Control of Mitochondrial Respiration by NO (cid:1) , Effects of Low Oxygen and Respiratory State*

Nitric oxide (NO (cid:1) ) inhibits mitochondrial respiration by binding to the binuclear heme a 3 /Cu B center in cyto- chrome c oxidase. However, the significance of this reaction at physiological O 2 levels (5–10 (cid:1) M ) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O 2 ], and [NO (cid:1) ] were measured simultaneously at physiological O 2 levels with constant O 2 delivery, to model in vivo respi - ratory dynamics. Under these conditions NO (cid:1) inhibited mitochondrial respiration with an IC 50 of 0.14 (cid:2) 0.01 (cid:1) M in state 3 versus 0.31 (cid:2) 0.04 (cid:1) M in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO (cid:1) is due to greater control over respiration by an NO (cid:1) -dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO (cid:1) in vivo and its implications for the control of vessel-parenchymal O 2 gradients. The interaction of nitric oxide (NO (cid:1) ) with mitochondria is now emerging as one of the major pathways through which NO (cid:1)

The interaction of nitric oxide (NO ⅐ ) with mitochondria is now emerging as one of the major pathways through which NO ⅐ can exert both physiological and pathological responses in a variety of cell types (1,2). The most sensitive and widely studied target for NO ⅐ in mitochondria is the terminal enzyme of the electron transport chain, cytochrome c oxidase (3)(4)(5)(6). The binuclear O 2 binding site of this enzyme (heme a 3 /Cu B ) is reversibly inhibited by NO ⅐ (3,7,8). This is controlled by O 2 at two levels: by a competition between NO ⅐ and O 2 at the enzyme's active site (9), and by the partition of both NO ⅐ and O 2 into mitochondrial membranes, which accelerates the direct reaction between these molecules (10,11).
Despite an acknowledged physiological existence of the NO ⅐ -cytochrome c oxidase interaction, its biological importance and function remain uncertain. Three of the likely physiological roles for this interaction are: (a) regulation of mitochondrial H 2 O 2 generation (2, 12), (b) regulation of cytochrome c release in apoptosis (13), and (c) prevention of hypoxia in respiring tissues by inhibiting mitochondrial respiration and extending O 2 diffusion gradients from blood vessels (14). In pathological situations, perturbation of the NO ⅐ -cytochrome c oxidase pathway can occur, resulting in enhanced apoptosis or loss of control over respiration, as has been demonstrated in several tissues (15)(16)(17). Critical to understanding the mechanisms regulating the NO ⅐ -cytochrome c oxidase pathway are fundamental biochemical questions that have not been addressed in detail. For example, different NO ⅐ -derived species bound to purified cytochrome c oxidase have been identified, including a putative heme a 3 -nitrite complex (7), but it is unknown which of these complexes is formed in respiring mitochondria. In addition, the sensitivity of mitochondrial respiration to NO ⅐ appears greater while actively respiring (state 3 in the isolated preparation of the organelle) than while quiescent (state 4) (18). This phenomenon represents an additional level of control over respiration. However, it is inherently linked to the regulation of tissue O 2 levels, because mitochondrial O 2 consumption is different in each respiratory state. Therefore it is essential to determine the control of state 3 and 4 respiration by NO ⅐ under conditions of O 2 delivery similar to those in vivo.
Mechanisms that could explain the greater sensitivity of state 3 respiration to NO ⅐ include: (a) the formation of different NO ⅐ -derived complexes in cytochrome c oxidase depending on electron flux (7), (b) different amounts of such complexes formed in each state, or (c) different levels of control over respiration exhibited by cytochrome c oxidase in each state (18,19). The concept of control (i.e. flux control coefficient (19 -22)) has received little attention in the context of NO ⅐ regulation of respiration. This is important to study because control is a changing parameter, with cytochrome c oxidase exhibiting more control over respiration in state 3 than in state 4 (19). In cells, it is likely that mitochondria can exist in a mixture of both respiratory states.
In this investigation, several of the above issues were addressed by measuring the effects of NO ⅐ on mitochondrial respiration under conditions of controlled O 2 delivery (23). In addition mitochondrial absorption spectra were acquired to provide information on the redox status of cytochromes and their NO ⅐ -derived complexes. The results indicate that mitochondrial state 3 respiration is indeed more sensitive to NO ⅐dependent inhibition than state 4 under controlled O 2 -delivery conditions. Analysis of the absorption spectra suggested a common NO ⅐ -dependent spectral intermediate is formed to a simi-lar extent in both respiratory states. Interestingly, the data suggest that the inhibition of respiration due to population of this species is greater in state 3. In addition, NO ⅐ -dependent inhibition of respiration elevated the local [O 2 ], as has been proposed to occur in vivo (14). The implications of these findings are discussed in the context of a role for NO ⅐ as a physiological regulator of both mitochondrial respiration and local O 2 gradients.

MATERIALS AND METHODS
Chemicals and Animals-Sucrose for mitochondrial preparations was ultra-pure grade from USB (Cleveland, OH). All other chemicals were from Sigma. Male Sprague-Dawley rats, 250 g in weight, were purchased from Harlan and housed according to standard humane procedures on a 12 h light/dark cycle with food and water available ad libitum. Pure solutions of NO ⅐ gas were prepared by bubbling NaOHscrubbed commercial NO ⅐ gas through deoxygenated (argon-purged) water in a gas-tight bulb (24). Solutions were sealed under NO ⅐ atmosphere, stored in the dark, and aliquots withdrawn through a gas-tight septum into a gas-tight syringe. The concentration of NO ⅐ in these stock solutions was determined spectrophotometrically by monitoring oxyhemoglobin to methemoglobin conversion and was typically ϳ1.5 mM. The NO ⅐ probe and NO ⅐ solutions were also calibrated using an acidified nitrite/potassium iodide method as per the manufacturer's instructions (WPI Inc., Sarasota, FL).
Isolation of Mitochondria-Rat liver mitochondria were isolated by standard differential centrifugation techniques in buffer comprising sucrose (250 mM), Tris (10 mM), and EGTA (1 mM), pH 7 (24). All steps were performed at 4°C, and mitochondria were stored on ice and used within 4 h of isolation. Mitochondria in this study had respiratory control ratio values of 4.7 Ϯ 0.1 with glutamate ϩ malate as substrates. Protein was determined using the Folin-phenol reagent against a standard curve constructed using bovine serum albumin (25).
Apparatus-Instrumentation was modified from Cole et al. (23). Optical spectra of mitochondrial suspensions were acquired with a Cary model 14 recording spectrophotometer equipped with a scattered transmission accessory (AVIV Associates, Lakewood, NJ). A dilute suspension of whole milk in the reference cuvette was used to attenuate the reference light beam and partially balance light scattering. The thermostatted cuvette, serving as the respirometry chamber, was a cylindrical quartz tube with a 16-mm light path, containing 5 ml stirred (460 rpm) sample with a 5-ml gas headspace. The floor of the chamber was a polarographic O 2 sensor (Orbisphere 2110, Geneva, Switzerland) and a NO ⅐ sensor (WPI ISO-NO, Sarasota, FL) was positioned through the cuvette wall into the liquid phase. Composition of the gas phase was set by mixing dry air and O 2 -free N 2 using a digital mass flow controller (Tylan, Carson, CA), humidifying the gas, and passing it through the cuvette headspace at a flow rate of 100 ml/min. All experiments were performed at 37°C.
Experimental Procedures-Five ml of respiration buffer comprising KCl (120 mM), sucrose (25 mM), HEPES (10 mM), EGTA (1 mM), KH 2 PO 4 (1 mM), MgCl 2 (5 mM), glutamate (15 mM), and malate (7.5 mM) pH 7.3, was added to the sample cuvette. The apparatus was assembled and purged with 100% N 2 to lower the liquid phase [O 2 ] prior to mitochondrial addition. After addition of mitochondria (1 mg protein/ml), the gas flow mixture was re-adjusted to give a steady-state [O 2 ] of ϳ5 M. For state 4 respiration (with glutamate plus malate alone, formally state 2), the mixture required 8% air, whereas state 3 respiration (with the additional presence of 2 mM ADP) required 39% air. In all cases, substrate and/or ADP concentrations were sufficient to maintain steady-state respiration for Ͼ25 min.
The range of cellular O 2 concentrations that are present in vivo have been investigated extensively, with precise values dependent upon the specific tissue, metabolic activity, and subcellular location. In this investigation, an initial value of ϳ5 M was chosen, being at the low range of values experienced by mitochondria (26,27).
After Average atmospheric pressure during the course of these experiments was 98.5 kPa. The concentration of O 2 in air-saturated mitochondrial respiration buffer at 37°C (C*) was thus calculated as 196.8 M (28). Throughout this paper [O 2 ] is referred to in M, calculated from the mV output of the polarographic O 2 sensor, because this is the term most commonly used in mitochondrial physiology. Fig. 1 shows a typical set of traces for a mitochondrial respiration experiment. Following addition of mitochondria and respiratory substrates, a new steady-state value of C 1 is established. Under these conditions, the rate of O 2 consumption (Q) is m(C* Ϫ C 1 ) from Equation 1.
Following addition of NO ⅐ , transient inhibition of respiration causes a rise in C 1 , and because the system is now no longer at steady-state ] is termed C* and is based on the O 2 content of the headspace gas (see "Materials and Methods"). C 1 is the measured liquid phase [O 2 ], from the O 2 sensor. Addition of mitochondria and respiratory substrates (as shown by the arrow) results in establishment of a new steady-state value for C 1 . At this steady-state, the rate of mitochondrial respiration is calculated using the equation m(C* Ϫ C 1 ). Upon inhibition of respiration by NO ⅐ , C 1 rises transiently. During the NO ⅐ inhibition, dC 1 /dt is factored into the rate calculation, as shown. Following disappearance of NO ⅐ , respiration recovers and C 1 returns to baseline levels.
tions (both steady-and non-steady-state) is as follows, where A is the rate of delivery of X, and B is the rate of consumption of X. In the current experimental system, A equates to O 2 delivery, i.e. m(C* Ϫ C 1 ), and B equates to mitochondrial O 2 consumption rate (Q). The intermediate X is the liquid phase O 2 concentration (C 1 ). Thus, substituting these terms into Equation 2 yields the following, and subsequent rearrangement of this equation yields a universal equation for the calculation of Q under all conditions (both steady-and non-steady-state).
Experimentally, dC 1 /dt is the slope of the measured liquid phase [O 2 ] trace (C 1 ). Examining Equation 4, it becomes clear that in the steadystate (dC 1 /dt ϭ 0), it simplifies to Equation 1. In fact, rather than considering Equation 4 as a derivative of Equation 1, it is somewhat beneficial to consider Equation 1 and the steady-state, as a subset of the conditions governed by the universal Equation 4.
In adapting these terms for application to the non-steady-state, the rate-limiting step in obtaining a value for C 1 is the response time of the liquid phase [O 2 ] to changes in Q, and this is governed by m. Neither the time constant of the O 2 sensor (6.5 Ϯ 0.1 s, n ϭ 6), nor the adjustment of mitochondria to a new respiration rate (ϳ100 ms (29)) are limiting factors. Supporting this, it was observed that C 1 changed almost instantaneously upon addition of NO ⅐ to respiring mitochondria ( Fig. 1) This correction takes into account any O 2 limitation in the system due to factors such as the K m of cytochrome c oxidase for O 2 , unstirred layers and diffusional barriers. Also at each steady-state, absorption spectra were acquired to determine cytochrome redox state as a function of [O 2 ] and in a separate experiment in the presence of sodium dithionite.
Statistics-Unless otherwise indicated, all data are means of 4 determinations Ϯ S.E. Statistical significance was determined by Student's t test, with significance set at p Ͻ 0.05.

RESULTS
This study represents the first use of an open-flow respirometry system to determine mitochondrial respiration rates under non-steady-state conditions. In the first series of experiments (Fig. 2) both O 2 and NO ⅐ were monitored simultaneously in respiring mitochondria in states 3 and 4 (thick line and thin lines, respectively). Liquid phase oxygen (C 1 ) was maintained at ϳ5 M prior to addition of a 1 M bolus of NO ⅐ (shown by the arrow), which then caused an increase in C 1 in both states. Following the decay of NO ⅐ , C 1 returned to a similar level as that seen before NO ⅐ addition, consistent with the reversal of respiratory inhibition. The increase in [O 2 ] upon addition of NO ⅐ was more rapid and reached a higher level in state 3 than in state 4, primarily because the proportion of air in the gas mixture used to support state 3 (39%) is greater than for state 4 (8%). This means that, for example, even if Q were identical for both states after NO ⅐ addition, the dissolved O 2 concentration will rise more rapidly for state 3 because the gas phase pO 2 is nearly 5-fold higher. The kinetics of NO ⅐ decay (Fig. 2, C and  D) were not significantly different between respiratory states.
From a series of identical experiments, mitochondrial respiration rates (Q) were calculated and are shown in Fig. 3 Interestingly, respiration rates increased to a value greater than the initial rate prior to addition of NO ⅐ at the later time points. We attribute this response to O 2 -limitation of mitochondrial respiration at low [O 2 ]. Upon addition of NO ⅐ , the inhibition of respiration results in an increase in [O 2 ] (see Fig. 2, A  and B). At later time points when most of the NO ⅐ has decayed, [O 2 ] is still higher than the initial level of 5 M. Thus the observed rate of respiration is greater than the value seen prior to NO ⅐ addition. Eventually, as [O 2 ] returns to baseline levels, the rate recovers to the initial value, because O 2 is once again limiting. The increase in Q above 100% is greater in state 3, because the increase in [O 2 ] is greater here than in state 4 (see Fig. 2).
As described under "Materials and Methods," to correct for this phenomenon, mitochondria respiring in state 3 were incubated in the absence of NO ⅐ at different values of C*, allowing the system to reach steady-state. The value of C 1 was then recorded, and the rate Q was calculated and expressed as a percentage of the maximal rate at non-limiting [O 2 ] (120 M). These data are shown in Fig. 4A and demonstrate that mitochondrial respiration is O 2 limited in this experimental system at Ͻ20 M O 2 (p50 ϳ2.5 M).
Absorption spectra were also acquired in this experiment, and difference spectra were obtained by subtracting the spectrum at 120 M O 2 from those obtained at lower values of [O 2 ]. The delta absorbance for the peak at 445 nm was then expressed as a percentage of the fully reduced spectrum obtained in the presence of sodium dithionite (Fig. 4B). This represents the fractional reduction of cytochromes aa 3 in cytochrome c oxidase. Consistent with inhibition of mitochondrial respiration at low [O 2 ], these data indicate that the respiratory chain is more reduced as [O 2 ] falls. This is in contrast to a previous criticism of open-flow respirometry, in which a mis-match between respiration rates and cytochrome redox status was reported (26).
Having quantified the O 2 saturation kinetics in this system (Fig. 4), it is thus possible to correct the respiration rates determined in Fig. 3, taking into account the variable [O 2 ] that occurs during transient NO ⅐ inhibition. This analysis is shown in Fig. 5, and results in elimination of the increase in respiration rate above initial values, during recovery from NO ⅐ inhibition. Also NO ⅐ dose-response curve (Fig. 5C) becomes left-shifted, with the IC 50 for inhibition of respiration being 0.14 Ϯ 0.01 and 0.31 Ϯ 0.04 M NO ⅐ in states 3 and 4, respectively. Fig.  5A shows absolute values of Q, demonstrating the magnitude to which this data manipulation has modified these rates (cf. Fig. 3A).
Irrespective of whether the varying O 2 concentration is included as a factor in calculating rates, mitochondrial respiration is more sensitive to NO ⅐ in state 3 than in state 4 (Figs. 3 and 5). This finding is consistent with previous observations performed over a range of higher O 2 concentrations (50 -250 M O 2 ) (18). Importantly, it is evident from this study that the IC 50 values are significantly lower than those reported in the range of ϳ0.5 M NO ⅐ , determined at saturating O 2 levels (9, 18).
Absorption spectra were acquired before, during, and after addition of NO ⅐ to mitochondria, in states 3 and 4 (Fig. 6, A and  B, respectively). These panels also show the difference spectra, generated by subtracting the baseline spectrum (pre-NO ⅐ addition) from those acquired in the presence of NO ⅐ . The dotted arrows indicate the direction of increasing NO ⅐ concentration. A distinct spectral species was formed with an absorption maximum at ϳ431 nm when NO ⅐ was added to mitochondria in either respiratory state.
To provide insight to the origin of this spectral species, a series of standard spectra were prepared under identical conditions, using mitochondrial respiratory inhibitors. The data in Fig. 7 show difference spectra (Ϯ inhibitor) for mitochondria in state 3, in the presence of antimycin A, CN Ϫ , or NO ⅐ . These spectra exhibit the expected characteristics for isolated mitochondria with these inhibitors (7, 30). For example, addition of the complex III inhibitor antimycin A results in minima at 420 and 550 nm, corresponding to oxidation of cytochrome c, whereas addition of CN Ϫ results in the appearance of a peak at 550 nm, corresponding to reduction of cytochrome c due to downstream inhibition of complex IV. Antimycin A also gives a peak at 432 nm, which can be attributed to reduction of the b cytochromes. In the case of CN Ϫ , the resultant peaks at 445 and 602 nm correspond to a combination of reduced cytochromes aa 3 , and the a 3 -CN derivative. The peak at 424 nm is likely due to a combination of reduced cytochromes b, c, and c 1 .
With NO ⅐ , a broad, high amplitude peak at ϳ431 nm is present. This peak is consistent with the formation of the a 3 -NO ⅐ complex at 430 nm (7), with a likely contribution from other reduced upstream cytochromes. However, any contribution from the b cytochromes is likely to be small under these conditions, because the maximum additional reduction of the b cytochromes attainable with antimycin A (see above) is significantly less than the peak at 431 nm with NO ⅐ . The remainder of the absorbance at 431 nm with NO ⅐ is likely due to a composite of reduced cytochromes c, c 1 , and aa 3 . However, the reduced aa 3 peak at 445 nm may be lost in the shoulder of the 432 nm peak, especially because the a 3 -NO ⅐ complex is blueshifted relative to a 3 . In particular, NO ⅐ exposure results in a peak at 550 nm, attributable to reduced cytochrome c, and thus it is likely that at least part of the Soret peak with NO ⅐ is due to reduction of this cytochrome as an indirect consequence of inhibition of complex IV.
By examining the data in Fig. 6 alongside those in Fig. 5, it is possible to gain further insight into the differences between NO ⅐ inhibition of state 3 and state 4 respiration. The magni- tude of NO ⅐ -dependent spectral peak at 431 nm was plotted as a function of the corresponding NO ⅐ concentration at the time of spectral acquisition (Fig. 8A). These data indicate that the extent of formation of this species is not significantly different between states 3 and 4, with half-saturation occurring at 0.08 -0.10 M NO ⅐ . Thus, the differential sensitivity of state 3 and 4 respiration to [NO ⅐ ] does not appear to be caused by different levels of formation of an NO ⅐ -dependent species.
Given the assignment of the NO ⅐ -dependent spectral species at 431 nm discussed above, it seems reasonable to assume that the height of this peak is representative of the degree of inhibition of cytochrome c oxidase by NO ⅐ . This assumption implies a constant proportionality between the contributions of each spectrally active intermediate, to the overall spectral peak, independent of the concentration of NO ⅐ . Acknowledging this limitation in the precise assignment of the peak, the relationship between the degree of NO ⅐ inhibition, and changes in spectra were used to construct control/threshold curves (i.e. the response of respiration to the inhibition of cytochrome c oxidase by NO ⅐ (see "Discussion")). This analysis is shown in Fig. 8B and illustrates that state 3 respiration is more inhibited for the same amount of the NO ⅐ -dependent spectral species than is state 4. From this we conclude that part of the greater sensitivity of state 3 respiration to NO ⅐ resides in the fact that cytochrome c oxidase has more control over respiration in state 3.

DISCUSSION
The primary findings of this study are: (i) mitochondrial respiration is more sensitive to NO ⅐ in state 3 than in state 4 at physiological O 2 levels, (ii) open flow respirometry (23) can be applied to examine mitochondrial function under non-steadystate conditions, (iii) mitochondrial respiration may be O 2limited at higher concentrations than previously thought, and (iv) inhibition of respiration by NO ⅐ is completely reversible in both state 3 and state 4. Additionally, mitochondrial respiratory inhibition by NO ⅐ appears to elevate local [O 2 ] in this constant O 2 -delivery system. In vivo, such a response may facilitate enhanced vessel-parenchymal O 2 diffusion, as previously proposed (14). Furthermore, the differential sensitivity of states 3 and 4 to NO ⅐ suggests that O 2 would be delivered to distances further from a vessel under conditions of increased work demand. This is because mitochondria in working tissues are in a state more akin to state 3, and thus more sensitive to NO ⅐ inhibition, as opposed to quiescent tissues, which would be in state 4 and thus less sensitive to NO ⅐ .
Because NO ⅐ -dependent inhibition of respiration is competitive with O 2 , previous reports (18) showing greater sensitivity of state 3 respiration to NO ⅐ were biased by the fact that these observations were made over a range of lower O 2 concentrations in state 3 than in state 4. In contrast, the open-flow respirometer yields more limited changes in [O 2 ] that are actually greater in state 3 (see Fig. 2), and thus competition with O 2 cannot account for the greater NO ⅐ sensitivity of state 3 respiration reported here.
Having confirmed that under physiological conditions, state 3 respiration is indeed more sensitive to NO ⅐ than is state 4, we sought to investigate the mechanisms involved. Fig. 6 suggests that the difference is not due to formation of a distinct NO ⅐derived species in each respiratory state, whereas Fig. 7 suggests that the amount of the common NO ⅐ -dependent spectral species formed is not different in each state.
Although the precise identity of the NO ⅐ -dependent spectral species is not known, it is likely a composite of both the heme a 3 -NO ⅐ complex and the upstream reduced cytochromes b, c, and c 1 . This assignment is based on the difference spectra acquired with respiratory inhibitors shown in Fig. 7. Unfortunately, acquisition of spectra over such a wide range of wavelengths (380 -640 nm at 0.5 nm resolution) takes ϳ2.5 min in this system, and this was incompatible with the rapidly changing kinetics of both [NO ⅐ ], [O 2 ], and mitochondrial respiration. Thus, a smaller window of wavelengths (400 -500 nm, 1 nm resolution) was examined in the NO ⅐ kinetic experiments, to maximize the time-resolution of this study. However in doing so, it is acknowledged that some spectral information is lost. Thus, although high resolution kinetic studies on purified cytochrome c oxidase have reported the formation of a cytochrome a 3 -nitrite complex within the enzyme, in addition to the a 3 -NO ⅐ complex (7), the lower relative resolution of the current spectral system does not allow us to address this point. However, our data does not preclude the possibility that in mitochondria and cells, different NO ⅐ -derived species could be formed at cytochrome c oxidase in different metabolic states.
Because the NO ⅐ -dependent spectral species in Fig. 6 appears to originate from an inhibited form of cytochrome c oxidase (see Figs. 6 and 7 and descriptions), it is possible to use these data to construct a control/threshold curve (20 -22). At this stage it is important to define what is meant by control in this context. In an integrated system such as mitochondrial respiration, the component steps have varying degrees of control over the respiration rate. These include but are not limited to: the tricarboxylic acid cycle, the respiratory chain, proton leak, the phosphorylation machinery (ATP synthase, adeninenucleotide translocase), and the various mitochondrial transmembrane solute carriers (19,31). Control can be examined by determining the response of the overall system (respiration) to the inhibition of one of these components. The greater the degree of control by a given component, the greater will be the effect of inhibiting it on respiration. The term threshold refers to a characteristic elbow-shape in control-analysis curves (see Refs. 21 and 22) and is typically used to define the excess capacity of individual components of a system. A component with a larger threshold is present in greater excess, and thus inhibiting it has relatively little effect on respiration. Generally, a larger threshold equates to a lower degree of control.
Plotting the population of the NO ⅐ -dependent spectral species against the respiration rate (Fig. 8B) reveals that this species has more control over respiration in state 3 than in state 4. These data are in agreement with previous control analysis studies (19,31) of mitochondria, which have shown that in state 4 much of the control over respiration lies outside the respiratory chain (e.g. in the proton leak of the inner membrane), whereas in state 3 it lies mostly within the respiratory chain (in particular at cytochrome c oxidase). We therefore conclude that the predominant reason why respiration is more sensitive to NO ⅐ in state 3 is due to a greater control over respiration by cytochrome c oxidase in this state.
Another important observation from the current data set is that mitochondrial respiration always returns to 100% of its initial rate following NO ⅐ decay. This property has been difficult to determine previously because in closed-chamber experiments the full recovery of respiration occurs at low [O 2 ], where the potency of NO ⅐ is increased, resulting in a progressive inhibition of the respiration rate near the end of the trace (see Ref. 10 for an example). The present study conclusively demonstrates that NO ⅐ is a fully reversible inhibitor of mitochondrial respiration.
With regard to the O 2 -sensitivity of mitochondrial respiration in this system (Fig. 4), the [O 2 ] for half-maximal saturation of respiration rate (p50) was ϳ2.5 M. This is ϳ10ϫ higher than previously published values for isolated mitochondria (26). However, several differences are present between this and previous experimental systems, including the use of an openflow versus a closed respirometry chamber, the temperature, the concentration and source of mitochondria, and the dimensions of the open-flow system. The wide range of values for O 2 p50 available in the literature (0.05-0.8 M O 2 ), as well as the current data, highlight the importance of determining p50 for each experimental system because no single value appears applicable to all conditions. Whether O 2 limitation of mitochondrial respiration occurs in vivo over this range of [O 2 ] (1-20 M) remains to be determined, although the implications for such a limitation are profound.
Using the information on O 2 -limitation contained in Fig. 4, a transformation was performed on the respiration rate data in Fig. 3, to give O 2 -corrected respiration rates, shown in Fig. 5. Although it is recognized that these data (Fig. 5) are not true values of Q because they do not originate from equation 1, nevertheless the outcome of this analysis is still the same, i.e. that respiration is more sensitive to inhibition by NO ⅐ in state 3 than in state 4.
The results of this paper, and in particular the rise in [O 2 ] following NO ⅐ addition to the chamber (Figs. 1 and 2), give further insight into the hypothesis that NO ⅐ -dependent inhibition of mitochondrial O 2 consumption, in the cells lining blood vessels, would allow O 2 to diffuse further from the vessels and facilitate parenchymal cell respiration (14). These experiments support this general principle, although it is recognized that in vivo, O 2 delivery is a complex variable that may itself respond to changes in respiration. In addition, other physiologic or pathologic functions could influence these responses including NO ⅐ regulation of O 2 gradients in O 2 -sensing tissues such as the carotid body.
In summary we have demonstrated that NO ⅐ -dependent inhibition of mitochondrial respiration is responsive to both respiratory state and O 2 tension. It is evident that NO ⅐ has an important role in the cell as an endogenous physiological regulator of both mitochondrial respiration and of potential mitochondrially linked cell signaling events (2).