Progression of subcellular changes during chemical hypoxia to cultured rat hepatocytes: A laser scanning confocal microscopic study

Authors

  • George Zahrebelski,

    1. Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
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  • Anna-Liisa Nieminen,

    1. Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
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  • Kristin Al-Ghoul,

    1. Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
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  • Ting Qian,

    1. Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
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  • Brian Herman,

    1. Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
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  • John J. Lemasters MD, PhD

    Corresponding author
    1. Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
    • Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina, Campus Box 7090, 236 Taylor Hall, Chapel Hill, NC 27599–7090
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Abstract

The aim of this study was to evaluate changes in the subcellular organelles of cultured hepatocytes by laser scanning confocal microscopy during chemical hypoxia with cyanide and iodoacetate, inhibitors of mitochon-drial respiration and glycolysis, respectively. Parameter-specific fluorophores used were calcein for cell topography and membrane permeability, rhodamine-dextran for lysosomes, rhodamine 123 and tetramethylrhodamine methylester (TMRM) for mitochondrial membrane potential (Δ Ψ) and propidium iodide for loss of cell viability. During the first 30 to 40 minutes of chemical hypoxia to cultured hepatocytes, numerous surface blebs formed and cell volume increased, but Δ Ψ decreased relatively little. Subsequently, the nonspecific permeability of mitochondrial membranes increased, and mitochondria depolarized. These events were followed a few minutes later by disintegration of individual lysosomes. After a few more minutes, viability was lost as indicated by bleb rupture, gross plasma membrane permeability to calcein, and nuclear labeling with propidium iodide. Thus, the following sequence of intracellular events occurred during chemical hypoxia: adenosine triphosphate (ATP) depletion, bleb formation with cellular swelling, onset of a mitochondrial permeability transition, disintegration of lysosomes, plasma membrane failure from bleb rupture, and cell death. Any explanation of the pathophysiology of hypoxic injury must take into account this unique sequence of events.

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