Some Characteristics of the Fluorescence Lifetime of Reduced Pyridine Nucleotides in Isolated Mitochondria, Isolated Hepatocytes, and Perfused Rat Liver In Situ

Wakita, Masayoshi; Nishimura, Goro; Tamura, Mamoru

doi:10.1093/oxfordjournals.jbchem.a125001

Cited by 104 publications

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“…However, changes in the free component have not been detected. Wakita et al (1995), in studies on rat liver mitochondria, report that the fluorescence signal is not due to changes in the environment (binding) but rather to changes in the amount of NAD(P)H. Kasimova et al (2006) find that the concentration of free NADH in isolated potato tuber mitochondria remains constant, regardless of the metabolic conditions or the total amount of NADH. When taken together, these considerations lead us to favor the idea that the troughs in the oscillatory signal represent an increase in NAD(P) 1 .…”

Section: Discussionmentioning

confidence: 99%

“…The troughs represent a decrease in NAD(P)H fluorescence, which could be due to two different processes. First, it could be due to a shift from bound to free NAD(P)H in which the bound form exhibits a much stronger fluorescence than that which is free (Wakita et al, 1995;Paul and Schneckenburger, 1996;Blinova et al, 2005). Second, it could be due to oxidation of NAD(P)H, where the oxidized form [NAD(P) 1 ] is nonfluorescent.…”

Section: Discussionmentioning

confidence: 99%

“…However, the troughs in the data are also thought to contain important information. It is known, for example, that the strongest fluorescent signal derives from proteinbound NAD(P)H, whereas free NAD(P)H has a substantially weaker fluorescent signal (Wakita et al, 1995;Paul and Schneckenburger, 1996;Blinova et al, 2005). The oscillation, therefore, could arise from a change between bound and free forms.…”

Section: Nad(p)h Fluorescence Oscillates During Pollen Tube Growth Osmentioning

confidence: 99%

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NAD(P)H Oscillates in Pollen Tubes and Is Correlated with Tip Growth

et al. 2006

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Nad(p)h Fluorescence Oscillates During Pollen Tube Growth Osmentioning

confidence: 99%

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NAD(P)H Oscillates in Pollen Tubes and Is Correlated with Tip Growth

et al. 2006

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“…However, the fluorescence of NAD(P)H increases 8-to 12-fold when it is bound as a coenzyme (13,(18)(19)(20). From estimates in yeast cells, hepatocytes, and isolated mitochondria, it is accepted that most of the NAD(P)H of normal cells is bound in vivo (18,19,(21)(22)(23), which would lead to a Ϸ10-fold overestimation of the NAD(P)H level. Thus, the unbound NADH standard curve does not rigorously apply here, but it can be used to determine an absolute upper limit for NAD(P)H changes.…”

Section: Methodsmentioning

confidence: 99%

Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet β cells

Patterson

Knobel

Arkhammar

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

262

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Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be Ϸ30 M. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be Ϸ7 M. Likewise, the NAD(P)H increase of the mitochondrial domain is Ϸ60 M and is delayed with respect to the change in cytoplasmic NAD(P)H by Ϸ20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.G lucose-induced insulin secretion is coupled to the metabolic state of the ␤ cell. After transport into the cell, glucose is phosphorylated and shunted into glycolysis, which increases metabolic flux. This altered metabolic state, which can be monitored by NAD(P)H autofluorescence increase, leads to an increase in the ATP͞ADP ratio that closes the plasma membrane-associated ATP-sensitive potassium (K ATP ) channel. Closure of this channel depolarizes the membrane, leading to the activation of voltage-sensitive calcium (Ca 2ϩ ) channels, Ca 2ϩ influx, and insulin secretion (1).Glucose usage in stimulated pancreatic ␤ cells is principally glycolytic, with the polyol pathway, glycogen synthesis, and pentose phosphate pathway accounting for Ͻ10% of total usage (2). Thus, the glucose metabolic signal is derived from glycolysis in the cytoplasm and pyruvate metabolism in the mitochondria. The absence of stimulated insulin secretion with nonmetabolizable glucose derivatives (1) and the abolition of glucosestimulated insulin secretion in a pancreatic ␤ cell line lacking mitochondrial DNA (3) suggest roles for both cytoplasmic and mitochondrial metabolism in normal secretion. Additionally, both glycolytic intermediates, such as glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (2), and mitochondrial substrates, such as leucine (4) or methyl pyruvate (5), stimulate insulin secretion, which further supports a role for metabolism in both compartments in glucose signaling.Attempts to resolve the glycolytic and mitochondrial contributions to glucose-stimulated insulin secretion have relied on nutrient secretagogues that couple at various points into glycolysis or Kreb's cycle or on pharmacological inhibition at various points along each pathway (1, 6). Although various nutrient supplements indicate that couplings of the pathway can lead to insulin secretion, they seldom mimic the effects of glucose. For example, pyruvate potentiates glucose-stimulated secretion but does not cause s...

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“…The ratio of free/bound NADH varies significantly from 1.5:1 20 to 1:4. 21 For example, Wakita et al 23 were unable to detect free NADH in rat liver mitochondria regardless of their respiratory state, whereas Blinova et al 20 observed a high proportion of free NADH in pig heart mitochondria using a similar technique. Interestingly, Kasimova et al suggested that the free NADH concentration in plant mitochondria is kept constant under different metabolic conditions.…”

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confidence: 99%