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

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 tetrameth-ylrhodamine methylester (TMRM) for mitochondrial membrane potential (A*) 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 Alk 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 pro-pidium iodide. Thus, the following sequence of intracel-lular events occurred during chemical hypoxia: adeno-sine 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 Abbreviations: ATP, adenosine triphosphate; KRH, Krebs-Ringer-HEPES (N-(2-hydroxyethyl)piperazine-N'-Z-ethanesulfonic acid) buffer containing 115 nmol/L NaCI, 5 mmol/L KCI, 2 mmoliL. CaCI,, 1 mmol/L KHpPOl, 1.2 mmoli L MgSO,, 25 mmol/I, NaHEPES buffer, pH 7.4; TMRM, tetramethylrhodamine methylester; calcein-AM, acetoxymethylester of calcein; A*, mitochondrial membrane potential. Exposure of cultured hepatocytes to cyanide and io-doacetate, inhibitors of oxidative phosphorylation and glycolysis, respectively, mimics the adenosine triphos-phate (ATPI-depletion and reductive stress of hy-poxia. 1-3 This type of metabolic inhibition, called chemical hypoxia, causes formation of protrusions of the plasma membrane called blebs. Blebs grow, coalesce, and ultimately rupture t o precipitate onset of cell death. A virtually identical sequence of plasma membrane changes occurs during anoxia in intact liver and cultured h e p a t ~ c y t e s. ~. ~ The subcellular events that cause bleb formation and lead to bleb rupture remain poorly understood. In recent years, a new optical imaging technique called laser-scanning confocal microscopy has emerged.6 Confocal microscopy creates micron-thin optical slices through thick specimens. Because light from other focal planes is rejected, superimposition of detail is eliminated , and the structure of individual organelles such as mitochondria and lysosomes can be resolved in living cells. Here, using laser-scanning confocal microscopy, we studied the relationship between lysosomal integrity , mitochondrial membrane potential (A*), and bleb rupture in cultured rat hepatocytes during chemical hypoxia. We found that mitochondrial depolarization followed by lysosomal disintegration immediately preceded bleb rupture and cell death. Release of hydrolytic enzymes from lysosomes may be the final event causing lysis of the plasma membrane and irreversible loss of cell viability. MATERIALS AND METHODS Hepatocyte Zsolation and Culture. Hepatocytes were isolated from fed male Sprague-Dawley rats (200 to 250 g) by collagenase perfusion of livers, as described previously.2 Cell viability routinely exceeded 90%, as determined by trypan blue exclusion. Aliquots of cell suspension (100,0OO/mL) in Waymouth's MB-742/1 medium containing 26.7 mmol/L NaHC03, 2 mmol/L L-glutamine, 10% fetal calf serum, 100 nmoVL insulin and 10 nmoVL dexamethasone were plated on collagen-coated glass coverslips in 60-mm Petri dishes. After culture for 18 to 24 hours in 5% CO,/air at 37"C., hepato-cytes were mounted on the stage of the laser scanning confo-1361