Maitotoxin (MTX) initiates cell death by sequentially activating 1) Ca2+ influx via non-selective cation channels, 2) uptake of vital dyes via formation of large pores, and 3) release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC) in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells.
Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC50 ≈ 1.9 and 0.66 μM, respectively) suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program.
Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms.
Infection of the bovine lung with Pasteurella haemolytica results in an acute respiratory disorder known as pneumonic pasteurellosis. One of the key virulence determinants used by this bacterium is secretion of an exotoxin that is specific for ruminant leukocytes (leukotoxin). At low concentrations, the leukotoxin can activate ruminant leukocytes, whereas at higher concentrations, it inhibits leukocyte functions and is cytolytic, presumably as a result of pore formation and subsequent membrane permeabilization. We have investigated the possibility that the activation-inhibition paradox is explained in part by leukotoxin-mediated apoptosis (i.e., activation-induced cell death) of bovine leukocytes. Incubation of bovine leukocytes with P. haemolytica leukotoxin caused marked cytoplasmic membrane blebbing (zeiosis) and chromatin condensation and margination, both of which are hallmarks of apoptosis. The observed morphologic changes in bovine leukocytes were leukotoxin dependent, because they were significantly diminished in the presence of an anti-leukotoxin monoclonal antibody. In addition, bovine leukocytes incubated with culture supernatant from a mutant strain of P. haemolytica that does not produce any detectable leukotoxin failed to exhibit the morphologic changes characteristic of cells undergoing apoptosis. These observations may represent an important mechanism by which P. haemolytica overwhelms host defenses, contributing to the fibrinous pleuropneumonia characteristic of bovine pasteurellosis.
Purinergic signaling is a crucial component of disease whose pathophysiological basis is now well established. This review focuses on P2X7, a unique bifunctional purinoreceptor that either opens a non selective cation channel or forms a large, cytolytic pore depending on agonist application and leading to membrane blebbing and to cell death either by necrosis or apoptosis.
Activation of P2X7 receptor has been shown to stimulate the release of multiple proinflammatory cytokines by activated macrophages, with the IL-1b to be the most extensively studied among them. These findings were verified by the use of knockout P2X7 (-/-) mice.
Update information coming from all fields of research implicate this receptor at the very heart of diseases such as rheumatoid arthritis, multiple sclerosis, depression, Alzheimer disease, and to kidney damage, in renal fibrosis and experimental nephritis.
Clinical studies are currently underway with the newly developed selective antagonists for P2X7 receptor, the results of which are eagerly anticipated. These studies together with data from in-vivo experiments with the P2X7 knockout mice and in-vitro experiments will shed light in this exciting area.
P2X7; NLRP3 inflammasome; IL-1b ATP; purinergic signaling; purinergic receptors; review
Cell permeabilization by electric pulses (EPs), or electroporation, has been well established as a tool to indiscriminately increase membrane flows of water solutes down the concentration and voltage gradients. However, we found that EPs of nanosecond duration (nsEPs) trigger formation of voltage-sensitive and inward-rectifying membrane pores. NsEP-treated cells remain mostly impermeable to propidium, suggesting that the maximum pore size is ~1 nm. The ion-channel- like properties of nsEP-opened nanopores vanish if they break into larger, propidium-permeable “conventional” pores. However, nanopores can be stable for many minutes and significantly impact cell electrolyte and water balance. Multiple nsEPs cause fast cell swelling and blebbing, whereas opening of larger pores with digitonin abolishes swelling and causes blebs to implode. The lipid nature of nsEP-opened nanopores is confirmed by fast externalization of phosphatidylserine residues. Nanopores constitute a previously unexplored ion transport pathway that supplements classic ion channels but is distinctly different from them.
electroporation; membrane lipids; electric pulses; membrane permeability; pores
Maitotoxin holds a special place in the annals of natural products chemistry as the largest and most toxic secondary metabolite known to date. Its fascinating, ladder-like, polyether molecular structure and diverse spectrum of biological activities elicited keen interest from chemists and biologists who recognized its uniqueness and potential as a probe and inspiration for research in chemistry and biology. Synthetic studies in the area benefited from methodologies and strategies that were developed as part of chemical synthesis programs directed toward the total synthesis of some of the less complex members of the polyether marine biotoxin class, of which maitotoxin is the flagship. This account focuses on progress made in the authors’ laboratories in the synthesis of large maitotoxin domains with emphasis on methodology development, strategy design, and structural comparisons of the synthesized molecules with the corresponding regions of the natural product. The article concludes with an overview of maitotoxin’s biological profile and future perspectives.
biotoxins; maitotoxin; natural product; polyether; synthesis
Cholera toxin, pertussis toxin, mastoparan, maitotoxin, and α-latrotoxin are complex protein or polyether-based toxins of bacterial, insect, or phytoplankton origin that act with high potency at the endocrine pancreas to stimulate secretion of insulin from β-cells located in the islets of Langerhans. The remarkable insulinotropic properties of these toxins have attracted considerable attention by virtue of their use as selective molecular probes for analyses of β-cell stimulus-secretion coupling. Targets of the toxins include heptahelical cell surface receptors, GTP-binding proteins, ion channels, Ca2+ stores, and the exocytotic secretory apparatus. Here we review the value of insulinotropic toxins from the perspective of their established use in the study of signal transduction pathways activated by the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). Our analysis of one insulinotropic toxin (α-latrotoxin) leads us to conclude that there exists a process of molecular mimicry whereby the ‘lock and key’analogy inherent to hormone-receptor interactions is reproduced by a toxin related in structure to GLP-1.
cholera toxin; pertussis toxin; mastoparan; maitotoxin; α-latrotoxin; insulin; GLP-1
Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to the development of peptic ulcer disease and gastric cancer. The secreted pore-forming toxin VacA is one of the major virulence factors of H. pylori. In the current study, we show that AZ-521 human gastric epithelial cells are highly susceptible to VacA-induced cell death. Wild-type VacA causes death of these cells, whereas mutant VacA proteins defective in membrane channel formation do not. Incubation of AZ-521 cells with wild-type VacA results in cell swelling, poly(ADP-ribose) polymerase (PARP) activation, decreased intracellular ATP concentration, and lactate dehydrogenase (LDH) release. VacA-induced death of these cells is a caspase-independent process that results in cellular release of histone-binding protein high mobility group box 1 (HMGB1), a proinflammatory protein. These features are consistent with the occurrence of cell death through a programmed necrosis pathway and suggest that VacA can be included among the growing number of bacterial pore-forming toxins that induce cell death through programmed necrosis. We propose that VacA augments H. pylori-induced mucosal inflammation in the human stomach by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins and may thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.
Atherosclerosis is still the leading cause of death in the western world. Besides known risk factors studies demonstrating Chlamydophila pneumoniae (C. pneumoniae) to be implicated in the progression of the disease, little is known about C. pneumoniae infection dynamics. We investigated whether C. pneumoniae induce cell death of human aortic endothelial cells, a cell type involved in the initiation of atherosclerosis, and whether chlamydial spots derive from inclusions.
Lactate dehydrogenase release revealed host cell death to be dependent on the amounts of Chlamydia used for infection. The morphology of lysed human aortic endothelial cells showed DNA strand breaks simultaneously with cell membrane damage exclusively in cells carrying Chlamydia as spots. Further ultrastructural analysis revealed additional organelle dilation, leading to the definition as aponecrotic cell death of endothelial cells. Exclusive staining of the metabolic active pathogens by chlamydial heat shock protein 60 labelling and ceramide incorporation demonstrated that the bacteria responsible for the induction of aponecrosis had resided in former inclusions. Furthermore, a strong pro-inflammatory molecule, high mobility group box protein 1, was shown to be released from aponecrotic host cells.
From the data it can be concluded that aponecrosis inducing C. pneumoniae stem from inclusions, since metabolically active bacterial spots are strongly associated with aponecrosis late in the infectious cycle in vascular endothelial cells and metabolic activity was exclusively located inside of inclusions in intact cells. Vice versa initial spot-like infection with metabolically inert bacteria does not have an effect on cell death induction. Hence, C. pneumoniae infection can contribute to atherosclerosis by initial endothelial damage.
Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic “release” assays which measure the extracellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known toxins from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity.
1536-well; NTP 1408 compound library; membrane integrity; cytotoxicity assay; protease release assay; qHTS; renal mesangial cells; HEK293 cells
The P2X7 ATP receptor mediates the cytotoxic effect of
extracellular ATP. P2X7-dependent cell death is heralded by
dramatic plasma membrane bleb formation. Membrane blebbing is a complex
phenomenon involving as yet poorly characterized intracellular pathways. We
have investigated the effect of extracellular ATP on HEK293 cells transfected
with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to
P2X7-transfected, but not to wt P2X7-less, HEK293 cells
caused massive membrane blebbing within 1–2 min. UTP, a nucleotide
incapable of activating P2X7, had no early effects on cell shape
and bleb formation. Bleb formation triggered by ATP was reversible and
required extracellular Ca2+ and an intact cytoskeleton.
Furthermore, it was completely prevented by preincubation with the P2X blocker
oxidized ATP. It was recently observed that the ROCK protein is a key
determinant of bleb formation. Preincubation of HEK293-P2X7 cells
with the ROCK blocker Y-27632 completely prevented P2X7-dependent
blebbing. Although ATP triggered cleavage of the ROCK I isoform in
P2X7-transfected HEK293 cells, the wide range caspase inhibitor
z-VAD-fluoromethylketone had no effect. These observations suggest that
P2X7-dependent plasma membrane blebbing depends on the activation
of the serine/threonine kinase ROCK I.
The cytolytic P2X7 purinoceptor is widely expressed on leukocytes and has sparked interest because of its key role in the activation of the inflammasome, the release of the pro-inflammatory cytokine IL-1β and cell death. We report here the functional characterisation of a R276A gain-of-function mutant analysed for its capacities to induce membrane depolarisation, calcium influx and opening of a large membrane pore permeable to YO-PRO-1. Our results highlight the particular sensitivity of R276A mutant to low micromolar adenosine triphosphate (ATP) concentrations, which possibly reflect an increased affinity for its ligands, and a slower closing kinetics of the receptor channel. Our findings support the notion that evolutionary pressures maintain the low sensitivity of P2X7 to ATP. We also believe that the R276A mutant described here may be useful for the generation of new animal models with exacerbated P2X7 functions that will serve to better characterise its role in inflammation and in immune responses.
P2X7 receptor; ATP; Inflammation; Apoptosis; Gain-of-function; Mutagenesis; Evolution
Smooth Brucella spp. inhibit macrophage apoptosis, whereas rough Brucella mutants induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current study, a random mutagenesis approach was used to create a mutant bank consisting of 11,354 mutants by mariner transposon mutagenesis using Brucella melitensis rough mutant 16MΔmanBA as the parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (release of 10% or less of the lactate dehydrogenase). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes in 51 mutants were identified by DNA sequence analysis, and the mutations included interruptions in virB encoding the type IV secretion system (T4SS) (n = 36) and in vjbR encoding a LuxR-like regulatory element previously shown to be required for virB expression (n = 3), as well as additional mutations (n = 12), one of which also has predicted roles in virB expression. These results suggest that the T4SS is associated with Brucella cytotoxicity in macrophages. To verify this, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase (ΔmanBA) and the T4SS (ΔvirB). As predicted, deletion of virB from 16MΔmanBA and 16M resulted in a complete loss of cytotoxicity in rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme multiplicities of infection (>1,000). Taken together, these results demonstrate that Brucella cytotoxicity in macrophages is T4SS dependent.
Bovine respiratory disease caused by Pasteurella haemolytica may be partially mediated by a leukotoxin secreted by the microorganism. We examined the effect of leukotoxic Pasteurella supernatants on leakage of the cytosol enzyme lactate dehydrogenase and the lysosomal enzyme arylsulfatase from bovine granulocytes. Lactate dehydrogenase release (94%) was much higher than arylsulfatase release (38%) over 30 minutes of incubation. The Pasteurella supernatants inhibited superoxide production by stimulated granulocytes at concentrations which also caused substantial cell death as measured by failure to exclude trypan blue. Both toxic effects were prevented by serum from aerosol-immunized calves, and protection appeared to be antibody-specific by comparison with fetal bovine serum or with serum absorbed against intact P. haemolytica. These findings suggest that the leukotoxin may selectively disrupt the granulocyte plasma membrane, and that antibody directed against a surface component of the microorganism is also capable of protecting against the leukotoxin effect.
Isoflurane preconditioning has been shown to protect endothelial cells against lipopolysaccharide and cytokines-induced injury. This study was designed to determine whether isoflurane preconditioning increased endothelial cell tolerance to ischemia.
Bovine pulmonary arterial endothelial cells were exposed or not exposed to various concentrations of isoflurane for 1 hr. After a 30-min isoflurane-free period, cells were subjected to oxygen-glucose deprivation (OGD) for 3 hr and reoxygenation for 1 hr. Lactate dehydrogenase (LDH) release from cells was used to measure cell injury. In some experiments, various protein kinase C (PKC) inhibitors and ATP-sensitive potassium channel (KATP channel) inhibitors were present from 30 min before isoflurane treatment to the end of isoflurane treatment.
Isoflurane preconditioning dose-dependently decreased the OGD-induced LDH release. This protection was inhibited by 2 μM chelerythrine, a general PKC inhibitor, or 10 μM GÖ6976, an inhibitor for the conventional PKCs. This protection also was inhibited by 0.3 μM glybenclamide, a general KATP channel inhibitor, and 500 μM 5-hydroxydecanoate, a mitochondrial KATP channel blocker. In addition, pretreatment with 100 μM diazoxide, a KATP channel activator, for 1 hr also reduced OGD-induced endothelial cell injury. This diazoxide-induced protection was inhibited by chelerythrine.
Our results suggest that isoflurane preconditioning induces endothelial protection against in vitro simulated ischemia. This protection may be mediated at least in part by conventional PKCs and mitochondrial KATP channels. Our results also indicate that PKCs may be downstream of KATP channels in causing endothelial protection.
endothelial cells; isoflurane; mitochondrial KATP channel; preconditioning; protein kinase C
An important but poorly understood feature of traumatic brain injury (TBI) is the clinically serious problem of spatiotemporal progression (“blossoming”) of a hemorrhagic contusion, a phenomenon we term progressive secondary hemorrhage (PSH). Molecular mechanisms of PSH are unknown and efforts to reduce it by promoting coagulation have met with equivocal results. We hypothesized that PSH might be due to upregulation and activation of sulfonylurea receptor 1 (SUR1)-regulated NCCa-ATP channels in capillary endothelial cells, predisposing to oncotic death of endothelial cells and catastrophic failure of capillary integrity. Anesthetized adult male rats underwent left parietal craniectomy for induction of a focal cortical contusion. The regulatory subunit of the channel, SUR1, was prominently upregulated in capillaries of penumbral tissues surrounding the contusion. In untreated rats, PSH was characterized by progressive enlargement of the contusion deep into the site of cortical impact, including corpus callosum, hippocampus, and thalamus, by progressive accumulation of extravasated blood, with a doubling of the volume during the first 12 h after injury, and by capillary fragmentation in penumbral tissues. Block of SUR1 using low-dose (non-hypoglycemogenic) glibenclamide largely eliminated PSH and capillary fragmentation, and was associated with a significant reduction in the size of the necrotic lesion and in preservation of neurobehavioral function. Antisense oligodeoxynucleotide against SUR1, administered after injury, reduced both SUR1 expression and PSH, consistent with a requirement for transcriptional upregulation of SUR1. Our findings provide novel insights into molecular mechanisms responsible for PSH associated with hemorrhagic contusions, and point to SUR1 as a potential therapeutic target in TBI.
contusion; glibenclamide; hemorrhage; sulfonylurea receptor 1; traumatic brain injury
Infection of human monocyte-derived macrophages in vitro with virulent Shigella flexneri resulted in cell death which involved rupture of the plasma membrane, cell swelling, disintegration of ultrastructure, and generalized karyolysis. These features bore resemblance to oncosis and are in striking contrast to previously described observations of mouse macrophages, where a similar infection by virulent Shigella resulted in cell death by apoptosis. Cell death by oncosis in human macrophages was confirmed by lactate dehydrogenase release, light microscopy, electron microscopy, terminal deoxynucleotidyltransferase end labeling of DNA ends, DNA fragmentation assays, and fluorescence-activated cell sorter analysis of propidium-labeled nuclei. Thus, the phenomena of cell death induced by virulent Shigella in human and mouse macrophages reflect different biochemical pathways. Interleukin-1beta (IL-1beta) was released in culture supernatants of human macrophages infected with virulent bacteria. Inhibition with IL-1beta-converting enzyme inhibitors indicated, however, that this release occurred as a passive event of cell lysis. The patterns of intracellular survival of Shigella strains within human and mouse macrophages reflect differences that exist not only between Shigella serotypes but also between the two different macrophage cell types.
Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.
We investigated interactions of human isolates of Acinetobacter calcoaceticus–baumannii complex strains with epithelial cells. The results showed that bacterial contact with the cells as well as adhesion and invasion were required for induction of cytotoxicity. The infected cells revealed hallmarks of apoptosis characterized by cell shrinking, condensed chromatin, and internucleosomal fragmentation of nuclear DNA. The highest apoptotic index was observed for 4 of 10 A.calcoaceticus and 4 of 7 A. baumannii strains. Moreover, we observed oncotic changes: cellular swelling and blebbing, noncondensed chromatin, and the absence of DNA fragmentation. The highest oncotic index was observed in cells infected with 6 A.calcoaceticus isolates. Cell-contact cytotoxicity and cell death were not inhibited by the pan-caspase inhibitor z-VAD-fmk. Induction of oncosis was correlated with increased invasive ability of the strains. We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death. Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨm). Bacterial infection caused generation of nitric oxide and reactive oxygen species. This study indicated that Acinetobacter spp. induced strain-dependent distinct types of epithelial cell death that may contribute to the pathogenesis of bacterial infection.
The nonhemolytic enterotoxin (Nhe) produced by Bacillus cereus is a pore-forming toxin consisting of three components, NheA, -B and -C. We have studied effects of Nhe on primate epithelial cells (Vero) and rodent pituitary cells (GH4) by measuring release of lactate dehydrogenase (LDH), K+ efflux and the cytosolic Ca2+ concentration ([Ca2+]i). Plasma membrane channel events were monitored by patch-clamp recordings. Using strains of B. cereus lacking either NheA or -C, we examined the functional role of the various components. In both cell types, NheA + B + C induced release of LDH and K+ as well as Ca2+ influx. A specific monoclonal antibody against NheB abolished LDH release and elevation of [Ca2+]i. Exposure to NheA + B caused a similar K+ efflux and elevation of [Ca2+]i as NheA + B + C in GH4 cells, whereas in Vero cells the rate of K+ efflux was reduced by 50% and [Ca2+]i was unaffected. NheB + C had no effect on either cell type. Exposure to NheA + B + C induced large-conductance steps in both cell types, and similar channel insertions were observed in GH4 cells exposed to NheA + B. In Vero cells, NheA + B induced channels of much smaller conductance. NheB + C failed to insert membrane channels. The conductance of the large channels in GH4 cells was about 10 nS. This is the largest channel conductance reported in cell membranes under quasi-physiological conditions. In conclusion, NheA and NheB are necessary and sufficient for formation of large-conductance channels in GH4 cells, whereas in Vero cells such large-conductance channels are in addition dependent on NheC.
Bacillus cereus; Pore-forming toxin; Nonhemolytic enterotoxin; Large-conductance channel; Vero cell; GH4 cell
High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1~2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 µM Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 µM of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca2+]i response; a transient [Ca2+]i peak and sustained [Ca2+]i increase secondary to ATP-stimulated Ca2+ influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.
P2X7 receptor; Membrane blebbing; ROCKI; Ca2+ influx
Background. Streptococcus pneumoniae causes serious diseases such as pneumonia and meningitis. Its major pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, which produces lytic pores at high concentrations. At low concentrations, it has other effects, including induction of apoptosis. Many cellular effects of pneumolysin appear to be calcium dependent.
Methods. Live imaging of primary mouse astroglia exposed to sublytic amounts of pneumolysin at various concentrations of extracellular calcium was used to measure changes in cellular permeability (as judged by lactate dehydrogenase release and propidium iodide chromatin staining). Individual pore properties were analyzed by conductance across artificial lipid bilayer. Tissue toxicity was studied in continuously oxygenated acute brain slices.
Results. The reduction of extracellular calcium increased the lytic capacity of the toxin due to increased membrane binding. Reduction of calcium did not influence the conductance properties of individual toxin pores. In acute cortical brain slices, the reduction of extracellular calcium from 2 to 1 mM conferred lytic activity to pathophysiologically relevant nonlytic concentrations of pneumolysin.
Conclusions. Reduction of extracellular calcium strongly enhanced the lytic capacity of pneumolysin due to increased membrane binding. Thus, extracellular calcium concentration should be considered as a factor of primary importance for the course of pneumococcal meningitis.
Pneumolysin has been identified as a virulence factor in Streptococcus pneumoniae disease. In addition to producing tissue injury through its cytolytic effect, pneumolysin might injure tissues indirectly by eliciting an inflammatory response. We demonstrate for the first time that pneumolysin is a rapid and potent activator of cellular phospholipase A in bovine pulmonary artery endothelial cells. In contrast to other toxin-activated phospholipases, pneumolysin-stimulated phospholipase A showed no substrate specificity among major cellular membrane phospholipids. Phospholipase A activation required the formation of functional transmembrane pores by pneumolysin rather than membrane lipid perturbation. Pneumolysin stimulation of phospholipase A was calcium dependent; however, pneumolysin did not appear to function simply as a calcium ionophore. Pneumolysin was capable of stimulating purified bee and snake venom phospholipase A2s against a phospholipid substrate isolated from endothelial cells. Thus, pneumolysin stimulates cellular phospholipase A and the resulting products might further injure tissues by direct cytolytic effect or by evoking inflammatory responses.
We examined the effects of hypoxia and reoxygenation in isolated, perfused rat livers. Hypoxia induced by a low rate of perfusion led to near anoxia confined to centrilobular regions of the liver lobule. Periportal regions remained normoxic. Within 15 min, anoxic centrilobular hepatocytes developed surface blebs that projected into sinusoids through endothelial fenestrations. Periportal hepatocytes were unaffected. Both scanning and transmission electron microscopy suggested that blebs developed by transformation of preexisting microvilli. Upon reoxygenation by restoration of a high rate of perfusion, blebs disappeared. Other changes included marked shrinkage of hepatocytes, enlargement of sinusoids, and dilation of sinusoidal fenestrations. There was also an abrupt increase in the release of lactate dehydrogenase and protein after reoxygenation, and cytoplasmic fragments corresponding in size and shape to blebs were recovered by filtration of the effluent perfusate. We also studied phalloidin and cytochalasin D, agents that disrupt the cytoskeleton. Both substances at micromolar concentrations caused rapid and profound alterations of cell surface topography. We conclude that hepatic tissue is quite vulnerable to hypoxic injury. The morphological expression of hypoxic injury seems mediated by changes in the cortical cytoskeleton. Reoxygenation causes disappearance of blebs and paradoxically causes disruption of cellular volume control and release of blebs as cytoplasmic fragments. Such cytoplasmic shedding provides a mechanism for selective release of hepatic enzymes by injured liver tissue.
During apoptosis, a number of physical changes occur in the cell membrane including a gradual increase in permeability to vital stains such as propidium iodide. This study explored the possibility that one consequence of membrane changes concurrent with early modest permeability is vulnerability to degradation by secretory phospholipase A2 (sPLA2). The activity of this hydrolytic enzyme toward mammalian cells depends on the health of the cell; healthy cells are resistant, but they become susceptible early during programmed death. Populations of S49 lymphoma cells during programmed death were classified by flow cytometry based on permeability to propidium iodide and susceptibility to sPLA2. The apoptotic inducers thapsigargin and dexamethasone caused modest permeability to propidium iodide and increased staining by merocyanine 540, a dye sensitive to membrane perturbations. Various sPLA2 isozymes (human groups IIa, V, X, and snake venom) preferentially hydrolyzed the membranes of cells that displayed enhanced permeability. In contrast, cells exposed briefly to a calcium ionophore showed the increase in cell staining intensity by merocyanine 540 without accompanying uptake of propidium iodide. Under that condition, only the snake venom and human group × enzymes hydrolyzed cells that were dying. These results suggested that cells showing modest permeability to propidium iodide during the early phase of apoptosis are substrates for sPLA2 and that specificity among isoforms of the enzyme depends on the degree to which the membrane has been perturbed during the death process. This susceptibility to hydrolysis may be important as part of the signal to attract macrophages toward apoptotic cells.
Application of volatile anesthetics during the onset of reperfusion reduced ischemia-induced cardiac and brain injury (anesthetic postconditioning). This study was designed to evaluate whether volatile anesthetics induced a postconditioning effect in endothelial cells. Bovine pulmonary arterial endothelial cell (BPAEC) cultures were exposed to oxygen-glucose deprivation, a condition to simulate ischemia in vitro, for 3 h. The volatile anesthetics isoflurane and desflurane were applied during the early phase of simulated reperfusion. Cell injury was quantified by lactate dehydrogenase (LDH) release and flow cytometrical measurement after annexin V and propidium iodide staining. Oxygen-glucose deprivation and the subsequent simulated reperfusion increased LDH release and annexin V-positive staining cells, a characteristic of cell apoptosis. Posttreatment with isoflurane, but not desflurane, reduced this cell injury. This protection was apparent even when 2% isoflurane was applied at 60 min after the onset of reperfusion. The isoflurane postconditioning effect was abolished by glybenclamide, a general ATP sensitive K+ (KATP) channel blocker, 5-hydroxydecanoate, a mitochondrial KATP channel blocker, and chelerythrine, a protein kinase C inhibitor. Diazoxide, a mitochondrial KATP channel activator, applied at the onset of reperfusion also decreased oxygen-glucose deprivation-induced endothelial cell injury. This diazoxide-induced protection was abolished by chelerythrine and 5-hydroxydecanoate. We conclude that isoflurane induced a postconditioning effect in BPAEC. The effective time window of isoflurane postconditioning was from 0 to 60 min after the onset of reperfusion. This isoflurane postconditioning effect may be mediated by mitochondrial KATP channels and PKC. PKC may be downstream of mitochondrial KATP channels for this isoflurane effect.
endothelial cells; postconditioning; volatile anesthetics