Preparation of rat enterocyte mitochondria.
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ABSTRACT:
SUBMITTER: Masola B
PROVIDER: S-EPMC1153358 | biostudies-other | 1984 Mar
REPOSITORIES: biostudies-other
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Project description:A technique for the rapid preparation of mitochondria from rat liver is described. Tissue fractionation is performed by a single centrifugation step with a discontinuous Percoll density gradient. Total preparation times of 5--6 min are achieved by using this method. The mitochondrial fraction obtained is relatively free of contaminating organelles, as judged by marker-enzyme activity determinations. Mitochondria isolated by Percoll-density-gradient centrifugation differ from mitochondria obtained by differential centrifugation [Taylor, Prpić, Exton & Bygrave (1980) Biochem. J. 188, 443--450] in that the former exhibit a higher acceptor control ratio and a higher calcium content. Values obtained for the protonmotive force are not significantly different between the two preparations. The technique described may be widely applicable for studies requiring the rapid preparation of functionally intact and relatively uncontaminated mitochondria.
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Project description:BackgroundMitochondria are the center of energy metabolism and the production of reactive oxygen species (ROS). ROS production results in a burst of "superoxide flashes", which is always accompanied by depolarization of mitochondrial membrane potential. Superoxide flashes have only been studied in the model plant Arabidopsis thaliana using a complex method to isolate mitochondria. In this study, we present an efficient, easier method to isolate functional mitochondria from floral tissues to measure superoxide flashes.MethodWe used 0.5 g samples to isolate mitochondria within <1.5 h from flowers of two non-transgenic plants (Magnolia denudata and Nelumbo nucifera) to measure superoxide flashes. Superoxide flashes were visualized by the pH-insensitive indicator MitoSOX Red, while the mitochondrial membrane potential (?? m) was labelled with TMRM.ResultsMitochondria isolated using our method showed a high respiration ratio. Our results indicate that the location of ROS and mitochondria was in a good coincidence. Increased ROS together with a higher frequency of superoxide flashes was found in mitochondria isolated from the flower pistil. Furthermore, a higher rate of depolarization of the ?? m was observed in the pistil. Taken together, these results demonstrate that the frequency of superoxide flashes is closely related to depolarization of the ?? m in petals and pistils of flowers.
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Project description:Aging represents a major risk for developing cardiac disease, including heart failure. The gradual deterioration of cell quality control with aging leads to cell death, a phenomenon associated with mitochondrial dysfunction in the heart. Apoptosis is an important quality control process and a necessary phenomenon for maintaining homeostasis and normal function of the heart. However, the mechanism of mitochondria-mediated apoptosis in aged hearts remains poorly understood. Here, we used male Fischer 344 rats of various ages, representing very young (1 month), young (4 months), middle-aged (12 months), and old (20 months) rats, to determine whether mitochondria-mediated apoptotic signals and apoptosis in the left ventricle of the heart are altered notably with aging. As the rats aged, the extramyocyte space and myocyte cross-sectional area in their left ventricle muscle increased, while the number of myocytes decreased. Additionally, mitochondrion-mediated apoptotic signals and apoptosis increased remarkably during aging. Therefore, our results demonstrate that aging promotes remarkable morphological changes and increases the degree of mitochondrion-mediated apoptosis in the left ventricle of rat hearts.
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Project description:Electron microscopic study of cardiomyocytes taken from healthy Wistar and OXYS rats and naked mole rats (Heterocephalus glaber) revealed mitochondria in nuclei that lacked part of the nuclear envelope. The direct interaction of mitochondria with nucleoplasm is shown. The statistical analysis of the occurrence of mitochondria in cardiomyocyte nuclei showed that the percentage of nuclei with mitochondria was roughly around 1%, and did not show age and species dependency. Confocal microscopy of normal rat cardiac myocytes revealed a branched mitochondrial network in the vicinity of nuclei with an organization different than that of interfibrillar mitochondria. This mitochondrial network was energetically functional because it carried the membrane potential that responded by oscillatory mode after photodynamic challenge. We suggest that the presence of functional mitochondria in the nucleus is not only a consequence of certain pathologies but rather represents a normal biological phenomenon involved in mitochondrial/nuclear interactions.
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Project description:The oxidation of L-glutamate and L-glutamine by enterocyte mitochondria was supported by malate. The stimulation of the rate of oxidation of the two amino acids by small amounts of added malate was 93% and 76% respectively. This could not be accounted for by the oxidation of the small amounts of malate added. Amino-oxyacetate added initially inhibited malate-supported oxidation of L-glutamate by 81% and that of L-glutamine by 38%. The inhibition of L-glutamate oxidation was partially reversed by L-glutamine. The dicarboxylate-carrier inhibitor 2-phenylsuccinate inhibited the malate-supported oxidation of both amino acids, but appeared to be slightly stimulatory to L-glutamine oxidation when added initially. The inhibition of L-glutamate oxidation was reversed by L-glutamine. The mitochondrial uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) inhibited malate-supported oxidation of L-glutamate by 78% when added initially. The oxidation of L-glutamine was completely inhibited. However, the uncoupler stimulated the oxidation of both amino acids when added finally. Pyruvate inhibited aspartate synthesis when either of these amino acids was the main substrate, alanine being synthesized. There was no effect on O2 uptake. Mitochondria did not swell in KCl solution, but swelled rapidly in water. Mitochondrial swelling in potassium phosphate and potassium acetate solutions was activated by valinomycin and to a lesser extent by the further addition of FCCP. With potassium malate, swelling was mainly activated by phosphate. The swelling of enterocyte mitochondria in potassium glutamate was slow. In glutamine solution, mitochondrial swelling was greater and appeared to be enhanced by the initial presence of small amounts of phosphate.
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Project description:The small intestine has an underappreciated role as a lipid storage organ. Under conditions of high dietary fat intake, enterocytes can minimize the extent of postprandial lipemia by storing newly absorbed dietary fat in cytoplasmic lipid droplets. Lipid droplets can be subsequently mobilized for the production of chylomicrons. The mechanisms that regulate this process are poorly understood. We report here that the milk protein Mfge8 regulates hydrolysis of cytoplasmic lipid droplets in enterocytes after interacting with the ?v?3 and ?v?5 integrins. Mice deficient in Mfge8 or the ?v?3 and ?v?5 integrins accumulate excess cytoplasmic lipid droplets after a fat challenge. Mechanistically, interruption of the Mfge8-integrin axis leads to impaired enterocyte intracellular triglyceride hydrolase activity in vitro and in vivo. Furthermore, Mfge8 increases triglyceride hydrolase activity through a PI3 kinase/mTORC2-dependent signaling pathway. These data identify a key role for Mfge8 and the ?v?3 and ?v?5 integrins in regulating enterocyte lipid processing.
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