Anna-Liisa Nieminen

Intracellular pH during "chemical hypoxia" in cultured rat hepatocytes. Protection by intracellular acidosis against the onset of cell death.

The relationships between extracellular pH (pHo), intracellular pH (pHi), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pHi was measured in single cells by ratio imaging of 2,7-biscarboxy-ethyl-5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pHo of 7.4, pHi decreased from 7.36 to 6.33 within 10 min. pHi remained at 6.1-6.5 for 30-40 min (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pHo produced a slightly greater drop in pHi, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H+ exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Cl- in buffer containing HCO3- abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress.

Irreversible injury in anoxic hepatocytes precipitated by an abrupt increase in plasma membrane permeability.

Using low‐light digitized video microscopy, the onset, progression, and reversibility of anoxic injury were assessed in single hepatocytes isolated from fasted rats. Cell‐surface bleb formation occurred in three stages over 1‐3 h after anoxia. Stage I was characterized by formation of numerous small blebs. In stage II, small blebs enlarged by coalescence and fusion to form a few large terminal blebs. Near the end of stage II, cells began to swell rapidly, ending with the apparent breakdown of one of the terminal blebs. Breakdown of the bleb membrane initiated stage III of injury and was coincident with a rapid increase of nonspecific permeability to organic cationic and anionic molecules. On reoxygenation, stages I and II were fully reversible, and plasma membrane blebs were resorbed completely within 6 min of reoxygenation without loss of viability. Stage III, however, was not reversible, and no morphological changes occurred on reoxygenation. The results indicate that onset of cell death owing to anoxia is a rapid event initiated by a sudden increase of nonspecific plasma membrane permeability caused by rupture of a terminal bleb. Anoxic injury is reversible until this event occurs.— Herman, B.; Nieminen, A.‐L.; Gores, G. J.; Lemasters, J. J. Irreversible injury in anoxic hepatocytes precipitated by an abrupt increase in plasma membrane permeability. FASEB J. 2: 146‐151; 1988.

Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: a multiparameter digitized confocal microscopic study

Exploiting the optical sectioning capabilities of laser scanning confocal microscopy and using parameter-specific fluorescent probes, we determined the distribution of pH, free Ca2+, electrical potential, and volume inside cultured adult rabbit cardiac myocytes during ATP depletion and reductive stress with cyanide and 2-deoxyglucose (chemical hypoxia). During normoxic incubations, myocytes exhibited a cytosolic pH of 7.1 and a mitochondrial pH of 8.0 (delta pH = 0.9 units). Sarcolemmal membrane potential (delta psi) was -80 mV, and mitochondrial delta psi was as high as -100 mV, yielding a mitochondrial protonmotive force (delta p) of -155 mV (delta P = delta psi - 60 delta pH). After 30 min of chemical hypoxia, mitochondrial delta pH decreased to 0.5 pH units, but mitochondrial delta psi remained essentially unchanged. By 40 min, delta pH was collapsed, and mitochondrial and cytosolic free Ca2+ began to increase. Mitochondrial and sarcolemmal delta psi remained high. as Ca2+ rose, myocytes shortened, hypercontracted, and blebbed with a 30% decrease of cell volume. After hypercontraction, extensive mitochondrial Ca2+ loading occurred. After another few minutes, mitochondrial depolarized completely and released their load of Ca2+. After many more minutes, the sarcolemmal permeability barrier broke down, and viability was lost. These studies demonstrate a sequence of subcellular ionic and electrical changes that may underlie the progression to irreversible hypoxic injury.

Calcium dependence of bleb formation and cell death in hepatocytes

Calcium dependence of bleb formation and cell death was evaluated in rat hepatocytes following ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). Cytosolic free Ca2+ was measured in single cells by ratio imaging of Fura-2 fluorescence using multiparameter digitized video microscopy. Cells formed surface blebs within 10 to 20 minutes after chemical hypoxia and most cells lost viability within an hour. An increase of cytosolic free Ca2+ was not required for bleb formation to occur. One to a few minutes prior to the onset of cell death, free Ca2+ increased rapidly in high Ca2+ buffer (1.2 mM) but not in low Ca2+ buffer (less than 1 microM). In either buffer, the rate of cell killing was the same. As the onset of cell death was approached in both high and low Ca2+ buffers, Fura-2 began to leak from the cells at an accelerating rate indicating rapidly increasing plasma membrane permeability. In high Ca2+ buffer, cytosolic free Ca2+ increased in parallel with dye leakage. No regional changes in cytosolic free Ca2+ were observed during this metastable period of increased membrane permeability. In many experiments, actual rupture of cell surface blebs could be observed which led to micron-size discontinuities of the cell surface and cell death. We conclude that a metastable period characterized by increasing plasma membrane permeability marked the onset of cell death in cultured hepatocytes which culminated in rupture of a cell surface bleb. An increase of cytosolic free Ca2+ was not required for the metastable state to develop or cell death to occur.

Mitochondria in pathogenesis

Preface. Acknowledgments. Evaluation of Mitochondrial Function in Living Cells. 1. Flow Cytometric Analysis of Mitochondrial Function H. Rottenberg. 2. Confocal Microscopy of Mitochondrial Function in Living Cells J.L. Lemasters, et al. 3. Primary Disorders of Mitochondrial DNA and the Pathophysiology of mtDNA-related Disorders E.A. Schon, S. DiMauro. 4. Transmission and Segregation of Mammalian Mitochondrial DNA E.A. Shoubridge. 5. Cardiac Reperfusion Injury: Aging, Lipid Peroxidation, and Mitochondrial Function L.I. Szweda, et al. Mitochondrial Ion Homeostatis and Necrotic Cell Death. 6. Ca2+-Induced Transition in Mitochondria: A Cellular Catastrophe? R.A. Haworth, D.R. Hunter. 7. Physiology of the Permeability Transition Pore M. Zoratti, F. Tombola. 8. Control of Mitochondrial Metabolism by Calcium-dependent Hormones P. Burnett, et al. 9. The Permeability Transition Pore in Myocardial Ischemia and Reperfusion A.P. Halestrap, et al. 10. Mitochondrial Calcium Dysregulation during Hypoxic Injury to Cardiac Myocytes E.J. Griffiths. Apoptosis. 11. Mitochondrial Implication in Cell Death P.X. Petit. 12. Role of Mitochondria in Apoptosis Induced by Tumor Necrosis Factor-alpha C.A. Bradham, et al. 13. The ATP Switch in Apoptosis D.J. McConkey. Mitochondria, Free Radicals and Disease. 14. Reactive Oxygen Generation by Mitochondria A.J. Kowaltowski, A.E. Vercesi. 15. The Role of the Permeability Transition in Glutamate-mediated Neuronal Injury I.J. Reynolds, T.G. Hastings. 16. Mitochondrial Dysfunction in the Pathogenesis of Acute Neural Cell Death G. Fiskum. 17. Varied Responses of CNS Mitochondria to Calcium N. Brustovetsky, J.M. Dubinsky. 18. Mitochondrial Dysfunction in Oxidative Stress, Excitotoxicity, and Apoptosis A.-L. Nieminen, et al. 19. Mitochondria in Alcoholic Liver Disease J.C. Fernandez-Checa, et al. 20. Mitochondrial Depolarization and Permeability Changes after Acute Alcohol H. Higuchi, H. Ishii. 21. Mitochondrial Dysfunction in Chronic Fatigue Syndrome B. Chazotte. Chemical Toxicity. 22. Bile Acid Toxicity G.J. Gores. 23. Reyes Related Chemical Toxicity L.C. Trost, J.J. Lemasters. 24. Purinergic Receptor-mediated Cytotoxicity J.F. Nagelkerke, J.P. Zoetewey. 25. Doxorubicin-induced Mitochondrial Cardiomyopathy K.B. Wallace. 26. Drug-induced Microvesicular Steatosis and Steatohepatitis D. Pessayre, et al.

Cytosolic Free Calcium and Cell Injury in Hepatocytes

Formation of cell surface blebs is an early event in hypoxic and toxic injury to liver (Lemasterset al., 1981, 1983; Jewellet al., 1982). Many authors have proposed that a rise of cytosolic free Ca2+ is the stimulus for bleb formation and the initiating factor in a sequence of events leading to irreversible injury and cell death (see Schanneet al., 1979; Trumpet al., 1980; Bellomo and Orrenius, 1985). Recently, we applied the technique of digitized video microscopy to quantitate changes in cytosolic free Ca2+ in relation to blebbing and other cellular parameters during “chemical hypoxia” with metabolic inhibitors in single cultured hepatocytes (Lemasterset al., 1987). Here we present new data examining the relation of bleb formation to the onset of cell death during cellular injury induced by a variety of toxic chemicals. We also determine whether changes in cytosolic free Ca2+ occur during cellular injury which might initiate bleb formation and lead to cell death. The results indicate that the onset of cell death is a very rapid event initiated by rupture of a large plasma membrane bleb leaving the cell in a hyperpermeable state. An increase in cytosolic free Ca2+ is not a necessity for the progression of bleb formation or the onset of cell death.

Cytotoxicity screening of surfactant-based shampoos using a multiwell fluorescence scanner: Correlation with Draize eye scores

The irritancy potential of seven shampoos was evaluated by a rapid cytotoxicity assay in cultured human keratinocytes and rat hepatocytes. Loss of cell viability was estimated from increases in propidium iodide fluorescence measured using a multiwell fluorescence scanner. The concentration of shampoo causing a 50% loss of cell viability after 15 min of incubation (V(50)) was determined by probit analysis. Log V(50) measured in human keratinocytes showed a strong negative correlation (r = -0.95; P <0.001) with Draize eye scores in rabbits. Log V(50) measured in rat hepatocytes did not show a statistically significant correlation with Draize eye scores. The results indicate that cytotoxicity screening of human keratinocytes using propidium iodide and a multiwell fluorescence scanner is highly predictive of Draize eye scores for surfactant-containing shampoos.

Blebbing, Cytosolic Free Calcium, and Mitochondrial Membrane Potential Following Toxic and Anoxic Injury in Hepatocytes

Ischemic and hypoxic injury to cells and tissues is probably the leading cause of death in human disease. For example, cardiovascular disease frequently produces ischemic and hypoxic injury to heart, brain, bowel, kidney, and liver. In liver, hypoxic injury is associated with congestive centrilobular necrosis, hepatic artery occlusion, and various shock syndromes. Thus, understanding the evolution of alterations in both the structural and functional properties of cells during ischemia may provide new and important information for the development of therapeutic strategies to protect cells from ischemic damage.

Relationships Between Extracellular pH, Intracellular pH, and Cell Injury During ‘Chemical Hypoxia’

Injury to hepatocytes, reversible and irreversible, is a common event in human disease. The essential mechanisms preceding and culminating in irreversible hepatocyte injury, a key phenomenon in all hepatic diseases, are still poorly understood. Therefore, experimental models of hepatocellular injury are critical to our understanding the cellular events producing irreversible liver injury.