J. J. Lemasters

Assessment of Fura-2 for measurements of cytosolic free calcium

Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Protection by acidotic pH against anoxic cell killing in perfused rat liver: evidence for a pH paradox.

Reperfusion of ischemic tissues causes a paradoxical injury. Here, we measured lactate dehydrogenase (LDH) release as an indicator of tissue damage in perfused rat livers during anoxia and reoxygenation. During anoxia, LDH release was substantially reduced at acidotic pH (pH 6.1–6.9). Using anoxia at pH 6.1 followed by reoxygenation at pH 7.3 to model ischemia and reperfusion, an abrupt release of LDH occurred after reperfusion. A similar release of LDH occurred when pH of anoxic livers was increased to 7.3 without reoxygenation, but LDH release did not occur after reoxygenation at pH 6.1. Thus, a rapid increase of pH rather than reoxygenation accounted for tissue injury after reperfusion of ischemic liver.—Currin, R. T.; Gores, G. J.; Thurman, R. G.; Lemasters, J. J. Protection by acidotic pH against anoxic cell killing in perfused rat liver: evidence for a pH paradox. FASEB J. 5: 207–210; 1991.

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.

Inhibition of Na+/H+ exchange preserves viability, restores mechanical function, and prevents the pH paradox in reperfusion injury to rat neonatal myocytes.

SummaryRat neonatal myocytes exposed to 2.5 mM CaCN and 20 mM 2-deoxyglucose at pH 6.2 (chemical hypoxia) quickly lose viability when pH is increased to 7.4, with or without washout of inhibitors — a ‘pH paradox’. In this study, we evaluated the effect of two Na+/H+ exchange inhibitors (dimethylamiloride and HOE694) and a Na+/Ca2+ exchange inhibitor (dichlorobenzamil) on pH-dependent reperfusion injury. Intracellular free Ca2+ and electrical potential were monitored by laser scanning confocal microscopy of rat neonatal cardiac myocytes grown on coverslips and co-loaded with Fluo-3 and tetramethylrhodamine methylester. After 30–60 min of chemical hypoxia at pH 6.2, mitochondria depolarized and Ca2+ began to increase uniformly throughout the cell. Free Ca2+ reached levels estimated to exceed 2 μM by 4h. Washout of inhibitors at pH 7.4 (reperfusion), with or without dichlorobenzamil, killed most cells within 60 min, despite a marked reduction of Ca2+ in dichloroben zamil-treated cells. Reperfusion at pH 7.4 in the presence of 75 μM dimethylamiloride or 20 μM HOE694, or at pH 6.2, prevented cell death. HOE694-treated cells placed into culture medium recovered mitochondrial membrane potential. In most cells, this occurred before normal Ca2+ was restored. Contracted myocytes re-extended over a 24-h-period. By 48 hours, most cells contracted spontaneously and showed normal Ca2+ transients. Our results indicate that Na+/H+ exchange inhibition protects against pH-dependent reperfusion injury and facilitates full recovery of cell function.

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.

Multiple microscopic techniques for the measurement of plasma membrane lipid structure during hypoxia

Alterations in plasma membrane structure and function are considered of primary importance in the pathogenesis of cell injury. Multiple microscopic techniques are employed to detail alterations in plasma membrane lipid structure during hypoxic injury in individual rat hepatocytes. Multiparameter digitized video microscopy, fluorescence quenching imaging, and fluorescence resonance energy transfer imaging are used to measure and monitor lipid domain formation and topography; laser tweezers are used to monitor the plasma membrane viscoelasticity. These microscopic techniques indicate that hypoxic injury in hepatocytes leads to alterations in plasma membrane lipid topography with the eventual formation of lipid domains. In concert with previous data generated with digitized fluorescence polarization microscopy and fluorescence recovery after photobleaching (FRAP), a model is proposed where formation of the distinct lipid domains promotes loss of the plasma membrane permeability barrier and cell death.

Measurements of plasma membrane architecture during hypoxia using multiple fluorescent spectroscopic techniques

Alterations in plasma membrane structure and function seem to be of primary importance in the pathogenesis of cell injury, calling for more understanding of the changes in plasma membrane lipid structure (e.g., lipid order, lateral diffusion, dependence of phase states, and viscoelasticity) during the evolution of hypoxic injury in hepatocytes using multiple fluorescent spectroscopic techniques. Following hypoxic injury, fluorescence recovery after photobleaching was used to monitor plasma membrane lipid diffusion, resonance energy transfer microscopy was used to detect the lipid topography (domain formation), and the laser trapping technique was used to measure the plasma membrane viscoelasticity. The use of these different kinds of fluorescent spectroscopic techniques coupled with the authors previous studies using digitized fluorescence polarization microscopy which was used to measure lipid order (fluidity) allowed the delineation of alterations in membrane structure during hypoxic injury and a model of membrane architecture during hypoxic injury, which could not be obtained from the use of any of these techniques alone. A model is proposed in which gel- and fluid-phase lipid islands form during hypoxic cell injury. Formation of these lipid domains promotes cell surface bleb formation, with eventual weakening of plasma membrane integrity, bleb rupture, and cell death. 11