PROGRESSION OF SUBCELLULAR CHANGES DURING CHEMICAL HYPOXIA TO CULTURED RAT HEPATOCYTES - A LASER-SCANNING CONFOCAL MICROSCOPIC STUDY

The aim of this study was to evaluate changes in the subcellular organ elles of cultured hepatocytes by laser scanning confocal microscopy du ring chemical hypoxia with cyanide and iodoacetate, inhibitors of mito chondrial respiration and glycolysis, respectively. Parameter-specific fluorophores used were calcein for cell topography and membrane perme ability, rhodaminedextran for lysosomes, rhodamine 123 and tetramethyl rhodamine methylester (TMRM) for mitochondrial membrane potential (Del ta Psi) and propidium iodide for loss of cell viability. During the fi rst 30 to 40 minutes of chemical hypoxia to cultured hepatocytes, nume rous surface blebs formed and cell volume increased, but Delta Psi dec reased relatively little. Subsequently, the nonspecific permeability o f mitochondrial membranes increased, and mitochondria depolarized Thes e events were followed a few minutes later by disintegration of indivi dual lysosomes. After a few more minutes, viability was lost as indica ted by bleb rupture, gross plasma membrane permeability to calcein, an d nuclear labeling with propidium iodide. Thus, the following sequence of intracellular events occurred during chemical hypoxia: adenosine t riphosphate (ATP) depletion, bleb formation with cellular swelling, on set of a mitochondrial permeability transition, disintegration of lyso somes, plasma membrane failure from bleb rupture, and cell death. Any explanation of the pathophysiology of hypoxic injury must take into ac count this unique sequence of events.