Extracellular PO2 Research Articles

Intracellular Oxygen Transient Quantification in Vivo During Ultra-High Dose Rate FLASH Radiation Therapy

Large, rapid extracellular oxygen transients (ΔpO2) have been measured in vivo during ultra-high dose rate radiation therapy; however, it has been unclear if they match intracellular oxygen levels. Here, the endogenously produced protoporphyrin IX (PpIX) delayed fluorescence signal was measured as an intracellular in-vivo oxygen sensor to quantify these transients, with direct comparison to extracellular pO2. Intracellular ΔpO2 is closer to the cellular DNA, the site of major radiobiological damage, and therefore should help elucidate radiochemical mechanisms of the FLASH effect and potentially be translated to human tissue measurement. PpIX was induced in mouse skin through intraperitoneal injection of 250 mg/kg of aminolevulinic acid. The animals were also administered a 50 µL intradermal injection of 10 µM oxyphor G4 (PdG4) for phosphorescence lifetime pO2 measurement. Paired oxygen transients were quantified in leg or flank tissues while delivering 10 MeV electrons in 3 µs pulses at 360 Hz for a total dose of 10 to 28 Gy. Transient reductions in pO2 were quantifiable in both PpIX delayed fluorescence and oxyphor phosphorescence, corresponding to intracellular and extracellular pO2 values, respectively. Reponses were quantified for 10, 22, and 28 Gy doses, with ΔpO2 found to be proportional to the dose on average. The ΔpO2 values were dependent on initial pO2 in a logistic function. The average and standard deviations in ΔpO2 per dose were 0.56 ± 0.18 mm Hg/Gy and 0.43 ± 0.06 mm Hg/Gy for PpIX and oxyphor, respectively, for initial pO2 > 20 mm Hg. Although there was large variability in the individual animal measurements of ΔpO2, the average values demonstrated a direct and proportional correlation between intracellular and extracellular pO2 changes, following a linear 1:1 relationship. A fundamentally new approach to measuring intracellular oxygen depletion in living tissue showed that ΔpO2 transients seen during ultra-high dose rate radiation therapy matched those quantified using extracellular oxygen measurement. This approach could be translated to humans to quantify intracellular ΔpO2. The measurement of these transients could potentially allow the estimation of intracellular reactive oxygen species production.

Sensitive simultaneous measurements of oxygenation and extracellular pH by EPR using a stable monophosphonated trityl radical and lithium phthalocyanine

The monitoring of acidosis and hypoxia is crucial because both factors promote cancer progression and impact the efficacy of anti-cancer treatments. A phosphonated tetrathiatriarylmethyl (pTAM) has been previously described to monitor both parameters simultaneously, but the sensitivity to tackle subtle changes in oxygenation was limited. Here, we describe an innovative approach combining the pTAM radical and lithium phthalocyanine (LiPc) crystals to provide sensitive simultaneous measurements of extracellular pH (pHe) and pO2. Both parameters can be measured simultaneously as both EPR spectra do not overlap, with a gain in sensitivity to pO2 variations by a factor of 10. This procedure was applied to characterize the impact of carbogen breathing in a breast cancer 4T1 model as a proof-of-concept. No significant change in pHe and pO2 was observed using pTAM alone, while LiPc detected a significant increase in tumor oxygenation. Interestingly, we observed that pTAM systematically overestimated the pO2 compared to LiPc. In addition, we analyzed the impact of an inhibitor (UK-5099) of the mitochondrial pyruvate carrier (MPC) on the tumor microenvironment. In vitro, the exposure of 4T1 cells to UK-5099 for 24 h induced a decrease in pHe and oxygen consumption rate (OCR). In vivo, a significant decrease in tumor pHe was observed in UK-5099-treated mice, while there was no change for mice treated with the vehicle. Despite the change observed in OCR, no significant change in tumor oxygenation was observed after the UK-5099 treatment. This approach is promising for assessing in vivo the effect of treatments targeting tumor metabolism.

Inhibition of Oxidative Phosphorylation Reverses Bone Marrow Hypoxia Visualized in Imageable Syngeneic B-ALL Mouse Model.

Abnormally low level of interstitial oxygen, or hypoxia, is a hallmark of tumor microenvironment and a known promoter of cancer chemoresistance. Inside a solid tumor mass, the hypoxia stems largely from inadequate supply of oxygenated blood through sparse or misshapen tumor vasculature whilst oxygen utilization rates are low in typical tumor's glycolytic metabolism. In acute leukemias, however, markers of intracellular hypoxia such as increased pimonidazole adduct staining and HIF-1α stabilization are observed in advanced leukemic bone marrows (BM) despite an increase in BM vasculogenesis. We utilized intravital fast scanning two-photon phosphorescence lifetime imaging microscopy (FaST-PLIM) in a BCR-ABL B-ALL mouse model to image the extracellular oxygen concentrations (pO2) in leukemic BM, and we related the extracellular oxygen levels to intracellular hypoxia, vascular markers and local leukemia burden. We observed a transient increase in BM pO2 in initial disease stages with intermediate leukemia BM burden, which correlated with an expansion of blood-carrying vascular network in the BM. Yet, we also observed increased formation of intracellular pimonidazole adducts in leukemic BM at the same time. This intermediate stage was followed by a significant decrease of extracellular pO2 and further increase of intracellular hypoxia as leukemia cellularity overwhelmed BM in disease end-stage. Remarkably, treatment of leukemic mice with IACS-010759, a pharmacological inhibitor of mitochondrial Complex I, substantially increased pO2 in the BM with advanced B-ALL, and it alleviated intracellular hypoxia reported by pimonidazole staining. High rates of oxygen consumption by B-ALL cells were confirmed by Seahorse assay including in ex vivo cells. Our results suggest that B-ALL expansion in BM is associated with intense oxidative phosphorylation (OxPhos) leading to the onset of metabolic BM hypoxia despite increased BM vascularization. Targeting mitochondrial respiration may be a novel approach to counteract BM hypoxia in B-ALL and, possibly, tumor hypoxia in other OxPhos-reliant malignancies.

Is the teleost heart oxygen limited? – Insights using “hyperoxic” incubations of contracting cardiac tissue from rainbow trout

Considerable effort has been devoted to understanding the negative effects of reduced PO2 on cardiac function. Much less is known about the impacts of elevated PO2 (hyperoxia) on cardiac performance and energetics, especially in fishes. The fish heart is of particular interest because cardiac dependence on oxygen is extremely variable between species and the early evolution of fish occurred when atmospheric PO2 was higher than current conditions. Although extracellular PO2 is variable and normally does not exceed 21 kPa, recent evidence suggests that teleost cardiac function is stimulated at supraphysiological PO2 values. The purpose of this study was to address whether cardiac contractility and energy metabolism is responsive to elevated PO2 values in sexually-immature female rainbow trout. Isometric force development (contractility) and oxygen consumption (V̇O2) were recorded in electrically-paced ventricular preparations. Contractility and V̇O2decreased when superfusate PO2 was decreased from ~70 kPa to 57 kPa or 45 kPa. However, PO2 calculated at the preparation core was always above 18 kPa. This estimate, along with complete recovery of contractility and V̇O2 at ~70 kPa, suggests that decreases observed in cardiac performance were not due to tissue hypoxia at the lower PO2 levels. In conclusion, the heart of female rainbow trout may be oxygen-limited in vitro and this study raises new questions about the choice of appropriate PO2 for experimentation, the relevance of elevated and varying PO2 to measurements of cardiac performance, and the possible existence of an oxygen sensor within rainbow trout cardiomyocytes.

A mitochondrial-targeted antioxidant improves myofilament Ca2+ sensitivity during prolonged low frequency force depression at low PO2.

Skeletal muscle contractile activity is associated with an enhanced reactive oxygen species (ROS) generation. At very low PO2, ROS generation by mitochondria can be elevated in intact cells. An elevated intracellular oxidant activity may affect muscle force development and recovery from fatigue. We treated intact single muscle fibres with a mitochondrial antioxidant and stimulated the fibres to contract at a low extracellular PO2 that is similar to the intracellular PO2 that is observed during moderate to intense exercise in vivo. The mitochondrial antioxidant prevented a sustained decrease in the myofibrillar Ca2+ sensitivity and improved muscle submaximal force development after fatigue at low extracellular PO2. Skeletal muscle can develop a prolonged low frequency-stimulation force depression (PLFFD) following fatigue-inducing contractions. Increased levels of reactive oxygen species (ROS) have been implicated in the development of PLFFD. During exercise the skeletal muscle intracellular PO2 decreases to relatively low levels, and can be further decreased when there is an impairment in O2 diffusion or availability, such as in certain chronic diseases and during exercise at high altitude. Since ROS generation by mitochondria is elevated at very low PO2 in cells, we tested the hypothesis that treatment of muscle fibres with a mitochondrial-targeted antioxidant at a very low, near hypoxic, PO2 can attenuate PLFFD. We treated intact single fibres from mice with the mitochondrial-specific antioxidant SS31, and measured force development and intracellular [Ca2+ ] 30min after fatigue at an extracellular PO2 of ∼5Torr. After 30min following the end of the fatiguing contractions, fibres treated with SS31 showed significantly less impairment in force development compared to untreated fibres at submaximal frequencies of stimulation. The cytosolic peak [Ca2+ ] transients (peak [Ca2+ ]c ) were equally decreased in both groups compared to pre-fatigue values. The combined force and peak [Ca2+ ]c data demonstrated that myofibrillar Ca2+ sensitivity was diminished in the untreated fibres 30min after fatigue compared to pre-fatigue values, but Ca2+ sensitivity was unaltered in the SS31 treated fibres. These results demonstrate that at a very low PO2, treatment of skeletal muscle fibres with a mitochondrial antioxidant prevents a decrease in the myofibrillar Ca2+ sensitivity, which alleviates the fatigue induced PLFFD.

MRI of brain tissue oxygen tension under hyperbaric conditions

The brain depends on a continuous supply of oxygen to maintain its structural and functional integrity. This study measured T1 from MRI under normobaric air, normobaric oxygen, hyperbaric air, and hyperbaric oxygen (HBO) conditions as a marker of tissue pO2 since dissolved molecular oxygen acts as an endogenous contrast agent. Brain tissue T1 decreased corresponding to increased pO2 with increasing inhaled oxygen concentrations, and tissue oxygenation was estimated from the T1 changes between different inhaled oxygen levels. Tissue pO2 difference maps between different oxygen conditions showed heterogeneous pO2 changes in the brain. MRI-derived tissue pO2 was markedly lower than the arterial pO2 but was slightly higher than venous pO2. Additionally, for comparison with published extracellular tissue pO2 data obtained using oxygen electrodes and other invasive techniques, a model was used to estimate extracellular and intracellular pO2 from the MRI-derived mean tissue pO2. This required multiple assumptions, and so the effects of the assumptions and parameters used in modeling brain pO2 were evaluated. MRI-derived pO2 values were strongly dependent on assumptions about the extra- and intracellular compartments but were relatively less sensitive to variations in the relaxivity constant of oxygen and contribution from oxygen in the cerebral blood compartment. This approach may prove useful in evaluating tissue oxygenation in disease states such as stroke.

Oxygen diffusion: an enzyme-controlled variable parameter.

Previous oxygen microelectrode studies have shown that the oxygen diffusion coefficient (DO₂) increases during extracellular PO₂ decreases, while intracellular PO₂ remained unchanged and thus cell function (spike activity of neurons). Oxygen dependency of complex multicellular organisms requires a stable and adequate oxygen supply to the cells, while toxic concentrations have to be avoided. Oxygen brought to the tissue by convection diffuses through the intercellular and cell membranes, which are potential barriers to diffusion. In gerbil brain cortex, PO₂ and DO₂ were measured by membrane-covered and by bare gold microelectrodes, as were also spike potentials. Moderate respiratory hypoxia was followed by a primary sharp drop of tissue PO₂ that recovered to higher values concomitant with an increase of DO₂. A drop in intracellular PO₂ recovered immediately. Studies on the abdominal ganglion of aplysia californica showed similar results.Heterogeneity is a feature of both normal oxygen supply to tissue and supply due to a wide range of disturbances in oxygen supply. Oxygen diffusion through membranes is variable thereby ensuring adequate intracellular PO₂. Cell-derived glucosamine oxidase seems to regulate the polymerization/depolymerisation ratio of membrane mucopolysaccharides and thus oxygen diffusion.Variability of oxygen diffusion is a decisive parameter for regulating the supply/demand ratio of oxygen supply to the cell; this occurs in highly developed animals as well as in species of a less sophisticated nature. Autoregulation of oxygen diffusion is as important as the distribution/perfusion ratio of the capillary meshwork and as the oxygen extraction ratio in relation to oxygen consumption of the cell. Oxygen diffusion resistance is the cellular protection against luxury oxygen supply (which can result in toxic oxidative species leading to mutagenesis).

Phosphonated Trityl Probes for Concurrent in Vivo Tissue Oxygen and pH Monitoring Using Electron Paramagnetic Resonance-Based Techniques

Previously we proposed the concept of dual function pH and oxygen paramagnetic probes based on the incorporation of ionizable groups into the structure of persistent triarylmethyl radicals, TAMs (J. Am. Chem. Soc.2007, 129, 7240-7241). In this paper, we synthesized an asymmetric monophosphonated TAM probe with the simplest doublet hfs pattern ideally suited for dual function electron paramagnetic resonance (EPR)-based applications. An extraordinary low line width of the synthesized deuterated derivative, p1TAM-D (ΔHpp ≤ 50 mG, Lorentz line width, ≤20 mG) results in high sensitivity to pO2 due to oxygen-induced line broadening (ΔLW/ΔpO2 ≈ 0.5 mG/mmHg or ≈400 mG/mM); accuracy of pO2 measurement, ≈1 mmHg). The presence of a phosphono group in the p1TAM-D structure provides pH sensitivity to its EPR spectra in the physiological range of pH from 5.9 to 8.2 with the ratio of signal intensities of protonated and deprotonated states being a reliable pH marker (accuracy of pH measurements, ± 0.05). The independent character of pH and [O2] effects on the EPR spectra of p1TAM-D provides dual functionality to this probe. The L-band EPR studies performed in breast tumor-bearing mice show a significant difference in extracellular pH and pO2 between tumor and normal mammary gland tissues, as well as the effect of animal breathing with 100% O2 on tissue oxygenation. The developed dual function phosphonated p1TAM-D probe provides a unique tool for in vivo concurrent tissue oxygen and pH monitoring.

Intracellular Hypoxia of Tumor Tissue Estimated by Noninvasive Electron Paramagnetic Resonance Oximetry Technique Using Paramagnetic Probes

Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO(2)) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO(2) in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO(2) were compared. The pO(2) in the blood can depend on the oxygen content in the breathing gas while tissue pO(2) was not directly influenced by pO(2) in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO(2) is similar to normal tissue in the peripheral region.

Environmentally Sensitive Paramagnetic and Diamagnetic Contrast Agents for Nuclear Magnetic Resonance Imaging and Spectroscopy

Even though alterations in the microenvironmental properties of tissues underlie the development of the most prevalent and morbid pathologies, they are not directly observable in vivo by Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS) methods. This circumstance has lead to the development of a wide variety of exogenous paramagnetic and diamagnetic MRI and MRS probes able to inform non invasively on microenvironmental variables such as pH, pO(2), ion concentration o even temperature. This review covers the fundamentals of environmental contrast and the current arsenal of endogenous and exogenous MRI and MRS contrast enhancing agents available to visualize it. We begin describing the physicochemical background necessary to understand paramagnetic and diamagnetic contrast enhancement with a special reference to novel magnetization transfer and (13)C hyperpolarization strategies. We describe then the main macrocyclic structures used to support the environmentally sensitive paramagnetic sensors, including CEST and PARACEST pH sensitive probes, temperature probes and enzyme activity or gene expression activatable probes. Finally we address the most commonly used diamagnetic contrast agents including imidazolic derivatives to reveal extracellular pH and tissue pO(2) values by MRS. The potential applications of these agents in multimodal and molecular imaging approaches are discussed.

Anoxic cell core can promote necrotic cell death in cardiomyocytes at physiological extracellular Po2

The physical law of diffusion imposes O2 concentration gradients from the plasma membrane to the center of the cell. The present study was undertaken to determine how such intracellular radial gradients of O2 affect the fate of isolated single cardiomyocytes. In single rat cardiomyocytes, mitochondrial respiration was moderately elevated by an oxidative phosphorylation uncoupler to augment the intracellular O2 gradient. At physiological extracellular O2 levels (2-5%), decreases in myoglobin O2 saturation and increases in NADH fluorescence at the center of the cell were imaged (anoxic cell core) while the mitochondrial membrane potential (DeltaPsim) and ATP levels at the anoxic cell core were relatively sustained. In contrast, treatment with 0.5 mM iodoacetamide (IA) to inhibit creatine kinase (CK) resulted in depletion of both DeltaPsim and ATP at the anoxic cell core. Even at normal extracellular Po2, actively respiring cardiomyocytes developed rigor contracture followed by necrotic cell death. Furthermore, such rigor was remarkably accelerated by IA, whereas cell injury was perfectly rescued by mitochondrial F1Fo inhibition by oligomycin. These results suggest that increases in radial gradients of O2 potentially promote cell death through the reverse action of F1Fo in mitochondria located at the anoxic cell core. However, in the intact cardiomyocyte, the CK-mediated energy flux from the subsarcolemmal space may sustain DeltaPsim at the cell core, thus avoiding uncontrolled consumption of ATP that can lead to necrotic cell death. Mitochondria at the anoxic core can cause necrotic cell death in cardiomyocytes at physiological extracellular Po2.

Low Extracellular Po2 Decreases Ca2+ Sensitivity During Fatigue In Single Xenopus Skeletal Muscle Fibers

PURPOSE We examined whether reduced O2 availability in working skeletal muscle, compared to conditions when O availability is greater, involves an alteration in myofilament Ca2+ sensitivity during fatigue. METHODS Relative force (P) and cytosolic Ca2+ concentration ([Ca2+]c) were measured simultaneously in isolated single Xenopusskeletal muscle fibers (n=8) stimulated at sequentially increasing frequencies (0.25, 0.33, 0.5 and 1 contraction/sec), at an extracellular PO2 of either 21 or 159 mm Hg. Stimulation was terminated when P<50% of initial peak tension values (Po). RESULTS No significant difference (p<0.05) in P was observed between treatments during the first 2 min of contractions. At 2 min, however, a significant difference (p<0.05) in P was observed between the low PO2 treatment (P/Po=0.81±0.03) and the high (P/PO=0.89±0.02), which continued until the end of stimulation. The fatigue end-point (P/P =0.5) occurred significantly sooner during the low PO treatment (4.6±0.4 min) than the high (5.3±0.4 min). Myofilament Ca2+ sensitivity was determined by examining the ratio of relative peak [Ca2+]c to relative force (i.e, [Ca2+]c /P). No difference in [Ca2+]c /P was observed between the two treatments during the initial 2 min. However, beyond this two-minute time point, a significant difference in [Ca2+] /P was observed between the low PO2 condition (1.16±0.03) and the high (1.09±0.03), showing a diminished force development for a given [Ca2+]c in the low extracellular PO2condition. CONCLUSIONS These results suggest that in contracting, single Xenopusskeletal muscle fibers, the development of fatigue during conditions of limited oxygen availability is associated with a decrease in myofilament Ca2+ sensitivity when compared to conditions of greater oxygen availability. Supported by NIH Grant AR40155

Low Extracellular Po2 Decreases Ca2+ Sensitivity During Fatigue In Single Xenopus Skeletal Muscle Fibers

PURPOSE We examined whether reduced O2 availability in working skeletal muscle, compared to conditions when O availability is greater, involves an alteration in myofilament Ca2+ sensitivity during fatigue. METHODS Relative force (P) and cytosolic Ca2+ concentration ([Ca2+]c) were measured simultaneously in isolated single Xenopusskeletal muscle fibers (n=8) stimulated at sequentially increasing frequencies (0.25, 0.33, 0.5 and 1 contraction/sec), at an extracellular PO2 of either 21 or 159 mm Hg. Stimulation was terminated when P<50% of initial peak tension values (Po). RESULTS No significant difference (p<0.05) in P was observed between treatments during the first 2 min of contractions. At 2 min, however, a significant difference (p<0.05) in P was observed between the low PO2 treatment (P/Po=0.81±0.03) and the high (P/PO=0.89±0.02), which continued until the end of stimulation. The fatigue end-point (P/P =0.5) occurred significantly sooner during the low PO treatment (4.6±0.4 min) than the high (5.3±0.4 min). Myofilament Ca2+ sensitivity was determined by examining the ratio of relative peak [Ca2+]c to relative force (i.e, [Ca2+]c /P). No difference in [Ca2+]c /P was observed between the two treatments during the initial 2 min. However, beyond this two-minute time point, a significant difference in [Ca2+] /P was observed between the low PO2 condition (1.16±0.03) and the high (1.09±0.03), showing a diminished force development for a given [Ca2+]c in the low extracellular PO2condition. CONCLUSIONS These results suggest that in contracting, single Xenopusskeletal muscle fibers, the development of fatigue during conditions of limited oxygen availability is associated with a decrease in myofilament Ca2+ sensitivity when compared to conditions of greater oxygen availability. Supported by NIH Grant AR40155

Effect of extracellular PO2 on the fall in intracellular PO2 in contracting single myocytes.

The purpose of this investigation was to study the effects of altered extracellular Po(2) (Pe(O(2))) on the intracellular Po(2) (Pi(O(2))) response to contractions in single skeletal muscle cells. Single myocytes (n = 12) were dissected from lumbrical muscles of adult female Xenopus laevis and injected with 0.5 mM Pd-meso-tetra(4-carboxyphenyl)porphine for assessment of Pi(O(2)) via phosphorescence quenching. At a Pe(O(2)) of approximately 20 (low), approximately 40 (moderate), and approximately 60 (high) Torr, tetanic contractions were induced at a frequency of 0.67 Hz for approximately 2 min with a 5-min recovery between bouts (blocked order design). The Pi(O(2)) response to contractions was characterized by a time delay followed by a monoexponential decline to steady-state (SS) values. The fall in Pi(O(2)) to SS values was significantly greater at each progressively greater Pe(O(2)) (all P < 0.05). The mean response time (time delay + time constant) was significantly faster in the low (35.2 +/- 5.1 s; P < 0.05 vs. high) and moderate (43.3 +/- 6.4 s; P < 0.05 vs. high) compared with high Pe(O(2)) (61.8 +/- 9.4 s) and was correlated positively (r = 0.965) with the net fall in Pi(O(2)). However, the initial rate of change of Pi(O(2)) (calculated as net fall in Pi(O(2))/time constant) was not different (P > 0.05) among Pe(O(2)) trials. These latter data suggest that, over the range of 20-60 Torr, Pe(O(2)) does not play a deterministic role in setting the initial metabolic response to contractions in isolated frog myocytes. Additionally, these results suggest that oxidative phosphorylation in these myoglobin-free myocytes may be compromised by Pe(O(2)) at values nearing 60 Torr.

Assessment of O2 uptake dynamics in isolated single skeletal myocytes.

The purpose of this research was to develop a technique for rapid measurement of O(2) uptake (Vo(2)) kinetics in single isolated skeletal muscle cells. Previous attempts to measure single cell Vo(2) have utilized polarographic-style electrodes, thereby mandating large fluid volumes and relatively poor sensitivity. Thus our laboratory has developed an approximately 100-microl, well-stirred chamber for the measurement of Vo(2) in isolated Xenopus laevis myocytes using a phosphorescence quenching technique [Ringer solution with 0.05 mM Pd-meso-tetra(4-carboxyphenyl)porphine] to monitor the fall in extracellular Po(2) (which is proportional to cellular Vo(2) within the sealed chamber). Vo(2) in single living myocytes dissected from Xenopus lumbrical muscles was measured from rest across a bout of repetitive tetanic contractions (0.33 Hz) and in response to a ramp protocol utilizing an increasing contraction frequency. In response to the square-wave contraction bout, the increase in Vo(2) to steady state (SS) was 16.7 +/- 1.3 ml x 100 g(-1) x min(-1) (range 13.0-21.9 ml x 100 g(-1) x min(-1); n = 6). The rise in Vo(2) at contractions onset (n = 6) was fit with a time delay (2.1 +/- 1.2 s, range 0.0-7.7 s) plus monoexponential rise to SS (time constant = 9.4 +/- 1.5 s, range 5.2-14.9 s). Furthermore, in two additional myocytes, Vo(2) increased progressively as contraction frequency increased (ramp protocol). This technique for measuring Vo(2) in isolated, single skeletal myocytes represents a novel and powerful investigative tool for gaining mechanistic insight into mitochondrial function and Vo(2) dynamics without potential complications of the circulation and other myocytes.

ASSESSMENT OF OXYGEN UPTAKE IN SINGLE FROG MYOCYTES

PURPOSE: At the transition to an elevated metabolic rate, ATP demand increases in square-wave fashion whereas readjustment of oxidative phosphorylation is delayed. Whether this metabolic inertia of mitochondrial ATP production resides within the exercising muscle or is dependent upon O2 delivery remains controversial. An isolated single fiber preparation would be effective in studying myocyte O2 exchange in the absence of poential limitation imposed by the microcirculatory control and muscle fiber type heterogeneity. However, previous attempts to measure single cell oxygen uptake (O2) have utilized the Clark electrode, thereby mandating large fluid Olumes and relatively poor sensitivity. METHODS: Our laboratory has developed an ∼100 μl Lucite chamber with dual infusion/withdrawal ports and transducer for assessment of force production for the measurement of O2 in isolated Xenopus laevis lumbrical myocytes using a phosphorescence quenching technique (Ringers solution with 1 mM palladium-meso-tetra (4-carboxyphenyl) porphine) to monitor the fall in extracellular PO2 (which is proportional to cellular O2). Single myocytes dissected from frog lumbrical muscles (n = 4) were studied at the transition from rest to repetitive tetanic contractions (1 contraction every 2 sec for 4 min). RESULTS: O2 during the final minute was 39.0 ± 2.1 ml/min/100 g. Time to 50% of the overall O2 response was 24 ± 5 s. CONCLUSION: This technique for measuring O2 represents a novel and powerful tool for gaining mechanistic insight into mitochondrial function and O2 dynamics in isolated skeletal myocytes. Supported, in part, by NIH (AR 40155).

Oxygenation and perfusion of rabbit tibialis anterior muscle subjected to different patterns of electrical stimulation.

Dual amperometric microelectrodes were used to measure local pO2 and perfusion at multiple sites in the fast-twitch tibialis anterior muscles of anaesthetized rabbits. Six muscles were stimulated continuously at 10, 5, or 2.5 Hz. For all three frequencies, perfusion declined to about 50% of resting levels and recovered after stimulation. These changes corresponded to a rise followed by a fall in extracellular pO2. The highest levels of pO2 were reached during stimulation at 10 Hz. Eight muscles were stimulated tetanically at 100 Hz for 200 ms with duty cycles that were varied between 1.3 and 20.0%. Perfusion rose to 8.7 +/- 2.0 ml s(-1) 100 g(-1) at a duty cycle of 5% and declined with further increases in duty cycle. pO2 was depressed for duty cycles less than 10% but rose above resting levels at higher duty cycles. It is suggested that the paradoxical combination of elevated pO2 and depressed perfusion is attributable to stimulation conditions that exceed the oxygen transport capacity of a fast muscle.

Are there significant gradients of pO2 in cells?

It is widely recognized that the intracellular oxygen tension (pO2) plays an important role in cellular function and metabolism. In experimental and theoretical consideration involving the role of the pO2 the values that are used usually are those for the pO2 at the exterior of cells, because these values can be more readily measured. Such an approach is based on the assumption that the intracellular pO2 is very similar to the extracellular pO2 because oxygen freely diffuses across cell membranes and within cells, at a rate that is similar to that in the extracellular media. For the past several years we have been developing and applying electron paramagnetic resonance (EPR) techniques to test directly whether there are intracellular gradients of pO2 and if so, where they occur and what factors determine them. We previously reported significant gradients between the average pO2 in the intracellular and extracellular compartments in cell suspensions (Glockner 1989). More recently we developed techniques that enabled us to measure simultaneously the concentration of oxygen within a specific compartment, the phagosomes of activated macrophages, and the extracellular compartment and found gradients of up to 48 microM under some conditions (James 1995). The precise mechanism of the intracellular-extracellular oxygen gradient remains uncertain. The possibilities include that the diffusion of oxygen is not as free as assumed (e.g. that the cell membrane can act as a barrier) and the occurrence of active transport of O2 out of the cells. We report here on the use of a simple theoretical approach to evaluate the values of three key parameters which might account for the observed intracellular-extracellular oxygen measurements: (1) oxygen consumption; (2) the diffusion coefficient of oxygen in the cytosol; (3) the solubility of oxygen in the cytosol. We used two different models for the relationship between the oxygen consuming compartment (assumed to be primarily the mitochondria) and the intracellular compartment in which the measurements were made (especially phagosomes): uniform and non-uniform distribution of the mitochondria. Using these models and consensus values from the literature, we were unable to account for the experimentally observed differences in pO2 between the intracellular and extracellular compartments. Also we found that with the variation of any one parameter we could not plausibly account for the measurements made in the phagosomal and extracellular compartments. There are at least three logical possibilities to account for these results: 1) this methodology is erroneous and/or produces artifacts in the system resulting in invalid results; 2) the observation of a gradient in oxygen concentration between these two compartments arises from significant simultaneous variations of more than one of the critical parameters which are used conventionally to calculate potential gradients in pO2; 3) there is another factor not considered in the model which accounts for the observation (e.g. active transport; significantly higher than expected barriers to oxygen diffusion in the membrane).

DIFFERENTIAL EXPRESSION OF SPECIFIC PROTEINS ASSOCIATED WITH APOPTOSIS (Bax) OR CELL SURVIVAL (Bcl-2, HSP70, HSP105) AFTER SHORT- AND LONG-TIME HYPOXIA IN CULTURED CENTRAL NEURONS • 233

We showed previously that a 6h-hypoxia followed by reoxygenation for 96h induces a delayed, mainly apoptotic cell death in cultured neurons from the fetal rat forebrain, whereas shorter hypoxia (3h) increases cell viability. To elucidate cellular events involved, the expression of specific proteins, either effector of apoptosis (Bax) or potentially related to cell recovery(Bcl-2, HSP70, HSP105), was analysed. After 6 days in vitro, neurons were exposed for 3 or 6h to hypoxia (95% N2-5% CO2) that decreased extracellular PO2 by 75-80%. Cells were then reoxygenated (95% air-5% CO2) for 96h. As a function of time, neuronal viability was assessed by the MTT procedure, while apoptosis was studied by the analysis of DNA integrity by gel electrophoresis and nuclear incorporation of the fluorescent dye DAPI. Protein synthesis was measured by incorporation of [3H]leucine, then neuronal proteins were separated by SDS-PAGE. Expression of specific proteins was investigated by immunohistochemistry and Western blot. All data reported are compared to controls kept in normoxia and are statistically significant(p<0.01). A 6h-hypoxia led to marked inductions of protein synthesis rates 1h after the onset of the insult (+77%, n=26 dishes) and 48h after reoxygenation (+72%, n=26). By 72h, synthesis rates decreased (-41%, n=24), concomitantly with reduced cell viability (-36%, n=30) and detection of 23% of apoptotic neurons (n=10). SDS-PAGE analysis showed specific alterations in the density of constitutive proteins during hypoxia and reoxygenation. Bax was overexpressed 1h after the onset of hypoxia, and then by 72 and 96h after reoxygenation. Bcl-2, HSP70 and HSP105 were also overexpressed at 1h, but decreased thereafter, with the development of apoptosis. Conversely, a 3h-hypoxia also led to increased protein synthesis after 1h, but not at 48h, and then to a late increase (+60%, n=10) by 72h post-reoxygenation, along with enhanced viability (+13%, n=10). During reoxygenation, Bax expression was not affected, while Bcl-2 and HSPs were overexpressed. Therefore, transient hypoxia induces early expression of specific proteins such as Bax, Bcl-2 and HSPs. Thereafter, depending on the duration of the insult, either apoptosis-related (Bax) or survival-related (Bcl-2, HSPs) proteins are overexpressed and may influence the final cell outcome.

Measurement of oxygen tensions in the abdominal cavity and in the skeletal muscle using 19F-MRI of neat PFC droplets.

Perfluorochemicals (PFCs) are not only of interest as artificial oxygen carriers but also as 19F-NMR (19F nuclear magnetic resonance) contrast agents for the determination of partial oxygen pressuresin vivo. Since the longitudinal relaxation rate R1 increases linearly with the partial oxygen pressure (pO2), pO2 values can be determined in regions of PFC accumulation. After the i.v. injection of a PFC emulsion, pO2 measurements are possible in large vessels until most of the PFC particles are removed from the circulation by macrophages. Then intracellular pO2 measurements can be performed in regions with high macrophage concentration, i.e. in the liver and spleen, and in pathological tissues with high macrophage activity, e.g. in tumors and in regions of inflammation. In many studies with PFC emulsions, negative apparent pO2 values were reported under hypoxic conditions (F1O2 < 0.21) [1-7]. Purpose of this work was to measure extracellular pO2 values in a depot of neat PFC in the abdominal cavity and in the skeletal muscle under hyperoxic, normoxic and hypoxic conditions, and further to determine whether neat PFC also yields negative apparent pO2 values under hypoxic conditions.KeywordsHypoxic ConditionAbdominal CavityPartial Oxygen PressureCalibration ConstantLongitudinal Relaxation RateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.