Biomedical subjects

M Kimland

Publications and source records attributed to M Kimland.

12 recordsLinked to original sources

PubMed1998

Cytochrome c release and caspase activation in hydrogen peroxide- and tributyltin-induced apoptosis.

The ability of H2O2 and tributyltin (TBT) to trigger pro-caspase activation via export of cytochrome c from mitochondria to the cytoplasm was investigated. Treatment of Jurkat T lymphocytes with H2O2 resulted in the appearance of cytochrome c in the cytosol within 2 h. This was at least 1 h before caspase activation was observed. TBT caused cytochrome c release already after 5 min, followed by caspase activation within 1 h. Measurement of mitochondrial membrane potential (delta psi(m)) showed that both H2O2 and TBT dissipated delta psi(m), but with different time courses. TBT caused a concomitant loss of delta psi(m) and release of cytochrome c, whereas cytochrome c release and caspase activation preceded any apparent delta psi(m) loss in H2O2-treated cells. Thus, our results suggest that different mechanisms are involved in triggering cytochrome c release with these apoptosis-inducing agents.

Apoptosis↗

PubMed1997

Disulfiram is a potent inhibitor of proteases of the caspase family.

We have recently shown that dithiocarbamate (DC) disulfides inhibit proteolytic processing of the caspase-3 proenzyme in Jurkat T lymphocytes treated with anti-CD95 (Fas/APO-1) antibody. Because the processing can be accomplished by caspase activity, we investigated the effect of DC disulfides, such as disulfiram (DSF), on active caspases. DSF showed a dose-dependent inhibition was prevented by including dithiothreitol (DTT) in the reaction buffer, thiol-disulfide exchange between inhibitor and target is suggested. Direct interaction of DSF with caspases was confirmed by its inhibition of the purified Ac-DEVD-AMC cleaving protease, caspase-3 (CPP32/apopain). An apparent rate constant (K(app)) for this inhibition was estimated to be 0.45 x 10(3)M(-1)s(-1). DSF was also observed to inhibit the purified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1 beta-converting enzyme, ICE), with a K(app) of 2.2 x 10(3) M(-1)s(-1). In this case protein mixed disulfide formation between DSF and caspase-1 was directly demonstrated using 35S-labeled DSF. The physiological disulfide GSSG was also observed to influence the activity of caspases. A glutathione buffer (5 mM) with a GSH:GSSG ratio of 9:1 decreased the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts by 50% as compared to GSH controls without GSSG. In conclusion, our study shows that caspases are quite sensitive to thiol oxidation and that DSF is a very potent oxidant of caspase protein thiol(s), being 700-fold more potent than glutathione disulfide.

Alcohol Deterrents↗

PubMed1995

Constitutive nuclear NF kappa B/rel DNA-binding activity of rat thymocytes is increased by stimuli that promote apoptosis, but not inhibited by pyrrolidine dithiocarbamate.

Rat thymocytes spontaneously undergo apoptotic death in cell culture, and are also sensitive to the induction of apoptosis by various stimuli. We show that unstimulated thymocytes constitutively express a p50-containing nuclear factor kappa B (NF kappa B)/rel DNA-binding activity in their nuclei. When the cells were fractionated by density-gradient centrifugation this activity was found to be most pronounced in immature CD4+8+ thymocytes, the cell population that undergoes selection by apoptosis in vivo and that is most sensitive to external inducers of apoptosis in vitro. The intensity of the NF kappa B/rel protein-DNA complex was significantly enhanced 30 min after exposing thymocytes to methylprednisolone or etoposide, two agents well known to induce apoptosis in these cells. Expression of this DNA-binding activity therefore correlates with the subsequent occurrence of apoptosis. By analogy to other systems, it has been suggested that antioxidants such as pyrrolidine dithiocarbamate (PDTC) inhibit thymocyte apoptosis by preventing the activation of an NF kappa B/rel transcription factor. However, we have found that etoposide induces a very similar enhancement of the NF kappa B/rel DNA-binding activity in the presence or absence of PDTC, despite a pronounced inhibition of apoptotic DNA fragmentation in the former situation. Dithiocarbamates therefore do not exert their anti-apoptotic activity in thymocytes by inhibiting the activation of this transcription factor.

Animals↗

PubMed1995

Dithiocarbamates induce apoptosis in thymocytes by raising the intracellular level of redox-active copper.

Dithiocarbamates are metal-chelating compounds that can exert either pro-oxidant or antioxidant effects in different situations. They have recently been found to potently inhibit apoptotic cell death, an activity attributed to their antioxidant action. However, when thymocytes were exposed to pyrrolidine dithiocarbamate, an oxidation of the glutathione pool occurred within 90 min. Longer incubation resulted in cell shrinkage, chromatin fragmentation, glutathione depletion, and eventual cell lysis, which is typical of apoptosis in these cells. These changes were inhibited by inclusion of non-permeable metal chelators in the incubation medium, suggesting that pyrrolidine dithiocarbamate exerts its toxic effect by transporting a redox-active metal into the cell. This was directly confirmed when sustained 8-fold elevations of intracellular copper were detected after addition of pyrrolidine dithiocarbamate. In agreement with this, supplementation of the incubation medium with submicromolar concentrations of copper significantly potentiated pyrrolidine dithiocarbamate toxicity. We conclude that pyrrolidine dithiocarbamate exerts a powerful pro-oxidant effect on thymocytes due to its ability to transport external redox-active copper into cells. The resulting increase in glutathione disulfide may also explain the temporary anti-apoptotic activity of this compound described in other systems.

Animals↗

PubMed1995

Nitrone spin traps and a nitroxide antioxidant inhibit a common pathway of thymocyte apoptosis.

Oxidative stress has recently been suggested to be a mediator of apoptotic cell death [Buttke and Sandstrom (1994) Immunology Today 15, 7-10], although evidence that this phenomenon is a widespread component of apoptosis is lacking. When rat thymocytes were exposed to the glucocorticoid methylprednisolone (MPS), a progressive increase in intracellular peroxides and a decrease in glutathione (GSH) were observed to accompany the onset of apoptosis. Using Percoll density gradients to isolate subpopulations of thymocytes at different stages of apoptosis, the increase in peroxide content was found to be restricted to apoptotic cells, while a significant depletion of GSH and reduced protein thiol was detected in both pre-apoptotic and fully apoptotic cells. To investigate the biological significance of these redox changes, the free radical spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide (TMPO), and the related nitroxide-radical antioxidant 2,2,6,6-tetramethyl-1-piperidinyl-1-oxyl (TEMPO) were tested as inhibitors of thymocyte apoptosis. The cell shrinkage and DNA fragmentation induced by four different initiators of apoptosis were reduced by each compound. TEMPO inhibition of both etoposide- and MPS-induced thymocyte DNA fragmentation was also found to correlate with an increase in intracellular GSH, providing support for the proposal that its antioxidant properties were responsible for the observed protective activity. We conclude that some form of intracellular oxidation (here measured indirectly by changes in intracellular GSH and peroxide levels) is required during thymocyte apoptosis even when this process is initiated by an agent that does not exert a direct oxidant action.

Animals↗

PubMed1994

Sensory neuropeptide-mediated bronchoconstriction of the guinea pig lung by diamide; a comparison to hydrogen peroxide.

The effect of the thiol oxidizing agent diamide on airway conductance, dynamic compliance and perfusion flow of isolated perfused and ventilated guinea pig lungs was investigated. When infused in the pulmonary circulation, diamide (100 microM) induced bronchoconstriction, but no effect on perfusion flow was observed. Although diamide exposure induced the formation of thromboxane A2, the thromboxane/prostaglandin endoperoxide receptor antagonist L-670,596 did not affect the decrease in conductance and compliance induced by diamide. Diamide induced the release of the sensory neuropeptide calcitonin gene-related peptide. The bronchoconstriction and the release of calcitonin gene-related peptide induced by diamide were abolished by capsaicin pretreatment of the guinea pigs. Combined pretreatment with the NK1 and NK2 receptor antagonists, CP-96,345 and SR-48968, attenuated the effect of diamide. Hydrogen peroxide-induced vaso- and bronchoconstriction was not affected by capsaicin-pretreatment, nor did hydrogen peroxide induce detectable release of calcitonin gene-related peptide. The results indicate that diamide activates sensory nerves and induces neuropeptide release and neurokinin receptor-mediated bronchoconstriction in the isolated perfused and ventilated guinea pig lung.

Animals↗

PubMed1994

Sulfur dioxide and sodium metabisulfite induce bronchoconstriction in the isolated perfused and ventilated guinea pig lung via stimulation of capsaicin-sensitive sensory nerves.

In this study the relationship between sulfur dioxide-induced sensory nerve activation and acute bronchoconstriction was assessed. We also studied the effects of sodium metabisulfite, an agent that is suggested to increase airway resistance via activation of sensory nerves. Sulfur dioxide (250 ppm) induced a characteristic biphasic bronchoconstriction. Concomitantly sulfur dioxide induced the release of calcitonin gene-related peptide (CGRP) from capsaicin-sensitive sensory nerves into the pulmonary circulation. In lungs of guinea pigs pretreated with a neurotoxic dose of capsaicin, the first phase of bronchoconstriction was reduced and the overflow of CGRP was not detectable. Tetrodotoxin abolished the initial phase of the bronchoconstriction induced by sulfur dioxide, indicating that a local neural reflex depending on sodium channels was operant. Inhibition of the vanilloid receptor with capsazepine slightly, although not significantly, reduced the contractile responses to sulfur dioxide. Sodium metabisulfite, when infused via the pulmonary circulation (3 mM), induced bronchoconstriction which was abolished by capsaicin pretreatment, but not significantly reduced by capsazepine. The results indicate that in the isolated guinea pig lung inhaled sulfur dioxide induces initial bronchoconstriction in part via sensory nerve activation, while other mechanisms are involved in the late effect. Sensory nerve activation appears to be the only mechanism for bronchoconstriction induced by infused sodium metabisulfite. A role for sensory nerve-mediated bronchoconstriction by sulfur dioxide or sodium metabisulfite via activation of the vanilloid receptor could not be conclusively demonstrated by this study using capsazepine.

Animals↗

PubMed1993

Perfluorooctane sulfonic acid is a potent inducer of peroxisomal fatty acid beta-oxidation and other activities known to be affected by peroxisome proliferators in mouse liver.

Perfluorooctane sulfonic acid is almost as potent as perfluorooctanoic acid in causing increases in peroxisomal fatty acid beta-oxidation, peroxisomal catalase activity, omega-hydroxylation of lauric acid, cytosolic epoxide hydrolase activity and cytosolic DT-diaphorase activity. Octane sulfonic acid was ineffective at doses used for perfluorooctane sulfonic acid and perfluorooctanoic acid. The results support the theory of co-regulation of these parameters and peroxisome proliferation. The fact that perfluorooctane sulfonic acid causes peroxisome proliferation challenges the hypothesis that the first step in this process is formation of a thioester between the proliferator (the carboxylic group) and coenzyme A.

Acyl Coenzyme A↗

PubMed1992

Intoxications with anticholinesterases: effect of different combinations of antidotes on the dynamics of acetylcholine in mouse brain.

Intoxications with organophosphorus compounds are normally treated with a mixture of atropine and an enzyme regenerating oxime. The addition of diazepam to the conventional drug therapy is reported to greatly improve the antidotal effect. The implication of the cholinergic system in such intoxications prompted us to study the effect of different combinations of antidotes on the acetylcholine (ACh) synthesizing system in mouse brain in vivo. The antidotes studied in this paper are diazepam, HI-6 and 1-hyoscyamine, the active enantiomer of atropine. Diazepam decreases the synthesis rate of ACh both when administered separately and in combination with 1-hyoscyamine and HI-6. This is in contrast to 1-hyoscyamine which, in addition to blocking muscarinic receptors, also increases the release and rate of synthesis of ACh, which probably is an unfavourable effect of the antidote. This might at least partly explain the advantage of combining 1-hyoscyamine and an oxime with diazepam in intoxications with anticholinesterases. Mice administered soman (0.75 x LD50), after pretreatment with the three-drug combination of antidotes, show no cholinergic symptoms despite a 50% increase in endogenous ACh. The rate of synthesis of ACh in these mice is in the same range as in animals administered diazepam alone. Mice administered the same dose of soman with no antidotal pretreatment suffer from severe tremor and salivation, and have a strongly reduced synthesis rate of ACh.

Acetylcholine↗

PubMed1992

Metrifonate and tacrine: a comparative study on their effect on acetylcholine dynamics in mouse brain.

Tetrahydroaminoacridine (THA) and metrifonate are cholinesterase inhibitors used in the treatment of Alzheimer's disease. In experimental animals they inhibit acetylcholinesterase activity and have been reported to increase levels of brain acetylcholine. This paper presents results from studies of their effect at two dose levels on the dynamics of acetylcholine in mouse brain. Metrifonate at two doses (10 and 30 mg/kg intraperitoneally), known to cause cholinesterase inhibition, had no effect on levels of acetylcholine or choline or on the rate of synthesis of acetylcholine. THA (3 mg/kg intraperitoneally) had no effect on levels of acetylcholine and choline but had a shortlasting decreasing effect on the synthesis rate of acetylcholine. THA (10 mg/kg intraperitoneally) increased levels of acetylcholine and choline and markedly decreased the synthesis rate of acetylcholine. At this dose, the animals showed severe cholinergic effects, e.g. tremor and salivation. It is suggested that a moderate cholinesterase inhibition in brain facilitates cholinergic nerve transmission which is obtained at a broader dose range for metrifonate than for THA.

Acetylcholine↗

PubMed1989

Immunoreactive anionic and cationic trypsin in human serum.

A simple method for the purification of anionic and cationic trypsinogen and trypsin from human pancreatic juice applying affinity chromatography on aprotinin coupled Sepharose is described together with the N-terminal amino acid sequences for both trypsinogens. In addition, enzyme-linked immunoabsorbent assay (ELISA) methods for the determination of anionic and cationic trypsin-like immunoreactivity (irAT and irCT) are described. Normal serum levels are 21.3 +/- 7.4 micrograms/l and 27.8 +/- 9.0 microgram/l for irAT and irCT respectively and the accuracy of these assays is 6-10%. In our population, the normal ratio between irCT and irAT in serum is 1.36 +/- 0.42. In normal serum trypsin-like immunoreactivity consists solely of trypsinogen. In acute pancreatitis there is an increase over normal of both irAT and irCT with a proportionally greater increase in irAT than irCT. Similar changes are also found in uremic patients.

Acute Disease↗

PubMedDate not supplied

Hydrogen peroxide-induced broncho- and vasoconstriction in the isolated perfused and ventilated guinea pig lung.

The effect of hydrogen peroxide on perfusion flow, airway conductance (Gaw) and dynamic compliance (Cdyn of isolated perfused and ventilated guinea pig lungs was investigated. Hydrogen peroxide (50 microM in the perfusion buffer) induced a decrease in Gaw and Cdyn and perfusion flow during 5 min. of exposure. Hydrogen peroxide also caused an increase in the levels of thromboxane in the perfusate of the lung. The constrictor effects as well as the formation of thromboxane were inhibited by the cyclooxygenase inhibitor ibuprofen (50 microM). The thromboxane/prostaglandin endoperoxide receptor antagonist L-670,596 (1 microM) abolished the effects of hydrogen peroxide on perfusion flow, Gaw and Cdyn, but did not affect the formation of thromboxane. The thromboxane-synthetase inhibitor carboxyheptylimidazole (100 microM) reduced both the hydrogen peroxide-induced formation of thromboxane and vaso- and bronchoconstriction, suggesting a predominant role for thromboxane A2 versus prostaglandin H2 in these effects. A role for platelet-activating factor in mediating the effect of hydrogen peroxide could not be supported, as the platelet-activating factor receptor antagonist WEB 2086 (10 microM) did not affect hydrogen peroxide induced vaso- and brochoconstriction. The results of this study show that hydrogen peroxide induces thromboxane A2 mediated vaso- and bronchoconstriction in the isolated perfused and ventilated guinea pig lung. Platelet-activating factor does not appear to play a significant role in the hydrogen peroxide-induced vaso- and bronchoconstriction. Our results also suggest that the perfused guinea pig lung is more sensitive to hydrogen peroxide than the perfused rat lung.

Animals↗