| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
This study is performed to verify a hypothesis, "ethanol-induced mitochondrial fusion and gigantism in the cultured myocardium is a compensatory system for ethanol-induced depression of mitochondrial respiration", and to elucidate its mechanism. For this aim, the mouse ventricular myocardium obtained by primary culture was exposed to 0, 50, 200 mM ethanol for 1, 3 and 24 hr, and effects of ethanol were analyzed by flow cytometry and fluorescence microscope.
1. [Digitalization of mitochondrial gigantism by flow cytometry] Three vital fluorescent dyes specific to mitochondria were used : JC-1 (ΔΨm (mitochondrial membrane potential) = orange fluorescence / green fluorescence ratio ; an index of ATP synthesis) ; Mito Tracker ^【R!○】 Green FM (MTG ; in proportion to mitochondrial volume) ; dihydrorhodamine 123 (DHR ; production of ROS (reactive oxygen species), an index of the function of mitochondrial respiratory chain)). The median of the fluorescence intensity of cells exposed to ethanol w
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as analyzed by comparing with that of the corresponding control cells. Increased ΔΨm means that the gradient of H^+ potential between inside and outside of mitochondrial membrane produced by oxidation of substrates in mitochondrial respiratory chain is not much used for the subsequent mitochondrial ATP synthesis.
The mitochondrial volume and size (MTG), together with ΔΨm (JC-1), of myocardial cells exposed to 200 mM ethanol and their isolated mitochondria gradually increased at 1 hr and thereafter, while the function of mitochondrial respiratory chain (DHR) was almost unchanged or rather slightly depressed until 3 hr but accelerated at 24 hr. It means that the exposure to 200 mM ethanol immediately induces a gradual augmentation in mitochondrial volume and size, a suppression of mitochondrial ATP synthesis and a transient and slight suppression of the mitochondrial respiratory chain, the last suppression recovering or rather accelerating at 24 hr. The ratio of light-scattering parameters, FSC (cell size) / SSC (granularity) (= an index of mitochondrial swelling), of myocardial cells decreased at 24 hr, though that of non-muscle cells slightly increased. It suggests that myocardial mitochondria ao not swell due to ethanol but rather increase. For 50 mM ethanol, the above-mentioned changes were generally smaller. The ethanol-induced increase of JC-1 orange fluorescence was also recognized by fluorescence microscope. Thus, a strategy of myocardial mitochondria for countering mitochondrial respiratory depression due to exposure to ethanol is mitochondrial increment and enlargement, being different from the case of the non-muscle cells, and resulting in the recovery of the respiratory chain and meeting the energy requirements for myocardial contraction. The hypothesis mentioned at the beginning has not been adequately verified, neither has it been denied.
2. [Signal transduction system] The amount of NF-κB was measured using FITC-labeled second antibody by flow cytometry. The 200-mM ethanol increased myocardial NF-κB p65 at 1 and 24 hr. while the 50-mM ethanol not. For non-muscle cells almost no change was recognized. Thus, these suggest a possibility that NF-κB has relation to mitochondrial gigantism, and also demonstrate that the flow cytometry is useful for detecting changes in the proteins of small-scale samples. Less
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