Cytosolic Ca2+ deregulation and blebbing after HgCl2 injury to cultured rabbit proximal tubule cells as determined by digital imaging microscopy.

Smith, Mary W.; Phelps, Patricia C.; Trump, Benjamin F.

doi:10.1073/pnas.88.11.4926

Cited by 43 publications

(36 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…When the cells entered the quiescent spherical phase, the mitochondria began to show varying degrees of matrical swelling, similar to that which occurs in oncosis in renal tubules after other types of prelethal injury, such as total ischemia in vivo, 22 or after exposure to many nephrotoxins, including mercuric chloride. 12 This swelling is known to be reversible and is usually associated with ATP depletion. 3 In the spherical phase the MMP initially remains intact, as measured by rhodamine 123 retention, demonstrating that the mitochondrial permeability transition had not occurred.…”

Section: Mitochondrial Changesmentioning

confidence: 99%

“…12 After loading, three to five fields of Fura-2-loaded uninjected control cells were selected, and image pairs were collected at 340/380-nm excitation. Two hours after microinjection of cytochrome c and Fura-2/AM loading, image pairs were collected at 20-minute intervals for up to 6 hours in fields having apoptotic cells.…”

Section: Examination and Evaluation Of Apoptotic Cellsmentioning

confidence: 99%

“…[Ca 2ϩ ] i calibrations were performed as described previously. 12 The ratioed image pairs were displayed using a pseudocolor or gray scale to indicate levels of [Ca 2ϩ ] i .…”

Section: Examination and Evaluation Of Apoptotic Cellsmentioning

confidence: 99%

See 2 more Smart Citations

Studies on the Mechanisms and Kinetics of Apoptosis Induced by Microinjection of Cytochrome c in Rat Kidney Tubule Epithelial Cells (NRK-52E)

Chang

Phelps

Berezesky

et al. 2000

The American Journal of Pathology

View full text Add to dashboard Cite

Recent reports substantiating the role of cytochrome c in the induction of apoptosis led us to examine the kinetics and mechanisms involved in this process as an extension of our ongoing studies of cell injury and cell death. Microinjection of cytochrome c into NRK-52E kidney cells produced rapid apoptosis, which usually began within 30 minutes and reached a maximum of 60 -70% by 3 hours. The changes that occurred included four phases: an initial shrinkage phase, an active phase, a spherical phase, and a necrotic phase. For morphological purposes, the progressive changes were followed by phase-contrast and fluorescence microscopy, transmission and scanning electron microscopy, and time-lapse video microscopy. Cells first showed shrinkage, then displayed multiple pseudopods, which rapidly extended and retracted, giving the cells a bosselated appearance. During this active phase there was chromatin condensation, mitochondria were swollen but retained membrane potential, and the endoplasmic reticulum was dilated. Within 2-4 hours, active-phase cells became spherical and smooth-surfaced but were still alive, the nuclei showed chromatin clumping, the mitochondria underwent high-amplitude swelling but retained membrane potential, the endoplasmic reticulum was highly dilated, and many large apical vacuoles were present. Elevation of [Ca 2؉ ] i was seen at the late spherical phase, shortly before cell death. Pretreatment with the caspase 3 inhibitor (Ac-DEVD-CHO) prevented apoptosis, whereas overexpression of Bcl-2 did not. Apoptosis is an important mechanism of cell death in the kidney during development and after many and diverse injuries.1 As part of our ongoing studies of cell injury and cell death in the kidney, we are exploring the detailed stages and mechanisms of apoptosis and comparing them with those of oncosis. As the prelethal changes of oncosis have been well characterized, we wished to carefully examine the changes of apoptosis before and after cell death. 2,3Cytochrome c release into the cytosol has been implicated as an intermediate event in the initiation of apoptosis after a variety of toxic and other stimuli and injury in several cell types 4 -9 ; it also serves as a useful model, inasmuch as we found that after microinjection, the early phase of apoptosis usually begins within 30 minutes. This means that the progression of changes in single injected cells can be followed by many important techniques. These include the use of time-lapse phase-contrast video microscopy, digital imaging fluorescence, and confocal microscopy to analyze mitochondrial membrane potential, mitochondrial permeability transition, cytosolic calcium concentration [Ca 2ϩ ] i , and cell death, as well as phase-contrast and electron microscopy for morphological characterization.The results clearly defined three principal prelethal stages or phases of apoptosis and characterized the morphology at the light and electron microscope levels. We also showed that ATP is required for the initiation of apoptosis, that the loss of mitocho...

show abstract

Section: Mitochondrial Changesmentioning

confidence: 99%

Section: Examination and Evaluation Of Apoptotic Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Studies on the Mechanisms and Kinetics of Apoptosis Induced by Microinjection of Cytochrome c in Rat Kidney Tubule Epithelial Cells (NRK-52E)

Chang

Phelps

Berezesky

et al. 2000

The American Journal of Pathology

View full text Add to dashboard Cite

show abstract

“…9). In addition, morphological changes, particularly bleb formation, have been widely reported by us (23,31,32) and others (4,22) (13,14,20 (21 ). Although several in vitro systems for predicting in vivo effects have been described (2,3,11,18,28) …”

Section: Cytotoxicitymentioning

confidence: 99%

“…Parameters such as cell viability (21), morphological changes (30), cell membrane integrity (29), cell detachment, and cloning efficiency and growth inhibition (27) (23,31,32) and may not only predict acute or chronic toxicity (13), but may also provide insight into the mechanisms involved (14).…”

Section: Introductionmentioning

confidence: 99%

Studies of Skin Toxicity In Vitro: Dose-Response Studies on JB6 Cells

et al. 1992

View full text Add to dashboard Cite

There are many reasons for developing in vitro tests of toxicity including cost, speed, studies of mechanisms, and studies utilizing human cells and tissues. The present study focuses on the development of in vitro tests to predict in vivo toxicity by comparing them to data from the literature. A broad spectrum of model toxic compounds was evaluated for toxicity on mouse skin JB6 cells in culture. These included mercuric chloride, sodium lauryl sulfate, formaldehyde, dimethyl sulfoxide, benzoyl peroxide, and ionomycin, all of which have been proven to be positive in the Draize test or in cutaneous toxicity studies. Cell viability was evaluated every 15 min for up to 1 hr, and then after 24 hr of treatment using the Trypan Blue exclusion method; morphological changes were evaluated using phase-contrast and transmission electron microscopy. Doseand time-dependent cell death and morphological changes were observed at concentrations ranging from 10 -14 to 10 -2 M. Arbitrary rankings were assigned based on 1) IC 50 value estimated from the present data, and 2) in vivo toxicity reported in the Registry of Toxic Effects of Chemical Substances. Good correlation between in vitro and in vivo toxicity based on arbitrary rankings was observed. Thus, these findings suggest that the JB6 cell culture model can be used for predicting in vivo toxicity. In the future, it may be possible to utilize this system for the study of intracellular ionized calcium ([Ca 2+ ] 1 ), and the expression of oncogenes as early indicators of toxicity.

show abstract

Early morphologic alterations in trophically deprived neuronal death in vitro occur without alterations in cytoplasmic Ca2+

Peña

Pilar

2000

J. Comp. Neurol.

View full text Add to dashboard Cite

Morphologic and functional parameters altered during neuronal death were investigated in chick ciliary ganglion (CG) neurons in vitro isolated from embryonic day 8 (E8, stage 34). Neurons were separated from nonneuronal cells to investigate their inherent cell death program and were cultured with or without trophic support (choroid, iris, pigment epithelium) from their appropriate target tissue. The cell death process was characterized with investigations focused on the earliest events at the onset of commitment to cell death at 11 hours after plating. Initial morphologic changes in the process of cell death were cytoplasmic; swelling, dendritic retraction, blebbing, vacuolization, which are all characteristics of necrosis. Later, nuclear chromatin condensation occurred, a characteristic of apoptosis. An increase in membrane permeability was measured earlier at 8 hours. During these alterations (associated with the initiation of cell death) single cell analysis was performed to evaluate mobile Ca(2+) changes in the same trophically deprived neurons during the course of the death process; Ca(2+) levels remained at 50 nM. Transient Ca(2+) entry was buffered in control and deprived cells at 13 hours but with different parameters. During the execution stage of death mobile Ca(2+) levels were variable. In this final stage of cell death, neurons demonstrated nuclear damage, cytosol disintegration, or morphology sharing both characteristics. These observations define embryonic CG cell death in vitro as neither purely apoptotic nor necrotic but a form of that exhibits features of both. These results also demonstrate a disassociation of changes in cytosolic Ca(2+) from both CG neuronal survival and trophically deprived cell death in vitro.

show abstract

Cytosolic Ca2+ deregulation and blebbing after HgCl2 injury to cultured rabbit proximal tubule cells as determined by digital imaging microscopy.

Cited by 43 publications

References 24 publications

Studies on the Mechanisms and Kinetics of Apoptosis Induced by Microinjection of Cytochrome c in Rat Kidney Tubule Epithelial Cells (NRK-52E)

Studies on the Mechanisms and Kinetics of Apoptosis Induced by Microinjection of Cytochrome c in Rat Kidney Tubule Epithelial Cells (NRK-52E)

Studies of Skin Toxicity In Vitro: Dose-Response Studies on JB6 Cells

Early morphologic alterations in trophically deprived neuronal death in vitro occur without alterations in cytoplasmic Ca2+

Contact Info

Product

Resources

About