Measurement of oxygen concentrations in the intact beating heart using electron paramagnetic resonance spectroscopy: A technique for measuring oxygen concentrationsin situ

Electron paramagnetic resonance (EPR) spectroscopy can be applied to measure oxygen concentrations in cells and tissues. Oxygen is paramagnetic, and thus it interacts with a free radical label resulting in a broadening of the observed linewidth. Recently we have developed instrumentation in order to enable the performance of EPR spectroscopy and EPR oximetry in the intact beating heart. This spectrometer consists of 1–2-GHz microwave bridge with the source locked to the resonant frequency of a specially designed lumped circuit resonator. This technique is applied to measure the kinetics of the uptake and clearance of different free radical labels. It is demonstrated that this technique can be used to noninvasively measure tissue oxygen concentration. In addition, rapid scan EPR measurements can be performed enabling gated millisecond measurements of oxygen concentrations to be performed over the cardiac cycle. Thus, low-frequency EPR spectroscopy offers great promise in the study of tissue oxygen concentrations and the role of oxygen in metabolic control.     

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.     

Electron paramagnetic resonance oximetry as a quantitative method to measure cellular respiration: a consideration of oxygen diffusion interference

Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO(2)-independent respiration at higher pO(2) ranges, pO(2)-dependent respiration at low pO(2) ranges, and a static equilibrium with no change in pO(2) at very low pO(2) values. The approach here enables one to comprehensively analyze all of the three zones together-where the progression of O(2) diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO(2) data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry.     

Vitamin C-induced activation of phospholipase D in lung microvascular endothelial cells: regulation by MAP kinases

Our earlier studies have shown that vitamin C at pharmacological doses (mM) induces loss of redox-dependent viability in bovine lung microvascular endothelial cells (BLMVECs) that is mediated by oxidative stress. Therefore, here, we investigated the vitamin C-induced activation of the lipid signaling enzyme, phospholipase D (PLD) in BLMVECs. Monolayer cultures of BLMVECs were treated with vitamin C (0-10 mM) for different time periods (0-2 h) and the activity of PLD was determined. Vitamin C induced activation of PLD in BLMVECs in a time- and dose-dependent fashion that was significantly attenuated by antioxidants, p38 mitogen-activated protein kinase (p38 MAPK)-specific inhibitor (SB203580), extracellular signal-regulated protein kinase (ERK)-specific inhibitor (PD98059), and transient transfection of cells with dominant-negative (DN)-p38 MAPK and DN-ERK1/ERK2. Vitamin C also induced phosphorylation and enhanced the activities of p38 MAPK and ERK in BLMVECs in a time-dependent fashion. It was also evident that vitamin C induced translocation of PLD(1) and PLD(2), association of p38 MAPK and ERK with PLD(1) and PLD(2), threonine phosphorylation of PLD(1) and PLD(2) and SB203580- and PD98059-inhibitable threonine phosphorylation of PLD(1) in BLMVECs. Transient transfection of BLMVECs with DN-p38 MAPK and DN-ERK1/ERK2 resulted in marked attenuation of vitamin C-induced phosphorylation of threonine in PLD(1) and PLD(2). We, for the first time, showed that vitamin C at pharmacological doses, activated PLD in the lung microvascular ECs through oxidative stress and MAPK activation.     

Cross-regulatory mechanisms in hormone signaling

Recent studies suggest that hormones act through a web of interacting responses rather than through isolated linear pathways. This signal integration architecture may be one mechanism for increasing the specificity of outcomes in different cellular contexts. Several common themes for cross-regulation between pathways can be observed. Here, we propose a classification scheme for different levels of signaling pathway cross-regulation. This scheme is based on which parts of the individual pathways are acting as information conduits between pathways. Examples from the recent plant hormone biology literature are used to illustrate the different modes of interaction. K. T. Kuppusamy and C. L. Walcher—co-first-authors.     

Structure-activity studies on the protection of Trimetazidine derivatives modified with nitroxides and their precursors from myocardial ischemia-reperfusion injury

Trimetazidine, the known anti-anginal and anti-ischemic drug, was modified by pyrroline and tetrahydropyridine nitroxides and their hydroxylamine and sterically hindered secondary amine precursors. The synthesized new compounds proved to be better superoxide scavenger molecules compared to the parent Trimetazidine in an in vitro experiment. This reactive oxygen species (ROS) scavenging activity was further supported by ischemia/reperfusion (I/R) studies on Langendorff-perfused rat hearts pretreated with Trimetazidine and with the modified Trimetazidine derivatives before ischemia. Two of the investigated compounds, containing 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole and 4-phenyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole substituents on the piperazine ring, provided significant protection from the cardiac dysfunction caused by I/R. The protective effect could be attributed to the combined anti-ischemic and antioxidant effects.     

Perchlorotrityl radical-fluorophore conjugates as dual fluorescence and EPR probes for superoxide radical anion

Perchlorotrityl radicals, mono-substituted with a fluorophore using an amide linker of varying chain length, were synthesized and characterized. Electron paramagnetic resonance (EPR) spectroscopic study indicated free-electron coupling with the aromatic hydrogen nuclei and long-range coupling with the methylene hydrogens of the linker group. Reactivity of the fluorophore-conjugated trityls with superoxide radical anion showed quenching of EPR signal and enhancement of fluorescence emission spectrum. This work presents the first example of a perchlorotrityl-fluorophore conjugate that can potentially be employed as a dual probe for the detection of superoxide under oxidative stress-mediated conditions in biological systems.     

Novel particulate spin probe for targeted determination of oxygen in cells and tissues

The synthesis and characterization of a new lithium octa-n-butoxy-substituted naphthalocyanine radical probe (LiNc-BuO) and its use in the determination of concentration of oxygen (oximetry) by electron paramagnetic resonance (EPR) spectroscopy are reported. The probe is synthesized as a needle-shaped microcrystalline particulate. The particulate shows a single-line EPR spectrum that is highly exchange-narrowed with a line-width of 210 mG. The EPR line-width is sensitive to molecular oxygen showing a linear relationship between the line-width and concentration of oxygen (pO(2)) with a sensitivity of 8.5 mG/mmHg. We studied a variety of physicochemical and biological properties of LiNc-BuO particulates to evaluate the suitability of the probe for in vivo oximetry. The probe is unaffected by biological oxidoreductants, stable in tissues for several months, and can be successfully internalized in cells. We used this probe to monitor changes in concentration of oxygen in the normal muscle and RIF-1 tumor tissue of mice as a function of tumor growth. The data showed a rapid decrease in the tumor pO(2) with increase of tumor volume. Human arterial smooth muscle cells, upon internalization of the LiNc-BuO probe, showed a marked oxygen gradient across the cell membrane. In summary, the newly synthesized octa-n-butoxy derivative of lithium naphthalocyanine has unique properties that are useful for determining oxygen concentration in chemical and biological systems by EPR spectroscopy and also for magnetic tagging of cells.     

Heat shock regulates the respiration of cardiac H9c2 cells through upregulation of nitric oxide synthase

Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium and cardiomyocytes against ischemic injury. In the present study, we have shown that heat shock regulates the respiration of cultured neonatal cardiomyocytes (cardiac H9c2 cells) through activation of nitric oxide synthase (NOS). The respiration of cultured cardiac H9c2 cells subjected to mild heat shock at 42 degrees C for 1 h was decreased compared with that of control. The O2 concentration at which the rate of O2 consumption is reduced to 50% was increased in heat-shocked cells, indicating a lowering of O2 affinity in the mitochondria. Western blot analyses showed a fourfold increase in the expression of heat shock protein (HSP) 90 and a twofold increase in endothelial NOS (eNOS) expression in the heat-shocked cells. Immunoblots of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) in the immunoprecipitate of HSP90 of heat-shocked cells showed that there was a sevenfold increase in eNOS and no changes in iNOS and nNOS. Confocal microscopic analysis of cells stained with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate showed higher levels of NO production in the heat-shocked cells than in control cells. The results indicate that heat shock-induced HSP90 forms a complex with eNOS and activates it to increase NO concentration in the cardiac H9c2 cells. The generated NO competitively binds to the complexes of the respiratory chain of the mitochondria to downregulate O2 consumption in heat-shocked cells. On the basis of these results, we conclude that myocardial protection by hyperthermia occurs at least partly by the pathway of HSP90-mediated NO production, leading to subsequent attenuation of cellular respiration.     

Copper redox-dependent activation of 2-tert-butyl(1,4)hydroquinone: formation of reactive oxygen species and induction of oxidative DNA damage in isolated DNA and cultured rat hepatocytes

The biotransformation of butylated hydroxyanisole (BHA), a possible carcinogenic food antioxidant, includes o-demethylation to 2-tert-butyl(1,4)hydroquinone (TBHQ) which can subsequently be oxidized to 2-tert-butyl(1,4)paraquinone (TBQ). In this study, we have examined the capacity of Cu, a nuclei- and DNA-associated transition metal, to mediate the oxidation of TBHQ. In phosphate buffered saline (PBS), autooxidation of TBHQ to TBQ was not detectable, while Cu(II) at micromolar concentrations strongly catalyzed the oxidation of TBHQ to TBQ. Oxidation of TBHQ by Cu(II) was accompanied by the utilization of O(2) and the concomitant generation of H(2)O(2). Using electron spin resonance spectroscopy, it was observed that Cu(II) mediated the one electron oxidation of TBHQ to a semiquinone anion radical. The formation of a semiquinone anion radical, the utilization of O(2) and the generation of H(2)O(2) and TBQ could be completely blocked by bathocuproinedisulfonic acid (BCS) and reduced glutathione (GSH), two Cu(I)-chelators. 4-Pyridyl-1-oxide-N-tert-butylnitrone (POBN)-spin trapping experiments showed that the reaction of TBHQ with Cu(II) resulted in the generation of POBN-CH(3) and POBN-CH(OH)CH(3) adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, suggesting the formation of hydroxyl radical or a similar reactive intermediate. The formation of POBN-CH(3) adduct from the TBHQ/Cu(II)+DMSO could be completely inhibited by catalase, GSH or BCS, indicating that the hydroxyl radical or its equivalent is generated from the interaction of H(2)O(2) with Cu(I). Incubation of supercoiled phiX-174 plasmid DNA with the TBHQ/Cu(II) resulted in extensive DNA strand breaks, which could be prevented by catalase or BCS. Incubation of rat hepatocytes with TBHQ in PBS led to increased formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in nuclear DNA. The TBHQ-induced formation of 8-OHdG was markedly reduced in the presence of cell permeable Cu(I)-specific chelator, bathocuproine or neocuproine, suggesting that a Cu(II)/Cu(I) redox mechanism may also be involved in the induction of oxidative DNA damage by TBHQ in hepatocytes. Taken together, the above results conclusively demonstrate that the activation of TBHQ by Cu(II) results in the formation of TBQ, semiquinone anion radical and reactive oxygen species (ROS), and that the ROS formed may participate in oxidative DNA damage in both isolated DNA and intact cells. These reactions may contribute to the carcinogenicity as well as other biochemical activities observed with BHA in animals. To our knowledge this study provides the first evidence that endogenous cellular Cu may be capable of bioactivating TBHQ, leading to oxidative DNA damage in cultured cells.