Role In Cell Injury Research Articles

Profile of 5-HT2A receptor involved in signaling cascades associated to intracellular inflammation and apoptosis in hepatocytes and its role in carbon tetrachloride-induced hepatotoxicity

Previously, we found that the 5-HT2A receptor plays a key role in cell injury. However, the mechanism by which the 5-HT2A receptor mediates intracellular processes remains unclear. In this study, we aimed to clarify this intracellular process in hepatocyte LO2 cells and evaluate its role in CCl4-induced hepatotoxicity in mice. In vitro, both the agonist and overexpression of 5-HT2A receptor could promote 5-HT degradation by upregulating the expression of 5-HT synthases and monoamine oxidase-A (MAO-A) to cause overproduction of ROS in mitochondria. We refer to this as the activation of the 5-HT degradation system (5DS) axis, which leads to the phosphorylation of JNK, p38 MAPK, STAT3, and NF-κB; upregulation of Bax, cleaved-caspase3, and cleaved-caspase9; and downregulation of Bcl-2, followed by apoptosis and oversecretion of TNF-α and IL-1β in cells. This phenomenon could be markedly blocked by the 5-HT2A receptor antagonist, MAO-A inhibitor, or gene-silencing MAO-A. Through protein kinases C epsilon (PKCε) agonist treatment and gene silencing of the PKCε and 5-HT2A receptor, we demonstrated that the 5-HT2A receptor controls 5-HT synthases and MAO-A expression via the PKCε pathway in cells. Unexpectedly, we discovered that PKCε-mediated phosphorylation of the AKT/mTOR pathway is also a consequence of the activation of the 5DS axis. Furthermore, we confirmed that the inhibition of the 5DS axis using the 5-HT2A receptor antagonist could prevent hepatotoxicity induced by CCl4 both in vitro and in vivo, inhibiting the aforementioned signaling cascades, inflammation, and apoptosis, and that the 5DS activation area overlapped the necrotic area of mouse liver. Taken together, we revealed a 5DS axis in hepatocytes that controls the signaling cascades associated with inflammation and apoptosis and confirmed its role in CCl4-induced hepatotoxicity.

AGING IN LIGNINOLYTIC FUNGI IN SPACE CONDITIONS

Aging is considered in the context of various abiotic stresses, such as water deficit, high temperature, salinity, cold, heavy metals, mechanical wounding, UV radiation, etc. It is well known that the extreme level of these factors can be reflected in sharply increased production of reactive oxygen species. Reactive oxygen species interact with cellular biomolecules, such as lipids, proteins and DNA and can play an important role in cell injury. At the same time, fungi are an appropriate model system in different research areas, including aging. Testing of ligninolytic fungi in space conditions is forthcoming. However, it is unknown how the conditions of microgravity would affect fungal cell aging and enzyme production. Determination of the long-term physiological responses and adaptation to the space environment is of great importance for fungal enzyme production as well.

FOXD3 inhibits SCN2A gene transcription in intractable epilepsy cell models

The expression of sodium voltage-gated channel alpha subunit 2 (SCN2A) is closely related to the development of epilepsy. This study investigated regulatory element of the SCN2A gene involved in epilepsy. An intractable epilepsy cell model was constructed using hippocampal primary neurons and the SH-SY5Y cell line. SCN2A protein and gene expression in cells as well as the level of lactic acid dehydrogenase (LDH) in the cell culture supernatants was detected. Potential regulatory factors of SCN2A and its upstream regulatory elements were identified using the dual-luciferase reporter assay. Finally, the role of the hypothetical transcription factor in epilepsy was examined by using its small interfering RNA (siRNA). Results found that levels of LDH and expression of the hypothetical transcription factor, Forkhead box D3 (FOXD3), was both increased in the model cells, whereas that of SCN2A was decreased. The results of dual-luciferase reporter assays revealed that an upstream region of SCN2A gene spanning from nucleotides −1617 to −1470 was a transcription factor binding region and a trans-acting factor role of FOXD3 was identified in the core region (GGCAAAATTAT). Then the FOXD3 binding site was further verified by the chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA). After SH-SY5Y cells were transfected with FOXD3 siRNA, the release of LDH into culture supernatants and the LDH expression levels in cells were significantly decreased. SCN2A expression in model cells was increased by knockdown of FOXD3. Therefore, this study demonstrated that FOXD3 is a trans-acting factor of SCN2A, and this mechanism may play a role in cell injury after epilepsy.

Effect of Activation of the Ca<sup>2+</sup>-Permeable Acid-Sensing Ion Channel 1a on Acid-Induced Vascular Endothelial Cell Injury of Henoch-Schönlein Purpura Children

Acidosis in local environment plays a critical role in cell injury. One key mediator of acidosis-induced cell injury is the acid-sensing ion channels (ASICs), particularly ASIC1a. Herein, we investigated the role of ASIC1a in acid-induced vascular endothelial cell injury of Henoch-Schonlein purpura (HSP) children. Acid-induced ASIC1a, Calpain and Calcineurin expression in vascular endothelial cells pretreated with IgA1 isolated from HSP were detected by real time quantitative polymerase chain reaction and western blot methods, respectively. Cell cytotoxicity was measured by interleukin-8 and nitric oxide production with ELISA. The results showed acid-induced ASIC1a, Calpain and Calcineurin expression in cells increased, especially at PH6.5. The cytotoxicity of vascular endothelial cells was increased by extracellular acidosis. Moreover non-specific or specific blockers of ASIC1a, Amiloride and PcTX-1 could remarkably decrease these parameters. These findings show that increased [Ca2+]i, mediated via ASIC1a, might contribute to acid-induced vascular endothelial cell injury of HSP.

Inhibition of ASICs reduces rat hepatic stellate cells activity and liver fibrosis: an in vitro and in vivo study.

Hepatic fibrosis is a chronic inflammation-associated disease, which is involved in the infiltration of inflammatory cells and releasing of proinflammatory cytokines. In the pathological process, protons are released by damaged cells and acidosis is considered to play a critical role in cell injury. Although the underlying mechanism (s) remain ill-defined, ASICs (acid-sensing ion channels) are assumed to be involved in this process. The diuretic, amiloride, is neuroprotective in models of cerebral ischemia, a property attributable to the inhibition of central ASICs by the drug. However, the effect of inhibition of ASICs by amiloride in the liver fibrotic process remains unclear. We found that amiloride (25, 50, or 100 μM) could restrain acid-induced HSCs at pH6 in vitro. In vivo experiments showed that amiloride could significantly alleviate liver injury, decreasing levels of profibrogenic cytokines, collagen deposition, and reducing pathological tissue damage. In summary, amiloride inhibits hepatic fibrosis in vivo and in vitro, which is probably associated with the downregulation of ASICs.

Obligatory role of heat shock protein 90 in iNOS induction

Inducible nitric oxide (NO) synthase (iNOS) plays an important role in cell injury and host defense. While undetectable in normal tissues, iNOS expression is induced by endotoxins and inflammatory cytokines primarily via the IκB kinase/nuclear factor-κB (IKK-NF-κB) and Janus kinase (JAK)-signal transducers and activators of transcription 1 (STAT1) pathways. Our previous studies found that heat shock protein 90 (Hsp90) associates with iNOS, and this association enhances iNOS activity. Here we show that Hsp90 is also essential for iNOS induction. With mouse macrophages, Hsp90 inhibition by geldanamycin or knockdown with small interfering RNA (siRNA) prevented lipopolysaccharide (LPS) or interferon-γ (IFN-γ)-stimulated iNOS protein expression. RT-PCR experiments showed that iNOS mRNA transcription was blocked by Hsp90 inhibition. Radicicol, another Hsp90 inhibitor whose structure is different from that of geldanamycin, also blocked iNOS mRNA transcription. These cell biology findings were confirmed in infarcted myocardium where iNOS expression was markedly attenuated by Hsp90 inhibition in vivo. Intriguingly, further analyses showed that inhibiting Hsp90 had no significant effect on the activation of either IKK-NF-κB or JAK-STAT1 in LPS/IFN-γ-stimulated cells. Neither was the nuclear transport of active NF-κB or STAT1 affected by Hsp90 inhibition. But Hsp90 inhibition markedly reduced the binding of active NF-κB and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that Hsp90 was essential for NF-κB and STAT1 bindings to iNOS promoters inside cells. These studies reveal that besides acting as an allosteric enhancer, Hsp90 is also required for transcriptional factor binding amid iNOS mRNA transcription. In view of the essential role of Hsp90 in iNOS gene transactivation, targeting Hsp90 may represent a new approach to intervene iNOS expression in diseases.

Protective effect of hydrogen sulfide on hypoxic respiratory suppression in medullary slice of neonatal rats

Hydrogen sulfide (H(2)S) has been shown to play a protective role in injury of cells induced by hypoxia. Little is known however about its effect on medullary hypoxia-induced rhythmic respiratory suppression. In the present study, a decrease in frequency of rhythmic discharge of hypoglossal rootlets was observed in medullary slices of neonatal rats perfused with 95% N(2)-5% CO(2) to produce hypoxia. Perfusion with NaHS (H(2)S donor) prevented the inhibitory effect of hypoxia on the burst activity of the rootlets, whereas such action of NaHS was suppressed by pretreatment with glibenclamide, a blocker of K(ATP) channels. In addition, the increase in malondialdehyde content and the up-regulation of c-fos mRNA expression of the slices induced by hypoxia was significantly reduced by NaHS. These results indicate that exogenous H(2)S may protect the medullary respiratory center against hypoxic injury via activation of K(ATP) channels, reduction of lipid peroxidation and down-regulation of c-fos.

Extra- and intra-cellular ice formation of red seabream ( Pagrus major) embryos at different cooling rates

The ice crystal formation is assumed as the most lethal factor for the failure of fish embryo cryopreservation and intracellular ice formation (IIF) plays a central role in cell injury during cooling. The objectives were to observe the morphological changes of red seabream ( Pagrus major) embryo during the cooling–thawing process, and to examine the effect of cryoprotectant and cooling rate on the temperatures of oil globule ice formation ( T OIF ), extra-cellular ice formation ( T EIF ) and intracellular ice formation ( T IIF ) using cryomicroscope. After thawing, the morphological changes of embryos were observed and recorded by the video attachment and monitor under the microscope. During the cooling process, three representative phenomena were observed: oil globule gradually turned bright firstly, then the whole field of view flashed and the embryo blackened. Cooling rate affect the temperature of both extra- and intra-cellular ice formations. T EIF and T IIF at high cooling rate were much lower than that at low cooling rate. And the value of T EIF − T IIF increased from 0.45 to 11.11 °C with the increase of cooling rate from 3 to130 °C/min. Taken together, our results suggested that high cooling rate with proper cryoprotectant would be a good option for red seabream embryo cryopreservation.

Membrane hydration correlates to cellular biophysics during freezing in mammalian cells

Cell survival during freezing applications in biomedicine is highly correlated to the temperature history and its dependent cellular biophysical events of dehydration and intracellular ice formation (IIF). Although cell membranes are known to play a significant role in cell injury, a clear correlation between the membrane state and the surrounding intracellular and extracellular water is still lacking. We previously showed that lipid hydration in LNCaP tumor cells is related to cellular dehydration. The goal of this study is to build upon this work by correlating both the phase state of the membrane and the surrounding water to cellular biophysical events in three different mammalian cell types: human prostate tumor cells (LNCaP), human dermal fibroblasts (HDF), and porcine smooth muscle cells (SMC) using Fourier Transform Infrared spectroscopy (FTIR). Variable cooling rates were achieved by controlling the degree of supercooling prior to ice nucleation (− 3 °C and − 10 °C) while the sample was cooled at a set rate of 2 °C/min. Membranes displayed a highly cooperative phase transition under dehydrating conditions (i.e. NT = − 3 °C), which was not observed under IIF conditions (NT = − 10 °C). Spectral analysis showed a consistently greater amount of ice formation during dehydrating vs. IIF conditions in all cell types. This is hypothesized to be due to the extreme loss of membrane hydration in dehydrating cells that is manifested as excess water available for phase change. Interestingly, changes in residual membrane conformational disorder correlate strongly with cellular volumetric decreases as assessed by cryomicroscopy. A strong correlation was also found between the activation energies for freezing induced lyotropic membrane phase change determined using FTIR and the water transport measured by cryomicroscopy. Reduced lipid hydration under dehydration freezing conditions is suggested as one of the likely causes of what has been termed as “solution effects” injury in cryobiology.

VEGF Stimulates the ERK 1/2 Signaling Pathway and Apoptosis in Cerebral Endothelial Cells After Ischemic Conditions

Cerebral endothelial cells that line microvessels play an important role in maintaining blood flow homeostasis within the brain-forming part of the blood-brain barrier. These cells are injured by hypoxia-induced reperfusion, leading to blood-brain barrier breakdown and exacerbation of ischemic injury. We investigated the roles of vascular endothelial growth factor (VEGF) and the downstream extracellular signal-regulated kinase (ERK) protein after oxygen-glucose deprivation (OGD) in primary endothelial cells. Primary mouse endothelial cells were isolated and subjected to OGD. Western analysis of VEGF and ERK 1/2 protein levels was performed. Cells were transfected with VEGF small interference RNA. A terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay and DNA fragmentation assay were used on mouse endothelial cells that overexpress copper/zinc-superoxide dismutase (SOD1). VEGF protein expression was induced and its receptor, Flk-1, was stimulated by OGD. Phosphorylation of ERK 1/2 protein levels was upregulated. Inhibition of phosphorylated ERK (pERK) expression by U0126 reduced endothelial cell death by OGD. Transfection of small interfering RNA for VEGF also inhibited an increase in pERK, suggesting that VEGF acts via ERK. The TUNEL and DNA fragmentation assays showed a significant decrease in TUNEL-positivity in the SOD1-overexpressing endothelial cells compared with wild-type cells after OGD. Our data suggest that OGD induces VEGF signaling via its receptor, Flk-1, and activates ERK via oxidative-stress-dependent mechanisms. Our study shows that in cerebral endothelial cells the ERK 1/2 signaling pathway plays a significant role in cell injury after OGD.

Islet transplantation and antioxidant management: A comprehensive review

Islet transplantation as a promising treatment for type 1 diabetes has received widespread attention. Oxidative stress plays an essential role in cell injury during islet isolation and transplantation procedures. Antioxidants have been used in various studies to improve islet transplantation procedures. The present study reviews the role of oxidative stress and the benefits of antioxidants in islet transplantation procedures. The bibliographical databases Pubmed and Scopus were searched up to November 2008. All relevant human and animal in-vivo and in-vitro studies, which investigated antioxidants on islets, were included. Almost all the tested antioxidants used in the in-vitro studies enhanced islet viability and insulin secretion. Better control of blood glucose after transplantation was the major outcome of antioxidant therapy in all in-vivo studies. The data also indicated that antioxidants improved islet transplantation procedures. Although there is still insufficient evidence to draw definitive conclusions about the efficacy of individual supplements, the benefits of antioxidants in islet isolation procedures cannot be ignored.

Administration of Glutamine After Hemorrhagic Shock Restores Cellular Energy, Reduces Cell Apoptosis and Damage, and Increases Survival

Hemorrhagic shock causes a rapid depletion of adenosine triphosphate (ATP) and an increase of the terminal metabolite xanthine. Free radicals generated from xanthine oxidase play a major role in cell injury. Programmed cell death, apoptosis, is a major pathway causing reperfusion injury. During apoptosis, cytosolic cytochrome-c is released from damaged mitochondria, and it further initiates activation of apoptosis as evidenced by the appearance of caspase-3. The bcl-2 protein serves as an antiapoptosis found on the mitochondrial membrane. Glutamine has been known as a conditionally essential nutrient and seems to have beneficial effects in critically ill patients. The hypothesis of the present study is that glutamine administered during resuscitation following hemorrhagic shock would restore the depletion of hepatic ATP, reduce cellular apoptosis, and increase survival. Male Sprague-Dawley rats were randomly assigned to 3 groups for resuscitation after the same pattern of hemorrhagic shock: Ringer's lactate (LR 21 ml/kg); Alanine-glycine (LR with alanine 0.15 gm/kg and glycine 0.18 gm/kg); and glutamine (LR with glutamine 0.3 gm/kg). Hepatic ATP and xanthine was measured at different time periods. Hepatic apoptosis was measured and the levels of cytosolic cytochrome-c, caspase-3 and bcl-2 were analyzed. Another group of rats were used for survival study. Glutamine administered during resuscitation following hemorrhagic shock partially restored the depletion of hepatic ATP, reduced cellular apoptosis, and increased survival. Glutamine administration during resuscitation significantly protected the liver from tissue damage caused by hemorrhagic shock. Glutamine supplementation may offer opportunities for therapeutic intervention during and after shock.

Induction of KLF4 in response to heat stress

Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. However, little is known about the response of KLF4 to heat stress. In this study, Western blot and reverse transcriptase-polymerase chain reaction were performed to determine the changes in KLF4 expression in response to heat stress. The results showed that heat stress up-regulated KLF4 messenger RNA and protein levels in a time-dependent manner in vivo and in 4 cell lines. Moreover, a study with heat shock transcription factor 1 (Hsf1) gene knockout mice indicated that the induction of KLF4 in response to heat stress was mediated by Hsf1. This process occurred rapidly, indicating that KLF4 is an immediate early response gene of heat stress. Next, the roles of KLF4 under heat stress conditions were analyzed for cells overexpressing or deficient in KLF4. The results showed overexpression of KLF4 increased the death rate of C2C12 cells, whereas KLF4 deficiency decreased the injury of C2C12 cells from heat stress conditions, suggesting that KLF4 might play an important role in cell injury induced by heat stress. KLF4 might be an immediate early response gene and could play an important role in cell injury induced by heat stress.

Suppression of oxidative stress-induced hepatocyte injury by Calpain antisense

Background. Calpain, a cytosolic Ca 2+-dependent proteinase, plays a pivotal role in cell injury. In this study, we investigated the effect of calpain-μ antisense oligonucleotide on oxidative stress induced-hepatocyte injury. Materials and methods. Hemagglutinating virus of Japan liposome complex with one of three types of antisense oligonucleotide (AS-1, AS-2, AS-3) or scramble oligonucleotide was added to the culture medium of HuH7 cells and incubated for 6 days. The expression of calpain-μ protein was examined by Western blotting. After the addition of tert-butyl hydroperoxide, bleb formation was examined by phase contrast microscopy, and cell viability was assessed by the release of lactate dehydrogenase. Results. Incubation of HuH7 cells with AS-2 resulted in a decrease in the amount of calpain on day 4 and a further decrease to almost undetectable levels on day 6, whereas scramble oligonucleotide had no effect. Bleb formation was observed 120 min after the addition of tert-butyl hydroperoxide in scramble oligonucleotide-treated cells as in untreated cells. In contrast, it was rarely observed in AS-2-treated cells. Lactate dehydrogenase release was significantly suppressed in AS-2-treated cells, compared with that in scramble oligonucleotide treated-cells. Conclusions. Our findings suggest that calpain activation is involved in the pathogenesis of oxidative stress injury and that transfection of calpain antisense may potentially protect against ischemia/reperfusion liver injury.

Expression and function of phospholipase A 2 in brain

Phospholipase A 2 (PLA 2) appears to play a fundamental role in cell injury in the central nervous system. We have investigated PLA 2 expression in the astrocytoma cell line 1231N1, and found that GIVA, GIVB, GIVC and GVI PLA 2 messages are expressed. PLA 2 activity is increased by inflammatory/injury stimuli such as interleukin-1β and lipopolysaccharide in these cells but with very different time courses. The arachidonic acid liberated is converted to prostaglandin E 2, possibly by cyclooxygenase-2, which is induced by inflammatory stimuli. This cell system emerges as a model to study injury/inflammation-related activation of the new PLA 2 forms GIVB and GIVC.

Xanthine oxidase-derived oxygen radicals play significant roles in the development of chronic pancreatitis in WBN/Kob rats.

Although oxygen-derived free radicals are known to play a role in cell injury and DNA alterations, the role of active oxidants in chronic pancreatitis has not been fully elucidated. Using WBN/Kob rats, which spontaneously develop chronic pancreatitis-like lesions, we investigated whether xanthine oxidase (XOD)-derived oxygen radicals are involved in pancreatic tissue injury. WBN/Kob rats were fed a control or a tungsten diet. The latter depletes XOD activity. Histologic al changes, glutathione (GSH) content and XOD and superoxide dismutase (SOD) activities were determined in pancreatic tissue. Pancreatic 8-hydroxy-deoxyguanosine (8-OH-dG) levels and lithostathine mRNA were also examined. In WBN/Kob rats, parenchymal destruction and fibrosis developed at approximately 12 weeks of age and progressed with each month. The activity of XOD was significantly higher in the early period (8-12 weeks), whereas the levels of GSH and SOD decreased after 16 weeks. Levels of 8-OH-dG in WBN/Kob rats were significantly elevated at 16 weeks. Lithostathine mRNA levels started to increase at 8 weeks, but were suppressed at 16 weeks. The tungsten diet significantly attenuated the histological changes in WBN/Kob rats. The increase in pancreatic XOD activity and 8-OH-dG content in WBN/Kob rats was significantly inhibited by the tungsten diet and lithostathine mRNA levels remained high at 16 weeks. These results suggest that oxygen radicals generated by XOD play an important role in oxidative DNA damage and the development of chronic pancreatic injury.

Mitochondria-mediated cell injury. Symposium overview.

Mitochondria have long been known to participate in the process of cell injury associated with metabolic failure. Only recently, however, have we come to appreciate the role of mitochondria as primary intracellular targets in the initiation of cell dysfunction. In addition to ATP synthesis, mitochondria are also critical to modulation of cell redox status, osmotic regulation, pH control, and cytosolic calcium homeostasis and cell signaling. Mitochondria are susceptible to damage by oxidants, electrophiles, and lipophilic cations and weak acids. Chemical-induced mitochondrial dysfunction may be manifested as diverse bioenergetic disorders and considerable effort is required to distinguish between mechanisms involving critical mitochondrial targets and those in which mitochondrial dysfunction is secondary and plays only a modulatory role in cell injury. The following paragraphs review a few important examples of chemical-induced cytotoxic responses that are manifested as interference with mitochondrial metabolism and bioenergetics, gene regulation, or signal transduction in the form of apoptosis and altered cell cycle control. Greater understanding of the molecular mechanisms of mitochondrial bioenergetics, ion regulation, and genetics will lead to numerous additional examples of mitochondria-mediated cell injury, revealing important new insight regarding the prediction, prevention, diagnosis, and treatment of chemical-induced toxic tissue injury.

D-Amphetamine-induced hepatotoxicity: possible contribution of catecholamines and hyperthermia to the effect studied in isolated rat hepatocytes.

Amphetamines are indirect-acting sympathomimetic drugs widely abused due to their physical and psychostimulating effects. However, the use of these drugs has been associated with numerous reports of hepatotoxicity. While glutathione depletion induced by amphetamines contributes to the exposure of hepatocytes to oxidative damage, other indirect effects attributed to amphetamines may have a role in cell injury. To examine this possibility, Wistar rats were used for plasma measurements of d-amphetamine and catecholamines (noradrenaline, adrenaline and dopamine) (15 min) after i.p. injection of d-amphetamine (5, 20 and 80 mg/kg). Freshly isolated rat hepatocytes were put into contact for 2 h with concentrations of d-amphetamine and catecholamines similar to those found in vivo. Since hyperthermia is a common consequence of acute amphetamine intake, the study using isolated hepatocytes was conducted at 37 degrees C and also at 41 degrees C in order to simulate high temperature levels. We found that hyperthermia was an important cause of cell toxicity: in vitro, a rise in incubation temperature from 37 to 41 degrees C causes oxidative stress in freshly isolated rat hepatocytes, as shown by a depletion of reduced glutathione (GSH; 23%), an increase of oxidized glutathione (GSSG; 157%), the induction of lipid peroxidation with 77% increase of thiobarbituric acid substances TBARS) and the consequent loss of cell viability (< or = 44%). Single treatment of isolated hepatocytes with catecholamines at 37 degrees C induced lipid peroxidation (29% increase of TBARS) but had no effect on glutathione or cell viability. Conversely, a single treatment with d-amphetamine induced glutathione depletion (< or = 24% depletion of GSH) with no effect on lipid peroxidation or cell viability. Also, d-amphetamine potentiated the induction by catecholamines of lipid peroxidation at 37 degrees C (< or = 48% increase of TBARS), while concomitant treatment of d-amphetamine and catecholamines potentiated cell death at 41 degrees C (< or = 56% of cell death) although no effect on viability was seen at 37 degrees C. It is concluded that the aforementioned modifications induced by d-amphetamine in vivo are cytotoxic to freshly isolated rat hepatocytes.

Mitochondria-Mediated Cell Injury

Mitochondria have long been known to participate in the process of cell injury associated with metabolic failure. Only recently, however, have we come to appreciate the role of mitochondria as primary intracellular targets in the initiation of cell dysfunction. In addition to ATP synthesis, mitochondria are also critical to modulation of cell redox status, osmotic regulation, pH control, and cytosolic calcium homeostasis and cell signaling. Mitochondria are susceptible to damage by oxidants, electrophiles, and lipophilic cations and weak acids. Chemical-induced mitochondrial dysfunction may be manifested as diverse bioenergetic disorders and considerable effort is required to distinguish between mechanisms involving critical mitochondrial targets and those in which mitochondrial dysfunction is secondary and plays only a modulatory role in cell injury. The following paragraphs review a few important examples of chemical-induced cytotoxic responses that are manifested as interference with mitochondrial metabolism and bioenergetics, gene regulation, or signal transduction in the form of apoptosis and altered cell cycle control. Greater understanding of the molecular mechanisms of mitochondrial bioenergetics, ion regulation, and genetics will lead to numerous additional examples of mitochondria-mediated cell injury, revealing important new insight regarding the prediction, prevention, diagnosis, and treatment of chemical-induced toxic tissue injury.

Cytoprotection of kidney epithelial cells by compounds that target amino acid gated chloride channels

Glycine, strychnine and certain chloride channel blockers were reported to protect cells against lethal cell injury. These effects have been attributed to interactions with membrane proteins related to CNS glycine gated chloride channel receptors. We have investigated the pharmacology of these actions. Madin-Darby canine kidney (MDCK) epithelial cells were depleted of adenosine triphosphate (ATP) by incubation in glucose free medium containing a mitochondrial uncoupler. Medium Ca2+ was adjusted to 100 nM in the presence of an ionophore such that intracellular Ca2+ did not increase, and Ca(2+)-related injury mechanisms were inhibited. This permitted more sensitive quantitation of protection against cell injury attributable to glycine or other agents whose actions might be related to those of the amino acid. Two classes of compounds showed cytoprotective activity in this system: (1) ligands at chloride channel receptors, such as glycine, strychnine and avermectin B1a; (2) chloride channel blockers, including cyanotriphenylboron and niflumic acid, both of which are known to bind to channel domains of CNS glycine receptors. Morphological and functional studies showed that the compounds preserved plasma membrane integrity, but permitted cell swelling. Substitution of medium chloride by gluconate, or chloride salts by sucrose, did not substantially modify lethal damage or its prevention by glycine or other drugs. The compounds did not modify ATP declines. At least for some compounds, cytoprotection appeared to be specific to structural features on the molecules. These observations are consistent with the hypothesis that a plasma membrane protein related to glycine-gated chloride channel receptors plays a significant role in cell injury, but indicate that the mechanisms of injury and protection by compounds active in this system are not related to chloride fluxes.