Regulation of oxidative phosphorylation in the mammalian cell.

Regulation

of oxidative

phosphorylation

in the mammalian

ROBERT

S. BALABAN

Laboratory

of Cardiac Energetics, National Institutes of Health, Bethesda, Maryland 20892

cell

BALABAN, ROBERTS. Regulation of oxidative phosphorylation in the mammalian cell. Am. J. Physiol. 258 (Cell Physiol. 27): C377-C389, 1990.The cell is capable of maintaining a steady-state flux of energy from mitochondrial oxidative phosphorylation, producing ATP, to the cytosolic adenosinetriphosphatases (ATPases), performing work. Considerable effort has been devoted to investigating the individual mechanisms involved in these two processes. However, less effort has been directed toward learning how these reactions of energy metabolism interact through the cytosol to maintain the observed steady state in the intact cell. The “classical” model for the cytosolic interaction of these two processes involves the feedback of ATP hydrolysis products, ADP and Pi, from the ATPases to oxidative phosphorylation. This model is based on data from isolated mitochondria in which the rate of oxidative phosphorylation is controlled by the concentration of ADP and Pi. Yet, recent data from intact tissues with high oxidative phosphorylation capacities (i.e., heart, brain, and kidney) indicate that the cytosolic concentration of ADP and Pi do not change significantly with work. These data imply that this simple feedback model is not adequate to explain the regulation of energy metabolism in these tissues. Other sites within the oxidative phosphorylation process must be playing a regulatory role or the kinetics of ATP synthesis must be very different than currently believed to establish the steady state. This review covers the potential sites within oxidative phosphorylation which may be regulated through cytosolic transducers to result in the necessary feedback network regulating the steady-state flow of energy in the cell. These sites will include substrate delivery to the cytochrome chain, the processes involved in the phosphorylation of ADP to ATP, and the delivery of oxygen. phosphate; oxygen; oxygen consumption; adenylate translocase; calcium; magnesium; blood flow; nuclear magnetic resonance; optical spectroscopy; mitochondria; metabolic substrates; carbohydrates; fats; adenosine 5’-triphosphate; adenosine Kdiphosphate; nicotinamide adenine dinucleotide; reduced nicotinamide adenine dinucleotide

USE OF ENERGY to maintain homeostasis and perform work is a basic property of all cells. In the cell, energy is converted, by intermediary metabolism, to a useful form which is delivered to energyrequiring processes. The free energy in ATP is believed to be the major cytosolic intermediate in this process (for review, see Refs. 16 and 17). ATP is produced from the energy obtained by the oxidation of metabolic substrates in glycolysis and oxidative phosphorylation. The energy in ATP is utilized by the controlled hydrolysis of ATP to ADP and Pi by various adenosinetriphosphatases (ATPases) in the cytosol to perform work. In this scheme, the rate of energy conversion (i.e., ATP production) and work (i.e., ATP hydrolysis) must be balanced to result in an effective steady state. This implies that a

THE CONTROLLED

control network between the work functions and energy conversion processes must be present in the cytosol. Therefore, the energy conversion and the work processes must possess appropriate cytosolic “transducers” to sense the respective rates of work and energy conversion to achieve a steady state. The purpose of this discourse is to review the information available on this cytosolic transduction process as it relates to the interaction of work and mitochondrial oxidative phosphorylation, the major source of ATP in most mammalian cells. There is a large body of information on the details of the mitochondrial oxidative phosphorylation reaction. These data are important for the elucidation of this phenomenon; however, relatively less effort has been expended in trying to understand how mitochondrial c377

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oxidative p’hosphorylation is controlled in the intact cell (for reviews, see Refs. 34, 41, 50, and 109). As will be pointed out in this review, this lack of information has resulted in a very uncertain picture with regard to the basic mechanisms involved in the regulation of oxidative phosphorylation in the intact cell. The current perception of the mechanisms involved in oxidative phosphorylation has been adequately reviewed in the literature (16,89,93,104) and is beyond the scope of this brief review. What will be discussed here are the potential sites in oxidative phosphorylation which may be influenced by cytosolic transduction processes, resulting in the control of cellular oxidative phosphorylation. To perform this task, a brief outline of oxidative phosphorylation and its relationship with the cytoplasm is necessary. Figure 1 is a cartoon depicting the interaction between the mitochondria and the cytosol with regard to the production and utilization of ATP. As seen in Fig. 1, the cytosol provides reducing equivalents to the mitochondria in the form of pyruvate, fats, and other reduced carbon chains. These reduced carbon chains are oxidized by various enzymes in ,&oxidation and the tricarboxylic acid (TCA) cycle and provide reducing equivalents to the cytochrome chain via NADH or FADH which “fuel” oxidative phosphorylation. Many of the enzymatic steps which result in the production of NADH or FADH may be regulated by numerous effecters linked to events in the cytosol. After the reducing equivalents are delivered to the respiratory chain, the free energy in NADH or FADH is used to create a proton electrochemical gradient (Ap) by the movement of protons, or net positive charge, out of the mitochondria at three putative sites along the chain. The mitochondrial inner membrane Ap is used to phosphorylate intramitochondrial ADP by coupling this energy-requiring reaction to the passive movement of protons into the mitochondrial matrix, down their electrochemical gradient (89, 93, 104). In light of the coupling of ATP production to the proton electrochemical gradient, modification of the mitochondrial proton electro-

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chemical gradient or proton permeability by a cytosolic event could be an effective modulator of ATP production. ADP and Pi are required in the mitochondria as substrates for the formation of ATP. ADP and Pi are generated in the cytosol by ATPases performing work, and in the steady state, the rate of ATP hydrolysis by these ATPases will determine the rate of oxidative phosphorylation. The complex delivery of ADP and Pi from the cytosol to the mitochondrial matrix is obviously under the control of cytoplasmic events as well as potential cytosolic “shuttles” of adenylates (60,88). Therefore, the ATP hydrolysis products are clearly potential controlling factors of oxidative phosphorylation. Indeed, these metabolites have been the basis of most classical models attempting to describe the regulation of oxidative phosphorylation in the cell. The final electron acceptor of the respiratory chain is molecular oxygen. Oxygen reacts with a high-affinity site on reduced cytochrome aa3 complex to form water and complete the reaction sequence in the cytochrome chain. Oxygen is delivered to the mitochondria from the capillaries through the cytosol, sometimes aided by facilitated diffusion by myoglobin (125). The Michaelis constant (Km) of oxygen for this process is very low and dependent on the metabolic state of the mitochondria or tissue. From this very brief outline of oxidative phosphorylation, it is evident that there are numerous sites that can be influenced by cytosolic events. I have taken the liberty of dividing the potential control sites of oxidative phosphorylation into the following three major groups: 1) the delivery of reducing equivalents to the cytochrome chain in the form of NADH and FADH, 2) the complex synthesis of ATP from ADP and Pi, and 3) the formation of water from oxygen and reduced cytochrome aa3. Each site in these groups will be briefly discussed along with the known cytosolic transducers that may affect this site. Because the synthesis of ATP from ADP and Pi was the first area studied in this regulatory process, it will be discussed first followed by the delivery of reducing equivalents and oxygen. ATP,

t Glucose

FIG.

intact

1. Brief cell.

ATP.Work,ADP

schematic

diagram

+ Pi

of oxidative

phosphorylation

in the

ADP,

AND

Pi

The effects of ADP and Pi on ATP production and respiratory control in isolated mitochondria were described by Lardy (81) and Chance (23) and co-workers in the early 1950s. In these studies it was shown that the rate of oxidative phosphorylation was dependent on the extramitochondrial concentration of ADP and Pi. These data demonstrated that the delivery of ADP and Pi could be an effective parameter in the regulation of mitochondrial oxidative phosphorylation in vitro. These observations led to the hypothesis that the cytosolic concentrations of ATP hydrolysis products are the cytosolic feedback mechanism between oxidative phosphorylation and ATPase activity in vivo (23, 81, 115). This hypothesis basically assumes that the cytosolic concentrations of ADP and Pi are proportional to the rate of ATP hydrolysis and work. Adjustment of cytosolic ADP and Pi concentrations by work is then believed to control the rate of oxidative phosphorylation via the kinetic mech-


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anisms observed in mitochondria in vitro, resulting in the steady-state flux of ATP. Subsequent studies attempted to establish the actual controlling parameters in the control of respiration by ADP and Pi in vitro; these models include ATP/ADP*Pi (34, 72, 120), ATP/ADP (29), and ADP and/or Pi alone (23,60,81). It should also be pointed out that Tager and co-workers (44, 109), as well as Wilson and co-workers (34), have proposed that multiple sites of action concerning ATP, ADP, Pi, and cytochrome redox states may be a more appropriate analysis of this process. This concept will be discussed later. The ATP/ADP*Pi model mostly is based on the concept that the majority of the mitochondrial respiratory chain (i.e., NADH to cytochrome c) is in near equilibrium with the cytosolic phosphorylation potential, and shifts in any of the equilibrium constituents (i.e., NADH, ATP/ ADP.Pi, and cytochrome c) result in alterations in cytochrome aa redox state which ultimately controls respiration through an irreversible step in the reduction of molecular oxygen. Several models using irreversible thermodynamics are also based on the free energy of ATP (112). The equilibrium model was one of the earliest models of respiratory control in isolated mitochondria (72) and was further developed by Wilson and co-workers (34, 120). The major problem with this model has been the lack of consistent evidence that either the adenylate translocase or the ATP synthesis reaction is actually at or near equilibrium in the cell. Studies from several laboratories have demonstrated that the adenylate translocase is not equilibrating intra- and extramitochondrial ATP and ADP in active mitochondria (109). This implies that the extramitochondrial phosphorylation potential is not in equilibrium with the intramitochondrial space. Erecinska and Wilson (34) suggest that this lack of equilibrium in the translocase is due to the conditions used in typical isolated mitochondria studies which may not be appropriate to model the intact cell. However, inhibitor studies done on intact cells are also consistent with the notion that the translocase is rate limiting (l), although this type of study in a complex enzyme system is difficult to interpret (64, 107). If the cytochrome chain and the phosphorylation potential are at equilibrium, then the unidirectional fluxes through the ATP synthesis and hydrolysis reactions would be much faster than the net flux through the system (2). Phosphorus-32 tracer studies on state 3 (active) mitochondria showed no evidence for rapid cycling (79). In addition, phosphorus-31 nuclear magnetic resonance (31P-NMR) studies have measured the unidirectional flux between ATP and Pi in intact tissues (71,84). These studies have shown that the unidirectional rate of ATP synthesis is equal to the calculated net flux of ATP production based on the oxygen consumption. Thus these results are inconsistent with an equilibrium condition involving ATP hydrolysis and the cytochrome chain. The ATP/ADP model is based on the consideration that the transport of ADP into and ATP out of the mitochondrial matrix by adenosine translocase is rate limiting for oxidative metabolism. Because ATP and ADP compete for binding and subsequent transport

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across the mitochondrial membrane, it has been assumed that the influx of ADP is responsive to the ratio of ATP to ADP. With the use of a variety of techniques, evidence has been collected which suggests that the translocase is potentially rate limiting (1, 44, 109). However, several studies on isolated mitochondria indicate that the ratio of the ATP to ADP is not the relevant parameter and that under isolated mitochondria conditions (i.e., high Pi, substrates, and oxygen), the ADP concentration alone is rate limiting (59). No clear picture has evolved on the actual mechanism of ADP, Pi, and ATP regulation of oxidative phosphorylation. The variety of conflicting results most likely reflects the complex nature of this interaction. Regardless of the mechanism of ADP and Pi regulation of oxidative phosphorylation, it is generally accepted that the halfmaximal concentration of ADP for stimulating ATP synthesis ([ADPI& in the presence of saturating concentrations of mitochondrial NADH or FADH and Pi is on the order of 30 PM (59). The half-maximal concentration of Pi ([Pi]so), in the presence of high concentrations of ADP and reducing equivalents, is on the order of 200800 PM (35). However, as stated earlier, there are complex kinetic interactions observed in isolated mitochondria between ATP, Ap, ADP, Pi, NADH, oxygen, and pH for driving oxidative phosphorylation. Thus each of these “apparent” affinities will have to be considered a function of all of these elements in various conditions in vitro and in vivo. However, it is interesting to note that recent 31P-NMR (2, 14, 38, 68, 69, 86, 126) and extraction (113) estimates of the intracellular concentrations of ADP and Pi in highly aerobic tissues in vivo are close to the [ADPI and [Pi]50 in isolated mitochondria. In contrast, within resting skeletal muscle, ADP and Pi are well below their [ADPI and [Pi]50 values (21, 77, 111). These data imply that ADP and Pi are in a concentration range in which they could effectively regulate oxidative phosphorylation. However, how accurate are our measurements of these metabolites? The NMR determined free cytosolic Pi is probably the most reliable value for this parameter at this time (2). However, the determination of free cytosolic ADP relies on the calculation of the creatine kinase or other enzyme system equilibrium in both extraction and NMR determinations. The reasons for this have been previously reviewed (2, 113) and are basically due to the very low concentration of free ADP [i.e., not directly detectable with NMR (2)], bound pools of ADP, and a rapid hydrolysis of ATP during extraction. The precise calculation of free ADP using the creatine kinase equilibrium could be in error because of in vivo modifications of the equilibrium constant, as well as the overestimation of the free creatine concentration (82, 102). However, it is a reasonable assumption that the cytosolic concentrations of ADP and Pi are lower or close to the in vitro [ADP]SO and and it is [Pi 150 values for oxidative phosphorylation, feasible that these compounds could be controlling the rate of oxidative phosphorylation. Is there a relationship between the cytosolic concentration of ATP, ADP, and Pi and the rate of oxidative phosphorylation during alterations in work in tissues as


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predicted by the “classical” model? From the earliest studies (53), when it was first proposed that ATP was the energy intermediate in cells, it has been extremely difficult to demonstrate a change in ATP or ADP during work increases in most tissues (37,58), although changes in Pi and creatine phosphate with work in skeletal muscle were easy to detect (see Ref. 58). Subsequent studies on heart tissue demonstrated that the changes in ATP, ADP, or Pi with work were difficult to detect (15, 91), with some exceptions (42). These observations have been greatly strengthened by the more recent studies using nondestructive 31P-NMR techniques, which have demonstrated in vivo that there is little correlation between the rate of oxidative phosphorylation in the steady state and cytosolic ADP, Pi, and ATP in heart (4, 69, 99), brain (A. P. Koretsky, personal communication), and kidney (126). The same phenomenon has also been observed in the heart in vitro depending on the perfusion substrates and work load conditions (14, 38, 66, 67, 86). Indeed, in anecdotal cases, the concentrations of ADP and Pi have been observed to decrease with work both in vitro and in vivo (66, 99). Thus it seems reasonable to assume that large increases in ADP and Pi are not occurring during increases in oxidative phosphorylation in tissues with relatively high oxidative phosphorylation capabilities. Indeed, as noted by Katz et al. (69), the only time significant changes in ADP or Pi were detected during work stress tests of the heart in vivo was when the work demands were greater than the overall oxidative capacity of the preparation, especially with regard to blood flow. These results suggest that the phosphorylation potential as well as the concentrations of ADP and Pi are remaining very stable in the heart despite large increases (d-fold) in ATP hydrolysis rates. It should be noted that smooth muscle tissue may also be able to increase the turnover of ATP with minimal changes in phosphorylation potential (see Ref. 98 for references). In contrast, studies on skeletal muscle have demonstrated that changes in ADP and Pi can be seen with work jumps in muscle in vivo using 31P-NMR (21, 111). Thus, in skeletal muscle, with less overall oxidative capacity [i.e., lower maximum velocity ( Vmax) for aerobic ATP production when compared with heart, brain, or kidney], the phosphorylation potential is not maintained with increases in work over a rather wide range. In this tissue ADP and Pi may play a significant role in the regulation of respiration (21), as well as other metabolic processes such as glycolysis as originally proposed. Training and development may also be important in this regulatory process. Clark et al. (26) demonstrated that with chronic stimulation skeletal muscle begins to take on the metabolic and morphological characteristics of heart tissue. That is, chronically stimulated skeletal muscles have very small alterations in the phosphorylation potential with increases in work. Although Portman et al. (99) demonstrated that the neonatal sheep heart in vivo cannot maintain a constant phosphorylation potential over physiological work loads, the ability to maintain a more constant phosphorylation potential emerges over the first few months of development. Thus the relationship between cellular phosphate metabolite concentra-

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tions and work can be a function of both training and development. The mechanisms involved in these alterations in metabolic responses are unknown but may provide important experimental insights. How large a change in ADP and Pi is required to regulate oxidative phosphorylation? The extent of ADP and Pi changes required to drive respiration in the heart using the classical in vitro [ADP]SO and [ Pi]50 values has been presented by Katz et al. (69) as well as Chance et al. (21) and demonstrates that simple Michaelis-Menten kinetics for ADP and Pi control of oxidative phosphorylation are not adequate to totally explain the regulation of respiration occurring in vivo. There are many possibilities to explain this result. These include 1) the kinetics of cytosolic ADP and Pi for stimulating oxidative phosphorylation are much different in vivo; 2) there are other effecters which magnify the effects of small cytosolic changes in Pi and ADP on oxidative phosphorylation; 3) other parameters (such as oxygen or reducing equivalent delivery) are dominating the activation of respiration; and 4) a combination of all of the effects above. Recent evidence in isolated pyruvate perfused hearts (38) and in vivo skeletal muscles (21,111) have indicated that the [ADPI for oxidative phosphorylation in these tissues is close to the in vitro data. Thus, in these cases, there does not appear to be an alteration in the kinetics of ADP regulation of respiration in the intact cell. The observations in highly aerobic tissues during work jumps indicate that the rate of ATP production can increase without significant cytosolic increases in the substrates, ADP and Pi, for this reaction. Thus the synthesis of ATP is occurring at a faster rate with essentially the same concentrations of extramitochondrial ADP and Pi. This is very analogous to what was observed with increases in NADH in isolated mitochondria (75) and is inconsistent with either the extramitochondrial ATP/ADP*P;, ATP/ADP, or Pi and ADP alone controlling respiration, as derived from the kinetics observed in isolated mitochondria studies. The increase in ATP synthesis under these conditions must be occurring by either increasing the apparent affinity for ADP and/or Pi or the maximum velocity of the oxidative phosphorylation. Although several studies have demonstrated alterations in the affinity of ADP and Pi for oxidative phosphorylation under some conditions (13, lO4), little evidence for this type of regulation is available. Thus mechanisms which alter the maximum velocity of oxidative phosphorylation without significant alterations in the phosphorylation potential may be more reasonable. Both the mitochondrial NAD redox state, as changed by alterations in substrate delivery or dehydrogenase activity, as well as oxygen delivery could regulate the maximum velocity of oxidative phosphorylation. Both of these possibilities will be discussed later in this review. Also related to the role of ADP and Pi in the regulation of oxidative phosphorylation is the creatine kinase reaction in muscle and brain tissue. This reaction was originally believed to serve as an energy buffer in the cell by producing the high-energy creatine phosphate, from


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ATP and creatine, as an energy reserve in the cell. This concept is still accurate, especially when this reaction is considered over short bursts of ATPase activity occurring, for example, in the heart. However, the discovery that creatine kinase is associated with both the mitochondrial membrane and several ATPases has led to the hypothesis that this reaction is serving as a shuttle for ADP and ATP in the cytosol (59,88). This shuttle could be in the form of simple diffusion (88) or more integrated aspects of mitochondrial control as suggested by Jacobus and others (59). Thus the creatine kinase reaction may serve as an important aspect in the “exchange” of ADP and ATP in the cytosol in some tissues. However, the key intermediates in this process are still the ADP and ATP concentrations at the mitochondria and ATPases. It is also interesting to note that in the kidney with very high rates of oxidative phosphorylation no creatine kinase-facilitated diffusion is required for the rapid communication of the cytosol with oxidative phosphorylation. This difference could be due to the particular kinetic requirements and geometry of these tissues. In summary, the delivery of ADP and Pi is able to control the rate of oxidative phosphorylation in vitro. Because ADP and Pi are produced by the hydrolysis of ATP, the cytosolic concentrations of ADP and Pi would provide an excellent feedback signal to oxidative phosphorylation during changes in work or ATPase activity. However, both classical and more recent NMR studies have indicated that the concentrations of ADP and Pi do not change significantly with large increases in oxidative phosphorylation in tissues with high oxidative phosphorylation capabilities. It is unreasonable to assume that cytosolic ADP and Pi, which are intimately linked to ATPase activity, do not play any role in the regulation of oxidative phosphorylation in these tissues. The small bidirectional changes in these metabolites observed with work may indicate that these parameters are the fine feedback control of a system relying on other aspects of the network to cause the larger increments. In skeletal muscle and neonatal heart, however, the simple classical model of respiratory control may be accurate, since appropriate large variations in ADP and Pi are observed with changes in work output. REDUCING

EQUIVALENT

DELIVERY

The energy to make ATP ultimately comes from the energy in the highly reduced substrates oxidized by intermediary metabolism. This energy is transmitted to the mitochondrial respiratory chain via NADH or FADH (see Fig. 1). To increase ATP synthesis there must be a proportional increase in reducing equivalent, or energy, delivery to the cytochrome chain to perform oxidative phosphorylation. Therefore, the net delivery of reducing equivalents from the cytosol to the cytochrome chain is a potential regulatory site of oxidative phosphorylation in the intact cell as implied by the equilibrium models of Wilson and co-workers (34). Can the rate of oxidative phosphorylation be controlled by the delivery of cytosolic reducing equivalents? Koretsky and Balaban (75), using fluorescence tech-

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niques, found that the maximum rate of ATP synthesis by isolated rat liver mitochondria is increased by raising the concentration of mitochondrial NADH. It should be noted that the mitochondrial NADH monitored using fluorescence techniques may be sampling a specific “bound” pool of NADH (24, 33, 75, 95). The effect of NADH levels on the maximum rate of ATP synthesis was not saturable under the conditions studied and was also observed over the “physiological” range of oxidative phosphorylation using exogenously added ATPases. These data demonstrate that the concentration of mitochondrial NADH is an important factor in determining the maximum rate of oxidative phosphorylation under physiological conditions. Similar results have been obtained in intact renal cells with the addition of exogenous substrates to these cells (6), as well as in other tissue types (18, 51, 61,95, 118). Indeed, it seems quite easy to modify the mitochondrial redox state in intact cells with substrates or hormones (see below) in contrast to isolated mitochondria in which the usual substrate regime results in near-maximum levels of NADH (75). This implies that the resting levels of NADH in tissues are much lower than in most isolated mitochondria preparations and may be more important in the regulation of respiration than appreciated in most in vitro mitochondria studies. With regard to the interaction of mitochondrial NADH and ATP metabolites, the increased or maintained ATP synthesis rate with augmented NADH concentrations occurred with a decrease or no change in the concentrations of extramitochondrial substrates for ATP synthesis reaction, ADP and Pi (75). Thus the effect of NADH is to increase the maximum velocity of oxidative phosphorylation, which effectively increases the rate of ATP synthesis at a given concentration of ADP and Pi. This results in a mechanism for stimulating ATP synthesis without significant changes in ADP and Pi concentrations. It should also be noted that the mechanism of increase in oxidative phosphorylation by increased substrate supply is still unclear. It could involve the interaction of the mitochondrial membrane potential (19), the equilibrium of the cytochrome chain (34), or simply the kinetic effects of increased mitochondrial NADH or FADH. Taken together, these data indicate that the delivery of reducing equivalents to the mitochondrial respiratory chain is a potential regulatory site in the control of oxidative phosphorylation. Most interesting was the observation that alterations in NADH can increase ATP synthesis without increases in the substrates, ADP and Pi, for this reaction. However, as mentioned earlier, the ultimate control of ATP synthesis by oxidative phosphorylation in the steady state must still be the rate of ATP hydrolysis used to perform work. For respiration to be regulated by reducing equivalent delivery, or to simply match substrate supply with demand, a cytosolic transduction system between the work process and metabolic substrate supply site or sites must be present. There are a myriad of metabolic and transport processes which could regulate the delivery of reducing equivalents to the mitochondria. Indeed, under the right con-


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ditions, they are probably all important. Many enzymes and reactions have been demonstrated to be controlled by various relevant factors in vitro. These studies have shown significant influences on enzymatic or transport activity by the absolute and relative concentrations of ADP, Pi, ATP, Ca2+, Mg2+, and numerous metabolites, such as citrate or acetyl CoA, as well as the NAD redox state, mitochondrial membrane potential, and pH. One of the many dangers of extrapolating in vitro data to in vivo metabolic regulatory processes is that the dependencies characterized in vitro may only be effective during very specialized conditions within the cell. For example, protective mechanisms for adjustment to hypoxia or ischemia may result in regulatory mechanisms that can be observed under experimental conditions in the test tube but have little bearing on the function of these enzymes during the normal function of the cell. To evaluate potential cytosolic transduction mechanisms that may regulate the mitochondrial NADH and FADH levels, the various stages of NADH and FADH generation will be discussed. The first step in the delivery of reducing equivalents into the cytochrome chain is the production of substrates in the cytosol in an appropriate form for entry into the mitochondria. For example, carbohydrates are generally transported into the mitochondria in the form of pyruvate, whereas fats are converted to carnitine esters for transport. Any process in the cytosol that could modify the concentration of these or other transported substrates could alter the delivery of reducing equivalents and NADH. This transduction process could include processes which activate glucose or fat uptake, glycolysis, or fatty acid acylation, producing more cytosolic pyruvate or fatty acid carnitine esters. The alteration of cytosolic substrate concentrations can affect mitochondrial NADH redox states and oxidative phosphorylation as observed in various preparations in which the extracellular substrate contents can modify the NADH redox state as well as the rate of oxidative phosphorylation (6, 18, 51, 61, 95, 118). The transport of the substrates could also be a ratelimiting step. An excellent review on this topic as well as how it may relate to metabolic control is provided by LaNoue and Schoolwerth (80). The major sources of reduced carbons transported into the mitochondria are pyruvate and fats, as stated above. Pyruvate enters the mitochondria via a cotransport process involving a proton symport with a K, for pyruvate of -0.5 mM. It is not clear whether any form of regulation other than the delivery of pyruvate to the transporter is important (80). However, the regulation of this transporter is still an area of active research. Fatty acids are converted to neutral carnitine esters before being transported into the mitochondrial matrix. This transport is extremely fast in isolated mitochondria and consequently not believed to be a potential ratelimiting step in the oxidation of fats under normal conditions (48, 80). Thus, if carnitine esters of fats are available in the cytosol, their transport into the mitochondria for P-oxidation does not seem to be rate limiting under normal conditions. The electrogenic glutamate-aspartate carrier is an im-

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portant transporter in the direct transport of reducing equivalents across the mitochondrial membrane. This electrogenic transport process is modulated by the mitochondrial membrane and is believed to be a ratelimiting step in the direct transfer of reducing equivalents between the cytosol and the mitochondrial matrix (80) Once in the mitochondria, substrates are oxidized by the TCA cycle or ,&oxidation, and NADH or FADH is formed. Citrate synthase, which inserts acetyl CoA into the TCA cycle, was one of the earliest sites to be proposed as a flux limitation site in the TCA cycle (76, 78). In many tissues, with the possible exception of the heart (28), the extracted citrate synthase activity is close to the maximum flux through the TCA cycle. The subsequent metabolites of this reaction do not normally accumulate in the matrix space, suggesting that this enzyme is rate limiting (108). As far as how this enzyme may be regulated in the cell, in vitro studies have demonstrated that citrate synthase is very sensitive to several potential feedback regulators such as NADH, ATP, and succinyl CoA (38). Several dehydrogenases of the TCA cycle have also been proposed to be important regulatory steps. These enzymes include pyruvate dehydrogenase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase. There are several recent reviews on these enzymes and their potential role in the regulation of metabolic rates by Denton and McCormack (30,31) and Hansford (49). The activities of these enzymes are very sensitive in vitro to Ca2+, Mg+, ATP, ADP, AMP, Pi, NADH, NAD, and numerous other metabolites, which may make effective transducers between a work process and these enzymes (for example, see Ref. 70). The classical model for the activation of substrate oxidation by work has basically proposed that during an increase in work the NADH/NAD decreases because of the increase in reducing equivalent flux down the cytochrome chain, as observed in numerous isolated mitochondria studies (23,75). This redox change in NAD and several metabolites then plays a critical role in the activation of TCA cycle flux (73, 74, 91, 108, 114). As discussed below, even this model may be a bit simplistic. Having established that there are several enzymatic steps that could control the delivery of NADH to the respiratory chain, is there any evidence that these enzymes are being regulated by the rate of ATPase activity in cells? Some of the earliest studies on pyruvate dehydrogenase demonstrated that this enzyme is activated by an increase in work or hormonal stimulation in several tissues (56, 74). These results suggest that this enzyme is being regulated in an appropriate direction to modify the net flux through TCA to keep up with oxidative phosphorylation. Later, Hansford (47) demonstrated in insect flight muscle mitochondria that an increase in ADP resulted in a net increase in mitochondrial NADH due to the activation of dehydrogenases by ADP and possibly Ca2+. This is in contrast to most studies on isolated mammalian mitochondria in which an increase in extramitochondrial ADP and Pi is usually associated with a net decrease in mitochondrial NADH and an


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oxidation of the cytochrome chain (with exception of cytochrome aas) in the classical state 4 (resting) to state 3 (active ATP synthesis) transition (23). This may be partially due to the methods used to isolate these mitochondria (see Refs. 34 and 49) or simply species differences. More recent studies have shown that the activation of several dehydrogenases is apparently due to an increase in mitochondrial Ca2’ (30, 31, 49). Blocking the entry of Ca2+ into the mitochondrial matrix can prevent the hormonal activation of pyruvate dehydrogenase (87) and the metabolic consequences of this activation (67). Other investigators have suggested that mitochondrial volume may be another mechanism of activating substrate utilization as well as other aspects of oxidative phosphorylation (45, 46). As mentioned earlier, for oxidative phosphorylation to increase in response to an increase in work, the flux through the TCA cycle must be increased. Thus a flux increase through the rate-limiting steps of the TCA cycle must occur, possibly involving some of the mechanisms discussed above or others. Is the increase in TCA activity enough to only match the required flux through oxidative phosphorylation or enough to actually increase t.he NADH redox state and result in an effective increase in the maximum velocity of oxidative phosphorylation? This is the key question as to whether or not substrate delivery is actually driving oxidative phosphorylation or simply following the removal of reducing equivalents by the cytochrome chain in the intact tissue. The best way to assess this problem is to evaluate the mitochondrial NADH redox state during a work transition to establish whether mitochondrial NADH is increasing or decreasing during the work transition. A description of this approach is provided for substrate additions in renal cells by Balaban and Mandel (6). However, it should be pointed out that this procedure only provides the net effect on the NADH redox state and, therefore, the net effect of the activation of the TCA cycle during a work increase. For example, if an increase in work and oxidative phosphorylation results in a decrease of NADH, then it is unlikely that NADH is the primary stimulus increasing oxidative phosphorylation. However, NADH could still be significantly contributing to the observed increase in respiration as well as activating the TCA cycle in this complex network. If an increase in NADH is observed with an increase in work, then the NADH level could be acting as a primary site of activation of oxidative phosphorylation. But again this does not eliminate the contribution of other factors influencing this process. Numerous studies on the effects of work increases on the mitochondria NADH content in several systems have provided an almost equal variety of results. In liver tissues stimulated to increase respiration by a hormone addition, it is clear that at least the initial portion of the stimulation is apparently driven by a calcium-dependent increase in mitochondrial NADH (3, 100, 107). These increases are likely due to the aforementioned Ca2+dependent activation of TCA dehydrogenases (3). Similar results have been recently obtained in smooth muscle

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tissue (98). In brain, direct stimulations of the cerebral cortex have been correlated with net increases or decreases in the mitochondrial NADH redox state (32). In excised skeletal muscle, very consistent data have indicated that the mitochondrial NADH content and reduction level of the cytochrome do decrease with an increase in work (20,61). These compare favorably with measurements of the phosphorylation potential, which also decreases with increases in work, favoring an ADP- and Pi-driven system as discussed above. Similar NADH results were obtained in the perfused frog kidney (5) and with large work transitions in mammalian renal tubules in the presence of minimal metabolic substrates (7). The greatest variety of results concerning mitochondrial NADH alterations with work has been obtained in heart. Early enzymatic equilibrium studies indicated that a net oxidation of mitochondrial NADH was being observed with increases in myocardial work (74,91,92,94, 103). This was subsequently supported by several studies using the NADH fluorescence signal from isolated perfused systems (24,114). However, several studies, including some very recent investigations, have demonstrated that the NADH fluorescence signal can increase in the glucose-perfused heart during a moderate increase in work with pacing (66) or with hormone additions (118), as well as in the blood-perfused dog heart (57). This variety of results with regard to the NADH levels in response to work indicates that it is still unclear what role the activation of substrate delivery to the mitochondria may play in the regulation of oxidative phosphorylation. There are many technical problems with regard to the actual measurement of mitochondrial NADH using enzyme equilibria (116) as well as fluorescence techniques (33) which may contribute to this variability. In addition, the precise conditions (i.e., metabolic substrates, hormones, temperature, and work loads) of the in vitro preparations used may also confuse the issue. Indeed, the variety of results obtained may be directly the result of the complex interaction of the oxidative phosphorylation control sites which are difficult to keep constant in the wide range of preparations evaluated. Very little information is available on the redox state of mitochondrial NADH, FADH, and cytochrome b in vivo, where the most consistent conditions may be obtained, because of the problems with freeze clamping and optical artifacts, which will be discussed in the section on oxygen. In summary, an increase in mitochondrial NADH or FADH can result in an increase in the maximum rate of oxidative phosphorylation in isolated mitochondria as well as in intact tissues. Thus reducing equivalent delivery is a potential regulatory site of oxidative phosphorylation. The supply of reducing equivalents to the cytochrome chain is increased or decreased in accordance with changes in work and oxidative phosphorylation in intact tissues, indicating that the rate of substrate oxidation is coupled to ATP hydrolysis. Potential cytosolic transduction mechanisms participating in this regulation include cytosolic ADP, Pi, ATP, Ca2+, and Mg? HOWever, it is still unclear to what extent the modulation of reducing equivalents supply may serve as a primary


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regulatory tissues.

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site of oxidative

phosphorylation

in intact

OXYGEN

Oxygen is the terminal oxidant of the cytochrome chain reacting with cytochrome cza3and must be present in adequate concentration for oxidative phosphorylation to proceed. The apparent K, of oxidative phosphorylation for oxygen is on the order of 0.01-0.3 PM in isolated mitochondria. This apparent K, changes according to the rate of reducing equivalent delivery to cytochrome aa3, [ATP], [ADP], and [Pi]:, as well as the intramitochondrial pH (34, 119-122). In the various cell suspensions, oxygen consumption has been shown to be constant with oxygen concentrations down to -20 PM, with an apparent K, of -1 PM (84,119). Because of this high affinity of oxidative phosphorylation for oxygen, cellular oxygen concentrations must be reduced to very low levels when compared’ with arterial or even venous blood to contribute to the rate limitation of oxidative phosphorylation. This could occur because of inefficient capillary oxygen delivery to the cell or putative diffusion barriers to oxygen within the cell (63). Evidence is also available in isolated heart cells that cytosolic oxygen delivery requires specialized delivery systems, involving myoglobin, to reach maximum respiratory rates (124). Some investigators have also suggested that the affinity of the cytochrome chain for molecular oxygen may be much lower in vivo than in vitro, making even normoxic oxygen tensions potentially rate limiting for oxidative phosphorylation (62, 101). The key question with regard to this review is whether or not oxygen delivery is contributing to the rate control of oxidative phosphorylation within the cell under “normoxie” conditions. Tissue oxygen content has been estimated using several different approaches, including microoxygen electrodes and optical spectroscopic studies of intracellular myoglobin and cytochromes or exogenously added indicators. Generally, oxygen electrode studies, because of their disruptive nature, usually report only gross oxygen tensions within tissue. However, some studies using intracellular oxygen electrodes have consistently found oxygen tensions in regions of tissues as low as O-5 Torr (83, 114). This concentration is significantly lower than found in venous blood. These results suggest that regional hypoxia could be occurring in the intact tissue under normal conditions. Other indicators of intracellular oxygen tension are the oxygenation state of myoglobin and the cytochrome redox state monitored using optical spectroscopy techniques pioneered by Chance and co-workers (19, 22, 97, 110). These optical techniques do not disrupt the integrity or vasculature of the tissue as occurs in microelectrode studies but do rely on in vitro calibrations of the chromophores studied. In studies of cytosolic myoglobin oxidation, several groups have obtained data from intact tissues or rapid-frozen samples that indicate that the PO* of tissue is at or greater than its K, for oxygen in the heart in vitro (110) as well as in skeletal muscle in situ (40). These measurements would put the cytosolic oxvgen tension in excess of five times the critical PO?for

REVIEW

oxidative phosphorylation determined from in vitro studies. Oxygen tensions on this order have also been found with other methods for determining myoglobin oxygenation (27). Cytochrome aa is the enzyme complex responsible for the reduction of oxygen to water using the reducing equivalents provided by the earlier portions of the cytochrome chain. If oxygen delivery is rate limiting, then the redox state of cytochrome aa should reflect this by being significantly reduced and responsive to small changes in oxygen delivery (for example, see Ref. 8). However, the fact that cytochrome aa is reduced does not indicate that the respiratory rate will be limited by oxygen (119), as will be discussed below. The optical results on the redox state of cytochrome aa in intact cells and tissues have been a bit more controversial. Jobsis and co-workers (62, 101,105) have claimed, based on optical studies, that cytochrome aa is highly reduced under normoxic conditions in tissues in vivo, implying that the oxygen tension within the cell is very low or the affinity of cytochrome aa is much lower in vivo than in vitro. The optical measurement of cytochrome aa redox state in vivo is difficult because of overlapping absorption lines of hemoglobin and myoglobin as well as lightscattering effects. However, numerous studies have been performed to attempt to validate this method even with these serious limitations (9, 54, 62). Even in simpler systems in vitro, numerous groups have found that the apparent redox K, of cytochrome aa for oxygen is higher in intact tissues than isolated mitochondria. Tamura et al. (110) proposed that this might be due to steep tissue oxygen concentration gradients. Jones et al. (for extensive review, see Ref. 63) have suggested that this apparent low affinity of cytochrome aa for oxygen observed in vivo and in some cell suspensions is the result of limited diffusion of oxygen in the cytosol surrounding the mitochondria because of mitochondrial clustering. These latter conclusions are based on studies on cell suspensions using a combination of optical and biochemical techniques, although conflicting results are present in the literature (65, 123, 125). Benson et al. (11) have shown that heterogeneous regions occur in pyrene-lbutyric acid fluorescence consistent with intracellular gradients of oxygen content or solubility. Exogenous electron paramagnetic resonance probes also reveal apparent oxygen gradients (90). However, the whole contention that barriers to molecular oxygen diffusion exist in the cytosol has been challenged on theoretical grounds by Clark et al. (25). Wilson and co-workers (119) have shown in some cell types that cytochrome c becomes steadily more reduced from oxygen tensions of -200 PM (10% reduced cytochrome c) to 20 PM (20% reduced cytochrome c) while the actual oxygen consumption rate remains constant. Although this has not been reproduced in all systems studied (8), these data indicate that the oxygen apparent K, values for the cytochrome redox state and oxidative phosphorylation are not necessarily the same. According to these data, the redox state of the cytochromes will change before a rate limitation of oxygen consumption is detected in a titration with oxvgen. Cvtochrome aan in


INVITED

vivo is on the order of lo-20% reduced, as deduced from several optical measurements (9,10, lOl), which is in the range in which the redox state of cytochrome may be sensitive to oxygen tensions but not the overall rate of oxidative phosphorylation. Thus the detection of reduced cytochrome in vivo does not necessarily mean that oxidative phosphorylation is oxygen limited. Wilson and coworkers suggested that the cytochrome chain redox state is adjusted during changes in oxygen concentration to maintain a constant respiratory rate through the complex reaction occurring at cytochrome aa3. It was hypothesized that the oxygen-sensitive cytochrome redox state is due to the dependency of the complex cytochrome reaction kinetics on other constituents of the reaction such as the phosphorylation potential and intramitochondrial pH. However, even in this scheme, the overall oxidative phosphorylation apparent K, for oxygen still remains in the region of 0.5 PM. In another approach to this problem, the effects of vasodilation or increased blood flow on resting oxygen consumpt.ion under control conditions can be evaluated. The rationale for this approach is that if oxygen is rate limiting, then an increase in oxygen delivery by an increase in blood flow should increase oxygen consumption. Gregg and colleagues (43) in studies of the heart were able to demonstrate that an increase in coronary blood flow was matched by a significant increase in coronary oxygen consumption (the so-called “Gregg phenomenon”). Although not reproduced in all models, this phenomenon has been reproduced in several laboratories (for references, see Ref. 36). In addition, studies with some vasodilators have shown a similar phenomenon (96)) although studies with a physiological vasodilator (i.e., adenosine) have been generally negative (36). In studies in which an increase in oxygen consumption was observed, the increases in oxygen consumption were modest relative to the increase in blood flow or oxygen delivery. That is, a 100200% increase in blood flow results in only an -30% increase in respiration (96). In the normal heart such an increase in blood flow with work would cause a 100200% increase in respiration (36, 69). However, the fact that oxygen consumption does increase with flow is consistent with the notion that blood flow or oxygen delivery is partially rate limiting for oxidative phosphorylation. With regard to how oxygen tensions may be regulated in intact cells by work, it has been known for many years that as the work, or respiration, of a tissue increases there is a proportional increase in blood flow and oxygen delivery. Thus at a tissue level there is an apparent transduction system between the rate of ATP hydrolysis and oxygen and substrate delivery via blood flow. The cytosolic transduction mechanisms involved in this process are an area of very active research. Some of the cytosolic transducers include tissue adenosine levels (12)) phosphorylation potential (X21), or nitric oxide (55), which may change with increases in ATP hydrolysis rates. However, as was seen in the earlier sections, the change in phosphorylation potential in most tissues with high oxidative capacities is small or nonexistent with physiological increases in ATPase rates. Thus it is un-

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C385

likely that the phosphorylation potential per se is controlling blood flow. Other parameters, such as adenosine which may be regulated independently from the phosphorylation potential, are more likely candidates as regulatory agents. In summary, it is apparent that there is still no consensuson whether or not oxygen is partially rate limiting for oxidative phosphorylation in intact tissues. Most of the conflicting results are based on technically difficult studies making measurements at very low oxygen tensions using optical or microelectrode techniques. Optical techniques, although potentially powerful, are very difficult to interpret because of the significant problems introduced by overlap in the absorbance spectra and light-scattering artifacts. In addition, the study of oxygen in submicromolar concentrations, even in vitro, is extremely difficult and requires very sensitive probes of oxygen tensions (122). These difficulties may contribute to the rather significant discrepancies in results from different laboratories. The lack of agreement in the literature on the presence or magnitude of intracellular and/or extracellular oxygen gradients and the redox state of cytochrome aa3 is the major source of the controversy. What is well established is that blood flow is regulated by the metabolic rate of tissue. Thus blood flow is appropriately modulated to participate in the control of oxidative phosphorylation. The mechanism resulting in the balancing of blood flow and work is unknown, just as is the mechanism balancing the rate of oxidative phosphorylation and work. These two processes may be related, even relying on the same cytosolic transduction network. SUMMARY

Oxidative phosphorylation occurring in the mitochondria can be influenced by the cytosol at all of the sites discussed above and most likely other sites involving different transducers yet to be discovered. A summary of the potential sites and transduction mechanisms which were discussed, and which have been experimentally verified in some fashion, are presented in Fig. 2. In this model, the rate of work can affect oxidative phosphorylation via the delivery of reducing equivalents, oxygen, and ATPase hydrolysis products. In addition, the stimulus for an increase in work, such as a hormone, may not only activate the work process alone but may simultaneously activate the other regulatory sites, providing a “balanced” stimulation of the tissue. In such a multistep process, it is unlikely that any one step is dominating the overall rate of reaction. Indeed, this distributed control of oxidative phosphorylation may be the cause of many of the discrepancies seen in the literature both on in vitro and in vivo studies in which investigators have evaluated “extreme” cases (i.e., hypoxia, substrate-limited or near-maximum work loads) and in which only one or two of these sites were dominating the control process. Kacser and Burns (64) have proposed a general control model for such complex reactions based on the early work of Higgins (52), which could be used to analyze the regulation of oxidative phosphorylation. These models tend to distribute the


INVITED

C386 Blood

Q&q

what is the cytosolic transducer network responsible for regulating this process to an effective steady state? One of the greatest problems in answering these questions is establishing the precise cytosolic environment that the mitochondria are experiencing and how it changes with alterations in cellular work. Hopefully, the use of nondestructive techniques that are designed to evaluate the intact cellular milieu and biochemistry will provide the necessary data concerning the intracellular events involved in this basic homeostatic network in the cell.

Flow -

jAdenosinet?)

I

“2 aa

I%-

e-0 /

Substrates (Fats, Lactate, Glucose)

Cell

I / Hormones, Stimulants

Proposed cytosolic feedback network resulting in the balancing of oxidative phosphorylation and cellular work. The cytosolic feedback system is limited to agents that have some experimental validation. I realize that this model is most likely incomplete; however, it forms a useful working hypothesis. FIG.

REVIEW

2.

control of the overall reaction at several sites, resulting in a well-damped but responsive system. Tager and colleagues (109) have attempted to use this approach on isolated mitochondria. A simple Michaelis-Menten model using multiple sites of control has also been recently proposed by Chance and co-workers (ZZ), but this approach is dependent on the reaction mechanisms, since it assumes simple Michaelis-Menten-type kinetics that are not occurring in this complex network as discussed in this review. Although we are a long way from establishing the relative control coefficients (64) of each step of oxidative phosphorylation in vivo or even in intact cells, it is worthwhile to establish which cytoplasmic transducers and mitochondrial regulatory sites in this process must be considered and then to develop the appropriate network model to describe its function. Clearly, the classical presentation of respiratory control in the intact cell involving the simple kinetic feedback of ATP hydrolysis products through the cytosol to the mitochondria is not adequate to completely explain the interaction of oxidative phosphorylation and work in most highly aerobic tissues. It is proposed in this review that the cytosolic ATP hydrolysis products play a fine control role in the regulation of respiration in these tissues with other parameters controlling aspects of Vmax. These other parameters include substrate and oxygen delivery as shown in Fig. 2. However, to what extent either of these parameters may be primary regulators of oxidative phosphorylation in the intact cell is controversial. Both of these alternative regulatory processes are appropriately modulated by alterations in tissue work, but the extent to which this modulation is responsible for regulating respiration and the cytosolic network controlling these processes is still unknown. Thus two major problems remain. What are the primary controlling factors of oxidative phosphorylation in the intact cell, and

I thank several investigators for their stimulating discussions concerning the material covered in this review, as well as for unpublished data in an attempt to keep this perspective up to date. These investigators include Drs. A. Koretsky, F. Heineman, E. Fiegl, M. Kushmerick, R. Lynch, K. LaNoue, D. Wilson, B. Wittenberg, J. Wittenberg, and L. Mandel. I also acknowledge the enthusiastic support of the late Dr. J. Orloff in the pursuit of this scientific problem. REFERENCES T. P. M., H. BOOKELMAN, AND J. M. TAGER. Control of ATP transport across the mitochondrial membrane in isolated rat liver cells. FEBS Lett. 74: 50-54, 1977. 2. BALABAN, R. S. The application of nuclear magnetic resonance to the study of cellular physiology. Am. J. Physiol. 246 (Cell 1. AKERBOOM,

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