16O2/18O2 analysis of oxygen exchange in Dunaliella tertiolecta. Evidence for the inhibition of mitochondrial respiration in the light

A mass spectrometric ¹⁶O₂/¹⁸O₂-isotope technique was used to analyse the rates of gross O₂ evolution, net O₂ evolution and gross O₂ uptake in relation to photon fluence rate by Dunaliella tertiolecta adapted to 0.5, 1.0, 1.5, 2.0 and 2.5 M NaCl at 25°C and pH 7.0.At concentrations of dissolved inorganic carbon saturating for photosynthesis (200 M) gross O₂ evolution and net O₂ evolution increased with increasing salinity as well as with photon fluence rate. Light compensation was also enhanced with increased salinities. Light saturation of net O₂ evolution was reached at about 1000 mol m^-2s^-1 for all salt concentrations tested. Gross O₂ uptake in the light was increased in relation to the NaCl concentration but it was decreased with increasing photon fluence rate for almost all salinities, although an enhanced flow of light generated electrons was simultaneously observed. In addition, a comparison between gross O₂ uptake at 1000 mol photons m^-2s^-1, dark respiration before illumination and immediately after darkening of each experiment showed that gross O₂ uptake in the light paralleled but was lower than mitochondrial O₂ consumption in the dark.From these results it is suggested that O₂ uptake by Dunaliella tertiolecta in the light is mainly influenced by mitochondrial O₂ uptake. Therefore, it appears that the light dependent inhibition of gross O₂ uptake is caused by a reduction in mitochondrial O₂ consumption by light.Abbreviations DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - DHAP dihydroxy-acetonephosphate - DIC dissolved inorganic carbon - DR_a rate of dark respiration immediately after illumination - DR_b rate of dark respiration before illumination - E₀ rate of gross oxygen evolution in the light - NET rate of net oxygen evolution in the light - PFR photon fluence rate - RubP rubulose-1,5-bisphosphate - SHAM salicyl hydroxamic acid - U₀ rate of gross oxygen uptake in the light     

O(2)-sensing signal cascade: clamping of O(2) respiration, reduced ATP utilization, and inducible fumarate respiration

These studies explore the consequences of activating the prolyl hydroxylase (PHD) O(2)-sensing pathway in spontaneously twitching neonatal cardiomyocytes. Full activation of the PHD pathway was achieved using the broad-spectrum PHD inhibitor (PHI) dimethyloxaloylglycine (DMOG). PHI treatment of cardiomyocytes caused an 85% decrease in O(2) consumption and a 300% increase in lactic acid production under basal conditions. This indicates a approximately 75% decrease in ATP turnover rate, inasmuch as the increased ATP generation by glycolysis is inadequate to compensate for the lower respiration. To determine the extent to which decreased ATP turnover underlies the suppressed O(2) consumption, mitochondria were uncoupled with 2,4-dinitrophenol. We were surprised to find that 2,4-dinitrophenol failed to increase O(2) consumption by PHI-treated cells, indicating that electron transport chain activity, rather than ATP turnover rate, limits respiration in PHI-treated cardiomyocytes. Silencing of hypoxia-inducible factor-1alpha (HIF-1alpha) expression restored the ability of uncoupled PHI-treated myocytes to increase O(2) consumption; however, basal O(2) uptake rates remained low because of the unabated suppression of cellular ATP consumption. Thus it appears that respiration is actively "clamped" through an HIF-dependent mechanism, whereas HIF-independent mechanisms are responsible for downregulation of ATP consumption. In addition, we find that PHD pathway activation enables mitochondria to utilize fumarate as a terminal electron acceptor when cytochrome c oxidase is inactive. The source of fumarate for this unusual respiration is derived from aspartate via the purine nucleotide cycle. In sum, these studies show that the O(2)-sensing pathway is sufficient to actively "clamp" O(2) consumption and independently suppress cellular ATP consumption. The PHD pathway also enables the mitochondria to utilize fumarate for respiration.     

Inhibition of brain mitochondrial respiration by dopamine: involvement of H(2)O(2) and hydroxyl radicals but not glutathione-protein-mixed disulfides

Examination of the downstream mediators responsible for inhibition of mitochondrial respiration by dopamine (DA) was investigated. Consistent with findings reported by others, exposure of rat brain mitochondria to 0.5 mm DA for 15 min at 30 degrees C inhibited pyruvate/glutamate/malate-supported state-3 respiration by 20%. Inhibition was prevented in the presence of pargyline and clorgyline demonstrating that mitochondrial inhibition arose from products formed following MAO metabolism and could include hydrogen peroxide (H(2) O(2) ), hydroxyl radical, oxidized glutathione (GSSG) or glutathione-protein mixed disulfides (PrSSG). As with DA, direct incubation of intact mitochondria with H(2) O(2) (100 microm) significantly inhibited state-3 respiration. In contrast, incubation with GSSG (1 mm) had no effect on O(2) consumption. Exposure of mitochondria to 1 mm GSSG resulted in a 3.3-fold increase in PrSSG formation compared with 1.4- and 1.5-fold increases in the presence of 100 microm H(2) O(2) or 0.5 mm DA, respectively, suggesting a dissociation between PrSSG formation and effects on respiration. The lack of inhibition of respiration by GSSG could not be accounted for by inadequate delivery of GSSG into mitochondria as increases in PrSSG levels in both membrane-bound (2-fold) and intramatrix (3.5-fold) protein compartments were observed. Furthermore, GSSG was without effect on electron transport chain activities in freeze-thawed brain mitochondria or in pig heart electron transport particles (ETP). In contrast, H(2) O(2) showed differential effects on inhibition of respiration supported by different substrates with a sensitivity of succinate > pyruvate/malate > glutamate/malate. NADH oxidase and succinate oxidase activities in freeze-thawed mitochondria were inhibited with IC(50) approximately 2-3-fold higher than in intact mitochondria. ETPs, however, were relatively insensitive to H(2) O(2). Co-administration of desferrioxamine with H(2) O(2) had no effect on complex I-associated inhibition in intact mitochondria, but attenuated inhibition of rotenone-sensitive NADH oxidase activity by 70% in freeze-thawed mitochondria. The results show that DA-associated inhibition of respiration is dependent on MAO and that H(2) O(2) and its downstream hydroxyl radical rather than increased GSSG and subsequent PrSSG formation mediate the effects.     

植物光合CO2响应模型对光下(暗)呼吸速率拟合的探讨

运用LI-6400便携式光合作用系统测定了不同光强(2000、1500、1000和500 μmol·m^-2·s^-1)和两种O₂浓度(21%和2%的O₂)下冬小麦(Triticum aestivum)灌浆期旗叶的CO₂响应曲线, 比较了现有CO₂响应模型(生化模型、直角双曲线模型和直角双曲线修正模型)拟合给出光下(暗)呼吸与测量值之间的差异。结果显示, 直角双曲线修正模型所给出的光下呼吸速率拟合值与测量值最为接近。植物光合作用对大气CO₂响应(A/C_a)的拟合结果优于光合作用对胞间CO₂浓度(A/C_i)的拟合。然而, 所有模型基于A/C_a拟合的光下(暗)呼吸在整体上与测量值存在显著差异(p < 0.05), 推测与现有模型没有考虑CO₂浓度对光呼吸和光下暗呼吸速率的影响有关。对小麦的试验结果表明, CO₂浓度对光呼吸和光下暗呼吸均有显著影响: 随着CO₂浓度的增加(0-1400 μmol·mol^-1), 不同光强下的表观光呼吸变化范围分别为5.035-11.670、4.222-11.650、4.330-10.999和3.263-9.094 μmol CO_2·m^-2·s^-1; 光下暗呼吸的变化范围分别为0.491-2.987、0.457-2.955、0.545-3.139和0.448-3.139 μmol CO₂·m^-2·s^-1。回归分析发现, 表观光呼吸和光下暗呼吸与CO₂浓度之间均存在较好的相关性。然而, 将该回归关系整合到现有模型中, 是否会优化模型, 从而提高模型对相关光合参数估算的准确性尚有待于进一步研究。     

Direct evidence for the control of mitochondrial respiration by mitochondrial creatine kinase in oxidative muscle cells in situ

The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g. cytosolic or mitochondrial intermembrane space, was evaluated in wild-type and creatine kinase (CK)-deficient mice. To activate respiration by endogenous production of ADP in permeabilized cells, ATP was added either alone or together with creatine. In cardiac fibers, while ATP alone activated respiration to half of the maximal rate, creatine plus ATP increased the respiratory rate up to its maximum. To find out whether the stimulation by creatine is a consequence of extramitochondrial [ADP] increase, or whether it directly correlates with ADP generation by mitochondrial CK in the mitochondrial intermembrane space, an exogenous ADP-trap system was added to rephosphorylate all cytosolic ADP. Under these conditions, creatine plus ATP still increased the respiration rate by 2.5 times, compared with ATP alone, for the same extramitochondrial [ADP] of 14 microM. Moreover, this stimulatory effect of creatine, observed in wild-type cardiac fibers disappeared in mitochondrial CK deficient, but not in cytosolic CK-deficient muscle. It is concluded that respiration rates can be dissociated from cytosolic [ADP], and ADP generated by mitochondrial CK is an important regulator of oxidative phosphorylation.     

四种作物光下暗呼吸速率降低的原因

以C₃作物(小麦, Triticum aestivum和大豆, Glycine max)和C₄作物(玉米, Zea mays和千穗谷, Amaranthus hypochondriacus)为例, 探讨了其光下暗呼吸速率降低的原因。结果表明, 2% O₂条件下, CO₂浓度为0时, 叶室CO₂浓度维持在0左右, 而胞间CO₂浓度(C_i)显著高于叶室CO₂浓度。分析认为这是由于此时植物的暗呼吸仍在正常进行。因此, 该测量条件下的表观光合速率应为CO₂浓度为0时的光下暗呼吸速率(R_d)。CO₂浓度为0时, 不同光强下的R_d均随光强的升高而降低, 且在低光强(50 μmol·m^-2·s^-1)和高光强(2000 μmol·m^-2·s^-1)之间存在显著差异, 说明光强对R_d具有较大影响。在2% O₂条件下, 经饱和光强充分活化而断光后, 以上4种作物叶片的暗呼吸速率(R_n)均随着时间的推移而下降, 说明光强并未抑制暗呼吸速率。试验结果表明, R_d的降低是由于CO₂被重新回收利用所导致, CO₂回收利用率随光强的升高而增大, 从低光强(50 μmol·m^-2·s^-1)到高光强(2000 μmol·m^-2·s^-1), 小麦、大豆、玉米和千穗谷的回收利用率范围变动分别为22.65%-52.91%、22.40%-55.31%、54.24%-87.59%和72.43%-90.07%。     

Photosynthetic refixing of CO2 is responsible for the apparent disparity between mitochondrial respiration in the light and in the dark

The net photosynthetic rate (P _N), the sample room CO₂ concentration (CO₂S) and the intercellular CO₂ concentration (C _i) in response to PAR, of C₃ (wheat and bean) and C₄ (maize and three-colored amaranth) plants were measured. Results showed that photorespiration (R _p) of wheat and bean could not occur at 2 % O₂. At 2 % O₂ and 0 μmol mol^?1 CO₂, P _N can be used to estimate the rate of mitochondrial respiration in the light (R _d). The R _d decreased with increasing PAR, and ranged between 3.20 and 2.09 μmol CO₂ m^?2 s^?1 in wheat. The trend was similar for bean (between 2.95 and 1.70 μmol CO₂ m^?2 s^?1), maize (between 2.27 and 0.62 μmol CO₂ m^?2 s^?1) and three-colored amaranth (between 1.37 and 0.49 μmol CO₂ m^?2 s^?1). The widely observed phenomenon of R _d being lower than R _n can be attributed to refixation, rather than light inhibition. For all plants tested, CO₂ recovery rates increased with increasing light intensity from 32 to 55 % (wheat), 29 to 59 % (bean), 54 to 87 % (maize) and 72 to 90 % (three-colored amaranth) at 50 and 2,000 μmol m^?2 s^?1, respectively.     

(Re)Greening photochemistry: using light for degrading persistent organic pollutants

Light is a cheap and abundant chemical reagent, capable of inducing highly selective reactions, some of which cannot be reasonably carried out by alternative ways nowadays. Photochemical processes may take place directly from the excited states or short-lived intermediates generated after light absorption or, alternatively, through reaction with species indirectly generated by the action of light on other chemicals present in the medium (catalysts or secondary reagents). As a consequence a range of possibilities are open to transform and/or degrade refractory pollutants or undesired chemicals or microbia in air, water, soil, on surfaces, etc., as well as other applications described below.     

Myxothiazol induces H(2)O(2) production from mitochondrial respiratory chain

Interruption of electron flow at the quinone-reducing center (Q(i)) of complex III of the mitochondrial respiratory chain results in superoxide production. Unstable semiquinone bound in quinol-oxidizing center (Q(o)) of complex III is thought to be the sole source of electrons for oxygen reduction; however, the unambiguous evidence is lacking. We investigated the effects of complex III inhibitors antimycin, myxothiazol, and stigmatellin on generation of H(2)O(2) in rat heart and brain mitochondria. In the absence of antimycin A, myxothiazol stimulated H(2)O(2) production by mitochondria oxidizing malate, succinate, or alpha-glycerophosphate. Stigmatellin inhibited H(2)O(2) production induced by myxothiazol. Myxothiazol-induced H(2)O(2) production was dependent on the succinate/fumarate ratio but in a manner different from H(2)O(2) generation induced by antimycin A. We conclude that myxothiazol-induced H(2)O(2) originates from a site located in the complex III Q(o) center but different from the site of H(2)O(2) production inducible by antimycin A.     

Leaf O(2) uptake in the dark is independent of coincident CO(2) partial pressure

Elevated CO(2), in the dark, is sometimes reported to inhibit leaf respiration, with respiration usually measured as CO(2) efflux. Oxygen uptake may be a better gauge of respiration because non-respiratory processes can affect dark CO(2) efflux in elevated CO(2). Two methods of quantifying O(2) uptake indicated that leaf respiration was unaffected by coincident CO(2) level in the dark.     

Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum

This study describes the O₂ uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O₂-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the “residual component”, operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O₂-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O₂ reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O₂ uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O₂ uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O₂ uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O₂ uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O₂-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the “residual component” and the peroxidase-mediated O₂ uptake. The peroxidase-mediated O₂ uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the “residual component” 24%.     

4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple sites

A destructive cycle of oxidative stress and mitochondrial dysfunction is proposed in neurodegenerative disease. Lipid peroxidation, one outcome of oxidative challenge, can lead to the formation of 4-hydroxy-2(E)-nonenal (HNE), a lipophilic alkenal that forms stable adducts on mitochondrial proteins. In this study, we characterized the effects of HNE on brain mitochondrial respiration. We used whole rat brain mitochondria and concentrations of HNE comparable to those measured in patients with Alzheimer's disease. Our results showed that HNE inhibited respiration at multiple sites. Complex I-linked and complex II-linked state 3 respirations were inhibited by HNE with IC50 values of approximately 200 microM HNE. Respiration was apparently diminished owing to the inhibition of complex III activity. In addition, complex II activity was reduced slightly. The lipophilicity and adduction characteristics of HNE were responsible for the effects of HNE on respiration. The inhibition of respiration was not prevented by N-acetylcysteine or aminoguanidine. Studies using mitochondria isolated from porcine cerebral cortex also demonstrated an inhibition of complex I- and complex II-linked respiration. Thus, in neurodegenerative disease, oxidative stress may impair mitochondrial respiration through the production of HNE.     

NO and N2O transformations of diverse fungi in hypoxia: evidence for anaerobic respiration only in Fusarium strains

Fungal denitrification is claimed to produce non-negligible amounts of N₂O in soils, but few tested species have shown significant activity. We hypothesized that denitrifying fungi would be found among those with assimilatory nitrate reductase, and tested 20 such batch cultures for their respiratory metabolism, including two positive controls, Fusarium oxysporum and Fusarium lichenicola, throughout the transition from oxic to anoxic conditions in media supplemented with . Enzymatic reduction of (NIR) and NO (NOR) was assessed by correcting measured NO- and N₂O-kinetics for abiotic NO- and N₂O-production (sterile controls). Significant anaerobic respiration was only confirmed for the positive controls and for two of three Fusarium solani cultures. The NO kinetics in six cultures showed NIR but not NOR activity, observed through the accumulation of NO. Others had NOR but not NIR activity, thus reducing abiotically produced NO to N₂O. The presence of candidate genes (nirK and p450nor) was confirmed in the positive controls, but not in some of the NO or N₂O accumulating cultures. Based on our results, we conclude that only the Fusarium cultures were able to sustain anaerobic respiration and produced low amounts of N₂O as a response to an abiotic NO production from the medium.     

Substitutions at the Asp-473 latch residue of chlamydomonas ribulosebisphosphate carboxylase/oxygenase cause decreases in carboxylation efficiency and CO(2)/O(2) specificity

The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO(2) and O(2). Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (V(max)/K(m)) and approximately 16% decreases in CO(2)/O(2) specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO(2)/O(2) specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco.     

Catalase regulates cell growth in HL60 human promyelocytic cells: evidence for growth regulation by H(2)O(2)

Reactive oxygen species (ROS) including hydrogen peroxide (H(2)O(2)) are generated constitutively in mammalian cells. Because of its relatively long life and high permeability across membranes, H(2)O(2) is thought to be an important second messenger. Generation of H(2)O(2) is increased in response to external insults, including radiation. Catalase is located at the peroxisome and scavenges H(2)O(2). In this study, we investigated the role of catalase in cell growth using the H(2)O(2)-resistant variant HP100-1 of human promyelocytic HL60 cells. HP100-1 cells had an almost 10-fold higher activity of catalase than HL60 cells without differences in levels of glutathione peroxidase, manganese superoxide dismutase (MnSOD), and copper-zinc SOD (CuZnSOD). HP100-1 cells had higher proliferative activity than HL60 cells. Treatment with catalase or the introduction of catalase cDNA into HL60 cells stimulated cell growth. Exposure of HP100-1 cells to a catalase inhibitor resulted in suppression of cell growth with concomitant increased levels of intracellular H(2)O(2). Moreover, exogenously added H(2)O(2) or depletion of glutathione suppressed cell growth in HL60 cells. Extracellular signal regulated kinase 1/2 (ERK1/2) was constitutively phosphorylated in HP100-1 cells but not in HL60 cells. Inhibition of the ERK1/2 pathway suppressed the growth of HP100-1 cells, but inhibition of p38 mitogen-activated protein kinase (p38MAPK) did not affect growth. Moreover, inhibition of catalase blocked the phosphorylation of ERK1/2 but not of p38MAPK in HP100-1 cells. Thus our results suggest that catalase activates the growth of HL60 cells through dismutation of H(2)O(2), leading to activation of the ERK1/2 pathway; H(2)O(2) is an important regulator of growth in HL60 cells.     

Phylogenetic evidence for role-reversals of gender-associated mitochondrial DNA in Mytilus (Bivalvia: Mytilidae)

Distinct gender-associated mitochondrial DNA (mtDNA) lineages (i.e., lineages which are transmitted either through males or through females) have been demonstrated in two families of bivalves, the Mytilidae (marine mussels) and the Unionidae (freshwater mussels), which have been separated for more than 400 Myr. The mode of transmission of these M (for male-transmitted) and F (for female-transmitted) molecules has been referred to as doubly uniparental inheritance (DUI), in contrast to standard maternal inheritance (SMI), which is the norm in animals. A previous study suggested that at least three origins of DUI are required to explain the phylogenetic pattern of M and F lineages in freshwater and marine mussels. Here we present phylogenetic evidence based on partial sequences of the cytochrome c oxidase subunit I gene and the 16S RNA gene that indicates the DUI is a dynamic phenomenon. Specifically, we demonstrate that F lineages in three species of Mytilus mussels, M. edulis, M. trossulus, and M. californianus, have spawned separate lineages which are now associated only with males. This process is referred to as "masculinization" of F mtDNA. By extension, we propose that DUI may be a primitive bivalve character and that periodic masculinization events combined with extinction of previously existing M types effectively reset the time of divergence between conspecific gender-associated mtDNA lineages.      

O2 versus N2O respiration in a continuous microbial enrichment

Despite its ecological importance, essential aspects of microbial N₂O reduction—such as the effect of O₂ availability on the N₂O sink capacity of a community—remain unclear. We studied N₂O vs. aerobic respiration in a chemostat culture to explore (i) the extent to which simultaneous respiration of N₂O and O₂ can occur, (ii) the mechanism governing the competition for N₂O and O₂, and (iii) how the N₂O-reducing capacity of a community is affected by dynamic oxic/anoxic shifts such as those that may occur during nitrogen removal in wastewater treatment systems. Despite its prolonged growth and enrichment with N₂O as the sole electron acceptor, the culture readily switched to aerobic respiration upon exposure to O₂. When supplied simultaneously, N₂O reduction to N₂ was only detected when the O₂ concentration was limiting the respiration rate. The biomass yields per electron accepted during growth on N₂O are in agreement with our current knowledge of electron transport chain biochemistry in model denitrifiers like Paracoccus denitrificans. The culture’s affinity constant (K_S) for O₂ was found to be two orders of magnitude lower than the value for N₂O, explaining the preferential use of O₂ over N₂O under most environmentally relevant conditions.

     

Visualization of myoglobin-facilitated mitochondrial O(2) delivery in a single isolated cardiomyocyte

The purpose of the present study was to visualize myoglobin-facilitated oxygen delivery to mitochondria at a critical mitochondrial oxygen supply in single isolated cardiomyocytes of rats. Using the autofluorescence of mitochondrial reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), the mitochondrial oxygen supply was imaged from approximately 1.4 microm inside the cell surface at a subcellular spatial resolution. Significant radial gradients of intracellular oxygenation were produced by superfusing the cell suspension with a mixed gas containing 2-4% oxygen while stimulating mitochondrial respiration with an uncoupler of oxidative phosphorylation. Augmentation of the NAD(P)H fluorescence started from the core of the cell (anoxic core) and progressively expanded toward the plasma membrane, as the extracellular Po(2) was lowered. Inactivation of cytosolic myoglobin by 5 mM NaNO(2) significantly enlarged such anoxic regions. Nitrite affected neither mitochondrial respiration in uncoupled cells nor the relationship between Po(2) and the NAD(P)H fluorescence in coupled cells. Thus we conclude that myoglobin significantly facilitates intracellular oxygen transport at a critical level of mitochondrial oxygen supply in single cardiomyocytes.