of rat liver mitochondria. The dotted line is the hyperbolic fitting of the data points (d[Oz]/dt) collected at 1 sec intervals after a second A D P pulse. Different symbols indicate different runs. The calculated [02]0.5 is 1.7 ixM. (From L. E. Costa, G. Mdndez, and A. Boveris, Am. J. Physiol. 273, C852 (1997).)
Determination of [02]0.5 for State 4 Resting Respiration The 02 dependence of resting respiration is determined in a reaction medium with 20-25/xM 02, reached by bubbling nitrogen under 02 electrode control. Substrate and mitochondrial preparation are added and the data points of O2 concentrations are stored. Two procedures can be used: (i) the direct transition from state 4 to state 5,2,3,13 or (ii) after a short period of anoxia (2-3 min), 100-150/xl of air-saturated medium is added to the reaction chamber. 3 The hyperbolic fitting of dO2/dt vs [O2] in (i) the transition from state 4 to state 5 (anoxia), and (ii) after the 02 pulse to
TABLE I K I N E T I C P A R A M E T E R S OF O X Y G E N U P T A K E OF R A T L I V E R A N D HEART
MITOCHONDRIA a
[02]0.5
Maximal rate of O2 uptake (nmol Oflsec m g protein)
(/xM)
Mitochondria
State 4
State 3
State 4
State 3
Rat liver Rat heart
0.17 _+ 0.02 0.19 ± 0.03
1.78 _+ 0.09 1.55 ± 0.09
0.30 _+ 0.06 0.25 _+ 0.08
1.69 _+ 0.09 1.45 ± 0.12
"Liver mitochondria were supplemented with 5 m M succinate and 2/xM rotenone; heart mitochondria were supplemented with 5 m M pyruvate and 5 m M malate. A D P when used was 300/xM.
194
BIOLOGICALACTIVITY
[20]
o,i\ A ~
g
Idt
o 410
510
610 710 Time [s]
810
910
FIG. 5. Oxygen dependence of the state 4 resting mitochondrial respiration. The time course of 02 concentration and its first derivative are shown during an 02 pulse to anaerobic rat liver mitochondria. The calculated [02]o.5is 0.45/xM. (From L. E. Costa, G. Mdndez, and A. Boveris, Am. J. Physiol. 273, C852 (1997).)
state 4 deenergized mitochondria (Fig. 5) usually result in similar values of [02]0.5 and state 4 02 uptake (V,nax).3 Eventual damage to heart mitochondria by the generation of H202 can be avoided by the addition of 1/xM catalase (liver has plenty of peroxisomal catalase) and evaluated by determining the respiratory control ratios at the end of every experiment. Mitochondrial preparations usually retain high respiratory control values, 6 to 8, after several reoxygenation cycles and for about 30-40 min. 3 The [02]0.5 values for the resting state for liver and heart mitochondria are given in Table I.
I n h i b i t i o n of C y t o c h r o m e O x i d a s e Activity a n d Mitochondrial R e s p i r a t i o n of Nitric Oxide Nitric oxide reacts with cytochrome oxidase in its reduced and oxidized forms; in the reduced form N O binds tightly to cytochrome a32+ and CUB+ acts as a second binding site with lower affinity, whereas that with the oxidized form N O binds to CUB2+.17,18 Reduced cytochrome oxidase forms with N O a distinct spectral complex with absorption maxima at 429 and 442 nm, and the enzyme is able to interact with N O during its catalytic cycle. 17'18 The cytochrome oxidase activity of the isolated enzyme, 7,18 and of rat heart submitochondrial particles, 9 as well as the active respiration of 17G. V. Brudvig, T. H. Stevens, and S. I. Chang, Biochemistry 19, 5275 (1980). 18j. Torres and M. T. Wilson, Methods Enzymol. 269, 3 (1996).
[20l
195
REGULATION OF M1TOCHONDRIAL RESPIRATION 100
80 "~. 03
'~
6O
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2O
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I
I
I
1
I
50
100
150
200
250
300
O2/NO Ratio FIG. 6. Inhibition of state 3 active mitochondrial respiration by NO. The inhibition of 0 2 uptake, normalized for the different experiments, is plotted against the ratio of Oz and NO concentrations. Rat liver mitochondria in the presence of: O, A D P (state 3), and II, 20/zM dinitrophenol (state 3u), data from ref. 8. A, Rat heart mitochondria in the presence of A D P (state 3). (From J. J. Poderoso, M. C. Carreras, C. Lisdero, N. Riob6, F. Schopfer, and A. Boveris, Arch. Biochem. Biophys. 328, 85 (1996).)
mitochondria isolated from rat muscle, 6 liver, 8 heart, 9 and brown adipose tissue 1° and of rat brain synaptosomes, v is effectively inhibited by 0.05-1 /~M NO. The binding and the inhibition of cytochrome oxidase by NO are reversible and removable by washing 6'9 or by addition of excess myoglobin or hemoglobin, s'9 The degree of inhibition of cytochrome oxidase by NO depends on the 02 concentration in the reaction medium. 7,s,I° Apparently, NO and 02 compete for a binding site at the binuclear center formed by cytochrome a3z+ and CuB + in the cytochrome oxidase: Then, the inhibition of mitochondrial respiration by NO can be expressed as a function of the ratio [ 0 2 ] / [ N O ] a s shown in Fig. 6. Half-maximal inhibition of the active state 3 respiration of rat liver and heart mitochondria is reached at a ratio of 150 O2 : 1 NO, which clearly indicates the very high affinity of NO for cytochrome oxidase. Similarly, ratios of 400-500 O2 : 1 NO and 500-1000 02 : 1 NO have been reported to inhibit by 50% the respiration of rat brain synaptosomes 7 and of rat brown adipose tissue, 1° respectively. Considering
196
BIOLOGICALACTIVITY
1201
14mi~ 2 pM.NO I
".o
700U.F.I FIG. 7. Simultaneous measurement of NO and O2 concentrations by the specific electrodes and parallel monitoring of mitochondrial membrane potential. An ISO-NOP 30 micro NO sensor was used. Fluorescence was measured at 503 nm (excitation) and 527 nm (emission) and expressed in arbitrary units of fluorescence (UF). Rat liver mitochondria (1.0 mg protein/ ml) suspended in 0.23 M mannitol, 0.07 M sucrose, 5 mM Na2HPO4, 4 mM MgC12, 0.2 mM EDTA, 20 mM Tris-HC1 (pH 7.4). For the fluorescence assay: 0.2/~M rhodamine 123. Temperature: 30 °. Succ: 8 mM succinate; 8/~M NO.
the second-order reaction constant of cytochrome oxidase and 0 2 (107-108 M -1 sec -t) and assuming a NO reactivity 100-150 times higher, it follows that the second-order reaction constant for NO and cytochrome oxidase will reach the Smoluchowski limit (101° M -1 sec 1) for diffusion-controlled reactions, implying that each molecular collision between NO and cytochrome oxidase will yield a N O - h e m o p r o t e i n complex. The concentrations of both 02 and NO can be monitored continuously and simultaneously with both electrodes in the same reaction chamber, since there is no interference of NO on the 02 electrode and of 02 on the NO electrode. K° Figure 7 shows one of this typical traces, in which a NO pulse produces a transient inhibition of the respiration of rat liver mitochondria. A parallel experiment shows the decrease in mitochondrial membrane potential monitored fluorometrically produced by the NO pulse. The recovery in the rate of 02 uptake and in membrane potential occur when NO is about to be exhausted. It is understood that NO is mainly utilized by a reduction to N O - (by a one-electron transfer from a component of the ubiquinone-cytochrome b area of the respiratory chain).
[201
REGULATION OF MITOCHONDRIAL RESPIRATION
197
Inhibition of Electron Transfer at Ubiquinone-Cytochrome b Region and Production of Superoxide Radicals Nitric oxide, in addition to its effect on cytochrome oxidase, also inhibits electron transfer in the mitochondrial respiratory chain at the ubiquinonecytochrome b region. Rat heart submitochondrial particles added with 0.40.5 /xM NO decrease their succinate-cytochrome c activity to one-half 9 and show increased reduction of cytochrome b. 5 This second effect of NO on the mitochondrial respiratory chain results in increased rates of 02 production in submitochondrial particles and of H202 in whole mitochondria, 9 being also in the 0.3-0.4/xM NO concentration range required for half maximal effects. The suitable assays for O{ and H202 have been already described in this series. 19 The interaction of NO with the NO-reactive component of the ubiquinone-cytochrome b area of the mitochondrial respiratory chain is also reversible but is not affected by the [O2]/[NO] ratio. It is understood that the increased production rate of 02 is afforded by ubisemiquinone autoxidation.2° Regulation of Mitochondrial Oxygen Uptake by Oxygen and Nitric Oxide The consideration of the regulation of cellular 02 uptake by the [O2]/ [NO] ratio is of physiological interest, since the 02 steady-state concentrations in mammalian organs, 5-25/xM 02, and especially in the heart, 3-8 /xM 02, are in a range in which 0.05-0.1/xM NO, a level reachable after endothelial NOS stimulation, will produce a 20-30% inhibition of mitochondrial respiration (Fig. 6). A better myocardial 02 distribution is developed by endothelial NO release in response to hypoxia or ischemia; NO increases 02 supply through vasodilation and attenuates the respiratory rate by inhibition of cytochrome oxidase, allowing 02 to diffuse further along its gradient and reaching more mitochondria and cells, lowering the steepness of the pO2 gradient in the normoxic-anoxic transition. 9 The NO-dependent mitochondrial production of 02 provides a regulatory mechanism to remove NO and the reversible inhibition of cytochrome oxidase. An increasing NO concentration will sequentially inhibit cytochrome oxidase activity and electron transfer in the ubiquinone-cytochrome b region of the mitochondrial respiratory chain. The increased O~ production and steady-state concentration, in turn, will set a feedback mechanism by clearing NO through the Beckman reaction (O~ + NO --* ONOO-).21 In accordance with this 19A. Boveris, Methods Enzymol. 105,429 (1984). 2oA. Boveris, E. Cadenas, and A. O. M. Stoppani, Biochem. J. 156, 425 (1976). 2aj. S. Beckman,Nature (London) 345, 27 (1990).
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BIOLOGICAL ACTIVITY
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hypothesis, isolated beating rat hearts perfused with a bradykinin pulse show a transient inhibition of the organ O2 uptake with simultaneous release of NO in the perfusate and a slightly delayed release of H202 in the same perfusate, s An active NO synthase located in the inner membrane of rat liver mitochondria has been described. 22,23 The enzyme uses L-arginine (Km 5 - 7 / x M ) and is activated by Ca2+; the activity is better measured in submitochondrial particles, or in toluene-permeabilized or broken mitochondria. It has been claimed that the NO produced by mitochondrial NO synthase regulates mitochondrial 02 uptake. 22'23
22p. Ghafourifar and C. Richter, F E B S Lett. 418, 291 (1997). 23C. Giulivi, J. J. Poderoso, and A. Boveris, J. Biol. Chem. 273, 11038 (1998).