Ultrastructure and fluorescence of chromatin: interaction of propidium iodide with DNA after different pretreatments.
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Animals↗
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Simultaneous fluorescence labeling of human fibroblast cells with fluorescamine and propidium iodide. An energy transfer study.
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Energy Transfer↗
Sequence-dependent cooperative interactions in A/T-containing oligo- and polydeoxyribonucleotides.
To evaluate the length and sequence dependence of the unusual interaction properties observed for nonalternating A/T sequences in deoxyribonucleic acid (DNA) [Wilson, W. D., Wang, Y. H., Krishnamoorthy, C. R., & Smith, J. C. (1985) Biochemistry 24, 3991-3999], we have synthesized the oligomers d(A-T)6, dA10 X dT10, and d(A6-T6) and evaluated their interaction with the intercalator propidium. Propidium visible spectral shifts on adding all three oligomers are quite similar. Low-temperature spectrophotometric binding measurements indicate that d(A-T)6 has a significantly larger binding constant for propidium than dA10.dT10, as with the analogous alternating and nonalternating DNA polymers. The oligomer dA10.dT10 displays positive cooperativity in its propidium binding isotherm, and its binding constant increases with increasing temperature while d(A-T)6 does not display positive cooperativity, and its binding constant decreases with temperature, again as with the analogous polymers. van't Hoff plots indicate that the propidium binding enthalpies are approximately -9 and +6 kcal/mol for the alternating and nonalternating DNA samples, respectively. The mixed-sequence self-complementary oligomer d(A6-T6) has an unusual low-temperature binding isotherm which suggests a single strong binding site and a larger number of weaker binding sites which bind propidium cooperatively. A van't Hoff plot indicates that the cooperative sites d(A-T)6 have binding constants and binding enthalpies similar to dA10.dT10. Similar rate constants are observed in the sodium dodecyl sulfate driven dissociation reaction of propidium from d(A-T)6 and d(A6-T6), but the association reaction of propidium is significantly slower with d(A6-T6) than with d(A-T)6.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Visualization of cell death in vivo during murine endotoxin-induced uveitis.
PURPOSE: To develop a technology to image cell death in the eye of a live mouse and to apply that technology to characterize the role of apoptosis and necrosis in the evolution of endotoxin-induced uveitis (EIU), a standard model of intraocular inflammation. METHODS: To induce EIU, 250 ng Escherichia coli 055:B5 lipopolysaccharide was injected into the vitreous body of BALB/c mice. At 0, 6, 10, 16, 20, 24, 48, 72, and 96 hours and on day 7 after endotoxin injection, annexin V and propidium iodide were injected into the anterior chamber of these mice, and labeled cells were observed by using intravital epifluorescence video microscopy. Iris and corneal wholemounts isolated from the mice were also evaluated with standard and confocal fluorescence microscopy. TUNEL staining was performed on iris wholemounts taken from additional mice similarly injected with endotoxin, to confirm the in vivo results. RESULTS: Uveitis was induced in all the mice that received an endotoxin injection. The percentages of annexin V(+) propidium(-) cells, annexin V(+) propidium(+) cells, and annexin V(-) propidium(+) cells in the iris tissues visible by intravital microscopy were comparable to those observed by TUNEL staining in vitro. In addition, intravital microscopy allowed observation of labeled cells in the aqueous humor and on the surface of the lens. Both the number and the pattern of labeled cells changed dramatically over time. The cells stained with annexin V had a variety of morphologies, including small and round, round with a lobulated or a kidney-shaped nucleus, dendriform, and irregular. CONCLUSIONS: A technique was developed to image cell death in the anterior segment of the eye in vivo and used to demonstrate that the number and proportion of early apoptotic (annexin V(+) propidium(-)) and late apoptotic or necrotic (annexin V(+) propidium(+) and annexin V(-) propidium(+)) cells change over the course of EIU. A variety of inflammatory cells and resident cells undergo apoptosis, or possibly necrosis, which may contribute to the rapid resolution of EIU. This in vivo technique will be a valuable tool for future studies on the resolution of ocular inflammation.
Animals↗
Ramiprilat attenuates hypoxia/reoxygenation injury to cardiac myocytes via a bradykinin-dependent mechanism.
Isolated rat neonatal cardiac myocytes were subjected to immersion in hypoxic (PO2 < 2 mm Hg), glucose-free Tyrode's solution for 5 h followed by concomitant reoxygenation and staining with the membrane-impermeant fluorophore, propidium iodide, in normoxic (PO2 > 150 mm Hg), serum-free culture media for 15 min in order to assess sarcolemmal damage indicative of myocyte viability due to hypoxia/reoxygenation injury. Prior to hypoxic exposure, cells were pretreated for 90 min with the angiotensin-converting enzyme inhibitor cyclopenta[b]pyrrole-2-carboxylic acid, 1-[2-[(1-carboxy-3-phenylpropyl)amino]-l-oxopropyl]octahydro-[2S-[1[R* (R*)]2 alpha, 3a beta, 6a beta]] (ramiprilat), concomitantly with ramiprilat and H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH (bradykinin B2 receptor antagonist HOE 140), the bioactive peptide Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin) or concomitantly with bradykinin and HOE 140. Hypoxia/reoxygenation injury to untreated control cardiac myocytes was characterized by a significant loss of sarcolemmal integrity measured at 75 +/- 4% of total cell fluorescence (mean +/- S.E., n = 42 cultures). Compared to propidium iodide staining of the above untreated control myocytes, those pretreated with 30 or 100 microM ramiprilat showed a significant reduction of propidium iodide staining to 45 +/- 9% and 40 +/- 8% (n = 9, P < 0.05) of untreated controls, respectively. Pretreatment with the protective concentrations of ramiprilat concomitant with 10 microM HOE 140 abolished the significant reduction in propidium iodide staining observed with ramiprilat alone. Similarly, pretreatment with 10 or 100 nM bradykinin significantly reduced propidium iodide staining to 35 +/- 5% and 60 +/- 10% (n = 6, P < 0.05) of the untreated hypoxic controls, respectively. In addition, concomitant pretreatment with protective concentrations of bradykinin and 10 microM HOE 140 also abolished the significant reduction in propidium iodide staining observed with bradykinin alone. The results indicate that the angiotensin-converting enzyme inhibitor ramiprilat has a protective effect on isolated cardiac myocytes exposed to hypoxia/reoxygenation and that this effect is most likely related to a local action of bradykinin on the cardiac myocyte via the activation of the kinin B2 receptor.
Analysis of Variance↗
Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives.
Propidium has been demonstrated in previous studies to be a selective ligand for the peripheral anionic site on acetylcholinesterase (EC 3.1.1.7). Its association with this site can be advantageously monitored by direct fluorescent titration. We have measured the ability of acetylcholine, acetylthiocholine, haloxon [di-(2-chloroethyl)3-chloro-4-methylcoumarin-7-ylphosphate] , and a coumarin derivative (3-chloro-7-hydroxy-4-methylcoumarin) to dissociate propidium from the peripheral anionic site of Torpedo californica acetylcholinesterase. Measurements were made by back-titration of propidium after complete inhibition of the active center with diisopropylfluorophosphate. Both acetylcholine and acetylthiocholine show substrate inhibition at high substrate concentrations. The concentrations required for occupation of the peripheral site, as ascertained by competition with propidium, correlated well with the concentration dependence for the kinetics of substrate inhibition. These observations are consistent with substrate inhibition being due to binding of acetylcholine or acetylthiocholine at a peripheral anionic site. Displacement of propidium by haloxon and coumarin indicated that these inhibitors also bind to the peripheral anionic site. The dissociation constants ascertained from peripheral site occupation are in agreement with the constants obtained from inhibition kinetics. Evidence is presented that competition with propidium obtained by direct fluorescence titrations, when combined with inhibition kinetics, provides a more reliable means for ascertaining site selectivity of various inhibitors than does a kinetic analysis alone.
Acetylcholine↗
Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures.
Excessive activation of excitatory amino acid receptors has been implicated in the neuronal degeneration caused by ischemia, hypoglycemia, and prolonged seizures. We have observed directly the time course and regional vulnerability of hippocampal neurons to glutamate receptor-mediated injury in organotypic hippocampal cultures, a preparation which combines accessibility and long-term survival with preservation of regional differentiation and neuroanatomic organization. Cultures were incubated with the fluorescent dye propidium iodide which selectively enters and stains cells only after membrane damage. After 5 to 10 min of a 30-min exposure to kainate (100 microM), large neurons in the hilus of the dentate were first to become brightly fluorescent. Propidium staining subsequently appeared in the other regions of the hippocampus and increased to a maximum over the first 6 h of recovery. NMDA (10 microM) caused propidium staining that was limited to CA1 and the dentate gyrus of the cultures, sparing CA3, consistent with the regions of highest NMDA receptor density in vivo. Glutamate (1 mM) caused a delayed, progressive pattern of staining that began in CA1 (2 to 4 h after exposure), then extended to include CA3 and finally the dentate gyrus over the next 24 h. Release of LDH activity into the media was slower and less sensitive than propidium staining. Histologic degeneration was limited to neurons 24 h after agonist exposure and was consistent with the propidium staining. NMDA, kainate, and glutamate each produced a unique pattern of neuronal injury. Most notably, glutamate had low potency as a toxin and its pattern of neuronal injury was not reproduced by NMDA.
Animals↗
A 24 h corticosterone exposure exacerbates excitotoxic insult in rat hippocampal slice cultures independently of glucocorticoid receptor activation or protein synthesis.
Elevations in circulating concentrations of glucocorticoids (GC) may increase the expression and/or sensitivity of ionotropic transmitter receptors in brain. For example, recent evidence suggests that acute and chronic GC exposure may alter the number and/or function of N-methyl-D-aspartate (NMDA)-type glutamate receptors, effects that may sensitize the brain to excitotoxic insults. The present studies examined the ability of short-term (24 h) corticosterone (CORT) exposure to potentiate NMDA-induced cytotoxicity in rat hippocampal slice cultures. Additional studies evaluated the role of mineralocorticoid (MR) and glucocorticoid receptor (GR) function, as well as de novo protein synthesis, in potentiation of toxicity by corticosterone exposure. Hippocampal slice cultures were exposed to NMDA (20 microM) for 24 h with cytotoxicity assessed by fluorescent detection of propidium iodide uptake. Exposure to NMDA caused significant propidium iodide uptake in each hippocampal region, while 24 h CORT (0.001-1 microM) exposure alone did not significantly increase propidium iodide uptake. Co-exposure of cultures to CORT and NMDA synergistically increased propidium iodide uptake in each hippocampal region, effects that were prevented by co-exposure to a non-toxic concentration of MK-801 (20 microM). In contrast, 24 h exposure with the MR antagonist spironolactone (1-10 microM), the GR antagonist RU-486 (1-10 microM), or the protein synthesis inhibitor cycloheximide (1 microM) failed to reduce the significant increase in propidium iodide uptake. These data suggest that relatively brief elevations in CORT levels may sensitize the hippocampus to injury independently of GC receptor activity and protein synthesis.
Analysis of Variance↗
DAPI (4',6-diamidino-2-phenylindole) binds differently to DNA and RNA: minor-groove binding at AT sites and intercalation at AU sites.
The interaction of DAPI and propidium with RNA (polyA.polyU) and corresponding DNA (polydA.polydT) sequences has been compared by spectroscopic, kinetic, viscometric, Tm, and molecular modeling methods. Spectral changes of propidium are similar on binding to the AT and AU sequences but are significantly different for binding of DAPI. Spectral changes for DAPI with the DNA sequence are consistent with the expected groove-binding mode. All spectral changes for complexes of propidium with RNA and DNA and for DAPI with RNA, however, are consistent with an intercalation binding mode. When complexed with RNA, for example, DAPI aromatic protons signals shift significantly upfield, and the DAPI UV-visible spectrum shows significantly larger changes than when complexed with DNA. Slopes of log kd (dissociation rate constants) versus-log [Na+] plots are similar for complexes of propidium with RNA and DNA and for the DAPI-RNA complex and are in the range expected for an intercalation complex. The slope for the DAPI-DNA complex, however, is much larger and is in the range expected for a groove-binding complex. Association kinetics results also support an intercalation binding mode for the DAPI-RNA complex. The viscosity of polyA.polyU solutions increases significantly on addition of both propidium and DAPI, again in agreement with an intercalation binding mode for both molecules with RNA. Molecular modeling studies completely support the experimental findings and indicate that DAPI forms a very favorable intercalation complex with RNA. DAPI also forms a very stable complex in the minor groove of AT sequences of DNA, but the stabilizing interactions are considerably reduced in the wide, shallow minor groove of RNA. Modeling studies,thus,indicate that DAPI interaction energetics are more favorable for minor-groove binding in AT sequences but are more favorable for interaction in RNA.
Adenine Nucleotides↗
Dynamic recording of cell death in the in vitro dorsal vagal nucleus of rats in response to metabolic arrest.
Anoxic/ischemic neuronal death is usually assessed in cell cultures or in vivo within a time window of 24 h to several days using the nucleic acid stain propidium iodide or histological techniques. Accordingly, there is limited information on the time course of such neuronal death. We loaded acute rat brain stem slices with propidium iodide for dynamic fluorometric recording of metabolic arrest-related cell death in the dorsal vagal nucleus. This model was chosen because dorsal vagal neurons show a graded response to metabolic inhibition: anoxia and aglycemia cause a sustained hyperpolarization, whereas ischemia induces a glutamate-mediated, irreversible depolarization. We found that the number of propidium iodide-labeled cells increased from 27% to 43% of total cell count within 1-7 h after preparation of slices. Compared with these untreated control slices, cyanide-induced anoxia (30 min) or aglycemia (1 h) did not cause further cell death, whereas 3-h aglycemia destroyed an additional 13% of cells. Ischemia (1 h) due to cyanide plus iodoacetate immediately labeled an additional 20% of cells, and an additional 48% of cells were destroyed within the following 3 h of postischemia. Continuous recording of propidium iodide fluorescence showed that loss of membrane integrity started within 25 min after onset of the ischemic depolarization and the concomitant intracellular Ca(2+) rise. The results show that propidium iodide can be used to monitor cell death in acute brain slices. Our findings suggest that pronounced cell death occurs within a period of 1-4 h after onset of metabolic arrest and is apparently due to necrotic/oncotic mechanisms.
Animals↗
Effects of changes in blood flow rate on cell death and cell proliferation in carotid arteries of immature rabbits.
Spontaneous and experimental changes in arterial blood flow rates affect tissue accumulation in developing arteries. To examine whether cell proliferation and/or cell death are affected by alterations in blood flow, we ligated the left external carotid artery of 3-week-old rabbits, which reduces left common carotid blood flow by 71%. In control arteries and after 2 days of flow reduction, agarose gel electrophoresis of DNA extracted from all carotid arteries resolved multiple low molecular weight bands characteristic of apoptosis; however, DNA fragmentation in arteries carrying reduced blood flow was 2.5-fold higher than that of control arteries. The effect of reduced blood flow on cell death subsequently waned but remained significant at 7 days. Cell death in carotid arteries was also detected by in vivo uptake of propidium iodide, a DNA-binding fluorescent dye that labels the nuclei of nonviable cells. Both smooth muscle and endothelial cells exhibited large and statistically significant increases in labeling index in the flow-reduced artery. Propidium iodide-labeled cells were cleared from the vessel wall within 1 to 4 hours of labeling, and nuclear staining displayed condensation (clumping) of chromatin in all labeled cells at later time points. This time course and nuclear morphology and the rapid clearance of labeled cells are consistent with death via apoptosis. Many propidium iodide-positive cells did not display chromatin condensation immediately after labeling; however, this was also true of cultured endothelial cells that were driven into apoptosis with sphingomyelinase treatment and then double-labeled with propidium iodide and the apoptosis marker annexin V. We infer that propidium iodide can label apoptotic vascular cells before these cells display chromatin condensation that is detectable with fluorescence labeling of DNA. Replication rates of smooth muscle and endothelial cells, determined by 5-bromo-2'-deoxyuridine uptake, were inhibited by >75% with decreased blood flow. The inhibition of proliferation was unabated after 7 days of reduced flow. These findings indicate that the coordinated regulation of cell death and cell proliferation, in response to changes in arterial blood flow rates, contributes to arterial remodeling during development.
Animals↗
A microfluorometric viability assay for isolated human and rat islets of Langerhans.
A microfluorometric viability assay for isolated human and rat islets of Langerhans has been developed using the fluorochromes fluorescein diacetate and propidium iodide. Fluoroscein diacetate causes live cells to fluoresce green under blue light excitation (490 nm); propidium iodide causes dead cells to fluoresce red under green light excitation (545 nm). The fluorescence intensity from the live and dead cells within a single islet was selectively measured by photometry using 520 nm and 610 nm barrier filters with blue and green light excitation respectively. All measurements were corrected for background fluorescence. It was necessary to incubate single islets with the fluorochrome mixture for 105 min in order to achieve maximum fluorescence intensity. It was found that when 50 microliters of a fluorochrome mixture containing 0.67 mumol/l fluorescein diacetate and 4.0 mumol/l propidium iodide was incubated with a single islet and the fluorescence from live (blue light excitation) and dead (green light excitation) cells measured, then the proportion of dead cells within the islet was equal to the (propidium iodide fluorescence)--(0.04 x fluorescein fluorescence) divided by the sum of the (fluorescein fluorescence) and (propidium iodide fluorescence--(0.04 x fluorescein fluorescence]. The proportion of dead cells within single human or rat islets measured by microfluorometry was found to correlate highly significantly (r = 0.99, p less than 0.001) with the proportion of dead cells measured by dissociating the same islet into a single cell suspension and counting the actual proportion of dead cells. This assay therefore provides a rapid, accurate and objective measurement of the proportion of dead cells within isolated human and rat islets.
Animals↗
Neutrophil adhesion to vascular prosthetic surfaces triggers nonapoptotic cell death.
OBJECTIVE: To test the hypothesis that neutrophil adhesion to expanded polytetrafluoroethylene (ePTFE) and Dacron triggers cell death. SUMMARY BACKGROUND DATA: Vascular prosthetic infections are intransigent clinical dilemmas associated with excessive rates of death and complications. Impaired neutrophil function has been implicated in the infection of implanted cardiovascular devices. ePTFE and Dacron are potent neutrophil stimuli able to elicit activation responses such as reactive oxygen species production independent of exogenous/soluble agonists. Reactive oxygen species that are released into the medium when neutrophils are challenged by soluble agonists are known to cause self-destruction. The authors therefore sought to examine whether neutrophil adhesion to prosthetic graft materials decreases neutrophil viability by means of reactive oxygen species production. METHODS: Neutrophils were adhered to surfaces for up to 6 hours. Cell viability was monitored with propidium iodide staining and lactate dehydrogenase release. RESULTS: Within 6 hours of adhesion to ePTFE and Dacron, respectively, 59% +/- 11% and 44% +/- 5% (n = 7) of the neutrophils were stained by propidium iodide. Indistinguishable results were obtained with plasma-coated ePTFE and Dacron. In contrast, less than 2% of the neutrophils adherent to fibrinogen-, immunoglobin-, or fetal bovine serum-coated polystyrene surfaces for 6 hours were positive for propidium iodide. The increase in membrane permeability to propidium iodide was accompanied by a two- to threefold increase in lactate dehydrogenase release. Pretreatment of neutrophils with N-acetyl-L-cysteine, cytochalasin D, or cyclosporin A significantly reduced the number of propidium iodide-positive ePTFE and Dacron adherent neutrophils. CONCLUSIONS: Neutrophil adhesion to ePTFE and Dacron triggers a rapid nonapoptotic cell death. The effect of ePTFE and Dacron on neutrophil viability appears to be caused by reactive oxygen species production. The premature death of graft-adherent neutrophils provides a novel explanation of the defect in neutrophil bacterial killing associated with vascular prosthetic grafts.
Blood Vessel Prosthesis↗
The regional vulnerability to hypoglycemia-induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK-801.
Profound hypoglycemia selectively damages CA1 and the dentate gyrus of the hippocampus. We have examined the time course of hippocampal neuronal injury in organotypic cultures following in vitro "hypoglycemia," using the fluorescent vital dye propidium iodide to observe directly the regional distribution of early neuronal membrane injury in living cultures. The in vivo hippocampal pattern of hypoglycemic injury was reproduced by a 2 hr exposure to glucose-free media, which resulted in simultaneous, selective propidium staining of CA1 and the dentate gyrus starting by 4 hr after exposure. After 24 hr of recovery, CA3 remained spared. A similar pattern of propidium staining was produced by incubation of cultures for briefer periods in glucose-free medium containing 5 mM 2-deoxyglucose (2-DG) to inhibit glycolysis. This "hypoglycemic" pattern and time course of neuronal injury was mimicked by 300 microM aspartate but not by glutamate. The NMDA receptor antagonists MK-801 and CPP, but not the relatively selective non-NMDA receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione, prevented the development of propidium staining. MK-801 protected against injury even if added to the recovery media 30 min after the insult, while TTX (10 microM) protected only if added by the end of the exposure. The appearance of propidium staining after 4-6 hr of recovery was well correlated with histological observation of pyknotic neuronal nuclei in the injured regions. The characteristic hippocampal regional vulnerability of CA1 and the dentate gyrus to injury following profound hypoglycemia can be reproduced in organotypic hippocampal culture and appears to be mediated both by an early TTX-sensitive component and by a more prolonged period of toxic NMDA receptor activation, extending for at least 30 min into the recovery period.
Animals↗
Site selectivity of fluorescent bisquaternary phenanthridinium ligands for acetylcholinesterase.
The synthesis of decidium and hexidium diiodides, their spectroscopic properties, and association with acetylcholinesterase from Torpedo californica are described and compared with those for propidium. Decidium, hexidium, and propidium, bisquaternary analogs of the fluorescent phenanthridinium ligand ethidium, contain 10, 6, and 3 methylene carbons, respectively, interposed between the exocyclic and endocyclic quaternary nitrogens. The three ligands exhibit linear competitive inhibition of enzyme carbamylation by N-methyl-7-dimethylcarbamoxyquinolinium. Dissociation constants for decidium, hexidium, and propidium are found by direct fluorescence titration to be 2.1 +/- 0.2 X 10(-8), 5.8 +/- 1.4 X 10(-7), and 3.7 +/- 0.4 X 10(-6) M, values in close accord with the inhibition constants obtained from kinetic analyses. Association of the three ligands is characterized by a stoichiometry of one fluorescent ligand per 80-kDa molecular weight subunit and occurs with respective 6.5-, 4.5-, and 3-fold increases in both quantum yield and fluorescence lifetime. Decidium and hexidium, in marked contrast with propidium, are dissociated by ligands selective for the active center and undergo pronounced reduction in affinity upon modification of the active center with pyrenebutyl methylphosphonofluoridate. Whereas the kinetics reveal no clear distinctions in inhibitory action of the three ligands, the fluorescence studies indicate that the alkyltrimethylammonium moiety of decidium and hexidium occludes the active center; propidium, in contrast, associates solely with the peripheral anionic site and does not occlude the active center. The temperature dependence of binding indicates that decidium association engenders a substantial increase (+55 eu) in entropy. The data indicate that the active center and peripheral anionic sites are separated by a crevice which can accommodate the hydrocarbon portion of extended n-alkyl cationic ligands, thereby affording entropic stabilization of complex formation. This stabilization is realized, however, only when the anionic subsite of the active center is not occluded, enabling electrostatic interaction between cationic ligand and the anionic active center.
Acetylcholinesterase↗
A method to detect leakage of DNA intercalators through liposome membranes.
A method to detect leakage of water-soluble intercalators such as ethidium bromide and propidium iodide from liposomes (egg-lecithin and egg-lecithin/cholesterol) by means of fluorescence is presented. The addition of excess DNA outside the liposomes brings about intercalation of released dye with the DNA. The increase in fluorescence intensity that results from the dye-DNA complex is used to detect the degree of dye release. Titration and surfactant-induced leakage measurements display the suitability of the method. Spontaneous leakage measurements show that propidium has much lower leaking rates than those of ethidium. This is probably due to the less lipophilicity of propidium as estimated from the partition coefficients between octanol and medium. Stabilizing the egg-lecithin membranes with cholesterol strongly reduces the leaking rates for ethidium while no effect is detected for the slow leaking rates of propidium. Lowering the temperature from 25 to 6 degrees C results in lower leaking rates for both dyes.
Cholesterol↗
Ethanol-acetylcholinesterase-inhibitor interactions: inhibitor hydrophobicity and site specificity dependence.
1. Depending on the hydrophobicity and the site specificity of an inhibitor, striking differences were found in ethanol-acetylcholinesterase (AChE)-inhibitor interactions. 2. AChE used was from electric eel and was purified by affinity chromatography. 3. Ethanol at 10-200 mM reduced the inhibitory ability of tetrabutylammonium bromide (Bu4NBr). 4. The observed reduction might be a result of Bu4NBr inhibition being partially compensated for by an ethanol activation effect. 5. In contrast to Bu4NBr, propidium and edrophonium are not involved in hydrophobic interaction with AChE. 6. Their abilities to inhibit AChE activity were enhanced by ethanol. 7. Such an enhancement could not result from combining individual perturbations from ethanol and propidium or edrophonium, since ethanol itself increased the AChE activity. 8. In the presence of ethanol, propidium which binds to the peripheral site of the enzyme remained as an uncompetitive inhibitor, while edrophonium which binds to the active site was changed from a competitive inhibitor to a mixed one. 9. The effect of ethanol was therefore greater in the inhibitor which is involved with the active-site binding. 10. Fluorescence quenching studies of propidium-bound enzyme and edrophonium-bound enzyme revealed that ethanol in the concentration less than or equal to 400 mM did not cause significant conformational change at both the peripheral and the active sites of the enzyme.
Acetylcholinesterase↗
Corticosterone and dexamethasone potentiate cytotoxicity associated with oxygen-glucose deprivation in organotypic cerebellar slice cultures.
Many patients display elevated levels of serum cortisol following acute ischemic stroke. Given that glucocorticoids may potentiate some forms of insult, these studies examined the effects of corticosterone or dexamethasone exposure on cytotoxicity following oxygen-glucose deprivation in the cerebellum, a brain region susceptible to stroke. In organotypic cerebellar slice cultures prepared from neonatal rat pups, 90-min of oxygen-glucose deprivation at 15 days in vitro resulted in significant cytotoxicity at 24-, 48-, and 72-h post-oxygen-glucose deprivation, as measured by uptake of propidium iodide. Exposure of cultures following oxygen-glucose deprivation to the antioxidant trolox (500 microM), but not to the glucocorticoid receptor antagonist RU486 (10 microM), completely blocked oxygen-glucose deprivation-induced cytotoxicity. Corticosterone (1 microM) or dexamethasone (10 microM) exposure alone did not significantly increase propidium iodide uptake above levels observed in control cultures. However, corticosterone or dexamethasone exposure after oxygen-glucose deprivation potentiated oxygen-glucose deprivation-mediated propidium iodide uptake at each time point. Trolox, as well as RU486, co-exposure of cultures to corticosterone or dexamethasone after oxygen-glucose deprivation abolished all cytotoxicity. In conclusion, these data demonstrated that glucocorticoid exposure modulated oxygen-glucose deprivation-mediated propidium iodide uptake, which likely involved glucocorticoid receptor activation and pro-oxidant effects.
Animals↗