Mitochondrial Reduced Nicotinamide Adenine Dinucleotide Research Articles

Two‐photon microscopy assessment of the overall energy metabolism alteration of amoeba in hypertonic environment

In this study, a two-photon fluorescence microscopic imaging technique is reported for assessment the effect of dynamic hypertonic environment on the overall energy metabolism alteration and adaptation of soil-living amoeba Dictyostelium discoideum. For that purpose the fluorescence intensity of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) was monitored and quantified in order to evaluate the corresponded metabolic state of monolayer cultured cells. The two-photon excitation of NADH with 720 nm near infrared irradiation produced blue fluorescence emission with maximum wavelength centered at 460 nm. The benefits of reported noninvasive microscopic technique are the significantly less cellular damage and avoiding the excitation of other biomolecules except of NADH. It enabled to acquire data for NADH levels of the observed cells on agar plate specimen and hypertonic nutrition media in a Petri dish. The method demonstrated also good sensitivity, reproducibility and the obtained results revealed that D. discoideum species form aggregation in hypertonic environment within several minutes with aim to survive. The formed aggregate had amorphous shape and it consisted from dozen amoeba cells, which kept their NADH amount in constant level for few hours. The reported imaging method might be applicable in various studies for characterization of metabolic events and assessment of the cell energy balance in hypertonic environment.

Early identification of acute hypoxia based on brain NADH fluorescence and cerebral blood flow

Hypoxia is closely related to many diseases and often leads to death. Early detection and identification of the hypoxia causes may help to promptly determine the right rescue plan and reduce the mortality. We proposed a new multiparametric monitoring method employing mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence, regional reflectance, regional cerebral blood flow (CBF), electrocardiography (ECG), and respiration under six kinds of acute hypoxia in four categories to investigate a correlation between the parameter variances and the hypoxia causes. The variation patterns of the parameters were discussed, and the combination of NADH and CBF may contribute to the identification of the causes of hypoxia.

Metabolic Imaging Using Two-Photon Excited NADH Intensity and Fluorescence Lifetime Imaging

Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

Gentamicin Rapidly Inhibits Mitochondrial Metabolism in High-Frequency Cochlear Outer Hair Cells

Aminoglycosides (AG), including gentamicin (GM), are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL) in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs) preferentially succumb to multiple HL pathologies while inner hair cells (IHCs) are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence during acute (1 h) GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.

Assessing the Metabolic State of Cultured Cells and the Intact Cochlea by Two-Photon Fluorescence Intensity and Lifetime Imaging

The ability to match energy production with demand is a fundamental determinant of the health of cells, tissues, and organs of the body. Metabolism and mitochondrial dysfunction have been broadly implicated in many disease states, including hearing disorders and noise-induced hearing loss. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) and flavoprotein (Fp) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, the fluorescence intensity has commonly been used as an indicator of the reduced NADH and/or oxidized Fp concentration in cells and tissue. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. This presents a new opportunity to track the cellular metabolic state as NADH binding changes with metabolism. To better understand what is revealed by variations in the fluorescence intensity and lifetime distributions, we have used mitochondrial uncouplers and inhibitors in conjunction with manipulation of substrate and oxygen concentrations to systematically adjust the metabolic state. We will compare the similarities and differences of the intensity- and FLIM-based assessment of metabolic rate in model systems of increasing complexity, ranging from single cells to intact tissue, highlighting the advantages and limitations of each technique. Supported by NIH DC 02053, NSF-EPSCoR EPS-0701892 (CFD 47.076), NIH P20 RR16469 from the National Center for Research Resources (NCRR), and NIH R15 GM085776.

A revision of the subgenusStegana(s.s.) (Diptera, Drosophilidae) from mainland China

The subgenus Stegana (s.s.) from mainland China is revised, with descriptions of five new species: Stegana (Stegana) apiciprocera Cao & Chen sp. nov., Stegana (Stegana)multispinataCao & Chen sp. nov., Stegana (Stegana) polyrhopalia Cao & Chen sp. nov., Stegana (Stegana) quadrata Cao & Chen sp. nov., and Stegana (Stegana) rotunda Cao & Chen sp. nov. A key to species examined based on morphological data is provided. Based on the mitochondrial reduced nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) gene sequences, the relationship amongst seven Chinese species is discussed, using two species of the subgenus Steganina as outgroups. In addition, a ‘molecular’ key to these Chinese species is also provided. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158, 726–739.

Using Two-photon Excited Fluorescence Intensity- and Lifetime-based NADH Imaging to Investigate Cochlea Metabolism

Metabolism and mitochondrial dysfunction are thought to be involved in many different hearing disorders including noise induced hearing loss and presbycusis. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to study the metabolic status of the different cell types in excised yet intact mouse organ of Corti preparations. Recent published studies employ fluorescence lifetime imaging (FLIM) to determine the ratio of the free to enzyme-bound fluorophores populations that occur during changes in metabolism. We have compared traditional intensity based methods to FLIM in order to evaluate the two different methods in both cultured cells and the excised organ of Corti. Treatment with both metabolic uncouplers and inhibitors caused systematic shifts in both the lifetime and populations of the free and bound pools of NADH, resulting in significant differences in the calculated concentration of NADH when compared to using intensity alone to calculate the same value. Mapping of the locations of the individual lifetimes, shows that the lifetime of NADH varies in different cellular locations as well as in different cell types. Possible implications for the study of hearing loss will be discussed.Supported by NIH DC 08895, NIH P20 RR016475 from the INBRE Program of the National Center for Research Resources., and NIH R15-GM085776.

Skeletal Muscle NAD(P)H Two-Photon Fluorescence Microscopy In Vivo: Topology and Optical Inner Filters

Two-photon excitation fluorescence microscopy (TPEFM) permits the investigation of the topology of intercellular events within living animals. TPEFM was used to monitor the distribution of mitochondrial reduced nicotinamide adenine dinucleotide (NAD(P)H) in murine skeletal muscle in vivo. NAD(P)H fluorescence emission was monitored (∼460nm) using 710–720nm excitation. High-resolution TPEFM images were collected up to a depth of 150μm from the surface of the tibialis anterior muscle. The NAD(P)H fluorescence images revealed subcellular structures consistent with subsarcolemmal, perivascular, intersarcomeric, and paranuclear mitochondria. In vivo fiber typing between IIB and IIA/D fibers was possible using the distribution and content of mitochondria from the NAD(P)H fluorescence signal. The intersarcomeric mitochondria concentrated at the Z-line in the IIB fiber types resulting in a periodic pattern with a spacing of one sarcomere (2.34±0.17μm). The primary inner filter effects were nearly equivalent to water, however, the secondary inner filter effects were highly significant and dynamically affected the observed emission frequency and amplitude of the NAD(P)H fluorescence signal. These data demonstrate the feasibility, and highlight the complexity, of using NAD(P)H TPEFM in skeletal muscle to characterize the topology and metabolic function of mitochondria within the living mouse.

Real-Time assessment of organ vitality during the transplantation procedure

Although organ transplantation has become well established as a treatment modality for many patients with organ failure, little attention has been given to the evaluation of organ quality during its various steps. A critical factor in the success of the actual engraftment procedure is the integrity of energy metabolism and oxygen balance (supply/demand) at the microvasculature and intracellular mitochondrial level. The supply of oxygen to the cells is dependent on the saturation of hemoglobin (HbO 2 ), tissue blood flow, and tissue oxygen partial pressure. The mitochondrial reduced nicotinamide adenine dinucleotide redox state represents oxygen balance in the tissue. Although these parameters can be monitored in all tissues of the body, demand for oxygen may be organ-specific. The various steps surrounding transplantation may require different techniques for the evaluation of tissue vitality. Assessment of blood flow or HbO 2 is not possible during preservation of the organ. On the other hand, because extracellular levels of potassium may represent the energy demand processes in many organs, monitoring of extracellular potassium as an indicator of ionic homeostasis may provide important information regarding the quality of the preservation techniques. Although a large number of relevant studies have been performed in small laboratory animals, real-time monitoring in patients needs more practical tools. We present here the principles of multiparametric monitoring by which tissue vitality may be measured in both experimental and clinical situations. Much of the relevant literature on the subject is limited to the monitoring of kidney and liver. There are also some data on the monitoring of skin flaps. We have reviewed the major published reports in which organ and tissue vitality and quality were assessed in real time and will describe tissue and organ oxygen balance, vitality principles, technologic features of the various monitoring techniques, the clinical or experimental tools available and the conceptual and technologic aspects of the multiparametric monitoring concept. We will also discuss both experimental results and preliminary clinical observations by using multiparametric monitoring.

Role of calcium in metabolic signaling between cardiac sarcoplasmic reticulum and mitochondria in vitro.

The role of Ca(2+) as a cytosolic signaling molecule between porcine cardiac sarcoplasmic reticulum (SR) ATPase and mitochondrial ATP production was evaluated in vitro. The Ca(2+) sensitivity of these processes was determined individually and in a reconstituted system with SR and mitochondria in a 0.5:1 protein-to-cytochrome aa(3) ratio. The half-maximal concentration (K(1/2)) of SR ATPase was 335 nM Ca(2+). The ATP synthesis dependence was similar with a K(1/2) of 243 nM for dehydrogenases and 114 nM for overall ATP production. In the reconstituted system, Ca(2+) increased thapsigargin-sensitive ATP production (maximum approximately 5-fold) with minimal changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH). NADH concentration remained stable despite graded increases in NADH turnover induced over a wide range of Ca(2+) concentrations (0 to approximately 500 nM). These data are consistent with a balanced activation of SR ATPase and mitochondrial ATP synthesis by Ca(2+) that contributes to a homeostasis of energy metabolism metabolites. It is suggested that this balanced activation by cytosolic Ca(2+) is partially responsible for the minimal alteration in energy metabolism intermediates that occurs with changes in cardiac workload in vivo.

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 ∼1.4 μm 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.

Determination of pyridine nucleotide fluorescence from the perfused heart using an internal standard.

Mitochondrial reduced nicotinamide adenine dinucleotide (NADH) is a key intermediate in energy metabolism in the heart, which can be qualitatively monitored using nondestructive surface fluorescence techniques. However, this optical technique is subject to artifacts from alterations in tissue absorbance, motion of the heart, and variations in excitation intensity. In this study rapid-scan fluorescence emission spectroscopy was used in conjunction with an internal fluorescence standard to compensate for these optical artifacts. The fluorescence spectra obtained from heart had a maximum at 460 nm and a shoulder at 415 nm. Dilution of heart homogenates resulted in a fluorescent spectrum characteristic of suspensions of mitochondria, indicating that absorption of fluorescence by tissue components produces an inner filter effect. This internal filter was characterized, and isobestic points with regard to O2 were found at 425 and 450 nm. Alterations in the inner filter effect due to changes in tissue oxygenation were eliminated by monitoring the NADH at 425 nm. Motion artifacts and excitation source fluctuations were corrected by loading heart cells with an internal fluorescent standard, 5(6)-carboxy-2',7'-dichlorofluorescein (ClCF). Motion of the heart and changes in excitation intensity altered the fluorescence detected from both NADH and ClCF. The use of the NADH-to-ClCF ratio detected at isobestic wavelengths (425 nm NADH and 520 nm ClCF) gives a relative measure of NADH fluorescence, which adequately compensates for both internal absorbance and motion artifacts.

Mitochondrial Reduced Nicotinamide—Adenine Dinucleotide Dehydrogenase is Transmembranous

Mitochondrial Reduced Nicotinamide—Adenine Dinucleotide Dehydrogenase is Transmembranous

Fluorometric studies of oxidative metabolism in isolated papillary muscle of the rabbit.

The fluorometric technique for measuring the levels of reduced pyridine nucleotides was used to study oxidative metabolism in isolated rabbit papillary muscle at 23 degrees C. The 100% standard level of tissue fluorescence was defined as that measured for muscles resting in oxygenated 10 mM pyruvate solution. This level increased 15% with anoxia and decreased 45% with stimulation in substrate-free solution. Thus, about one-half of the standard tissue fluorescence was metabolically labile and this labile fraction is suggested to be mitochondrial in origin. Decreased tissue fluorescence following mechanical activity was identified with increased oxidation of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) owing to stimulation by adenosine diphosphate (ADP), released during activity, of mitochondrial respiration. The kinetics of the fluorescence transients were slowed fourfold by removal of pyruvate. This effect was not significantly reversed by addition of 10 mM glucose. The time integrals of the fluorescence transients were linearly related to the amounts of mechanical activity in the presence, but not in the absence, of pyruvate. A positive correlation was observed between the steady-state peak tension at constant stimulus rate and the resting level of reduction of pyridine nucleotides in various media. The fluorometric results are interpreted to be indicative of the steady and transient states established by the substrate dehydrogenases and the respiratory chain during oxidative phosphorylation in mitochondria.