calyculin-a and Necrosis

We examined the possibility that p38 mitogen-activated protein kinase and caspase-3 would be activated for execution of apoptosis and excitotoxicity, the two major types of neuronal death underlying hypoxicischemic and neurodegenerative diseases. Mouse cortical cell cultures underwent widespread neuronal apoptosis 24 h following exposure to 10-30 nM calyculin A, a selective inhibitor of Ser/Thr phosphatase I and IIA. Activity of p38 was increased 2-4 h following exposure to 30 nM calyculin A. Addition of 3-10 microM PD169316, a selective p38 inhibitor, partially attenuated calyculin A neurotoxicity. Activity of caspase-3-like proteases was increased in cortical cell cultures exposed to 30 nM calyculin A for 8-16 h as shown by cleavage of DEVD-p-nitroanilide and phosphorylated tau. Proteolysis of tau was completely blocked by addition of 100 microM N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), a broad-spectrum inhibitor of caspases, but incompletely by 10 microM PD169316. Calyculin A neurotoxicity was partially sensitive to 100 microM z-VAD-fmk. Cotreatment with 10 microM PD169316 and 100 microM z-VAD-fmk showed additive neuroprotection against calyculin A. Neither PD169316 nor z-VAD-fmk showed a beneficial effect against excitotoxic neuronal necrosis induced by exposure to 20 microM NMDA. Thus, caspase-3-like proteases and p38 likely contribute to calyculin A-induced neuronal apoptosis but not NMDA-induced neuronal necrosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspases; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Drug Synergism; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Fetus; Imidazoles; Marine Toxins; Mice; Mitogen-Activated Protein Kinases; N-Methylaspartate; Necrosis; Neurons; Neurotoxins; Oxazoles; p38 Mitogen-Activated Protein Kinases; Receptors, N-Methyl-D-Aspartate; tau Proteins

Microcystin-induced ser/thr protein phosphatase (PP) inhibition and toxicity were examined in the little skate (Raja erinacea), an evolutionarily primitive marine vertebrate. As in mammals, PP inhibition and toxicity were exclusively hepatocellular, but were much more persistent in the skate. A dose of 63 microg/kg given iv to adult male skates resulted in the near complete inhibition of hepatic PP activity at 24 h. PP activity was still 95% inhibited 7 days after dosing in skates given 125 microg/kg microcystin. Mortality occurred at doses of 500 microg/kg or more. Hepatic lesions were only seen in animals with fully inhibited PP activity in liver. The histological changes seen at 125 microg/kg were mild periportal inflammatory changes increasing in severity together with hepatocyte necrosis at higher doses of microcystin. Microcystin persisted and could be detected in plasma up to 7 days after dosing. This finding shows that, in the skate, as in mammals, the liver is the only organ capable of uptake of microcystin, since there was no significant inhibition of PP activity in the rectal gland and small decreases in PP activity of the kidney that were not time or dose dependent. In vitro microcystin caused dose-dependent inhibition of PP activity in isolated skate hepatocytes, while it was without effect in cultured rectal glands. Uptake of microcystin and the accompanying inhibition of PP activity in skate hepatocytes was prevented by the addition of a series of organic dyes and bile acids. The spectrum of inhibitors of microcystin uptake in skate is similar to that seen in the rat, indicating common features of the carrier(s) in these diverse species.

Topics: Animals; Cell Adhesion; Cell Size; Cells, Cultured; Cholic Acids; Coloring Agents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Hemorrhage; Kidney; Liver; Male; Marine Toxins; Microcystins; Necrosis; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Salt Gland; Sharks; Skates, Fish

The cytotoxic effects of anthrax lethal toxin purified from an avirulent strain were examined on mouse macrophage-like J774A.1 cells. Cell death induced by high concentration of purified lethal toxin had the characteristics of necrosis. At lower concentrations, the toxin caused no morphological change and most of the cells were viable. Interestingly, apoptotic cells were observed when the cells were preincubated with a serine/threonine phosphatase inhibitor, calyculin A, and then exposed to a toxin concentration of 0.1 microg/ml. This is the first report that lethal toxin of the anthrax bacillus can induce both necrosis and apoptosis and that protein phosphatases are implicated in the regulation of bacterial toxin-induced apoptosis.

Topics: Animals; Apoptosis; Bacillus anthracis; Bacterial Toxins; Macrophages; Marine Toxins; Mice; Necrosis; Oxazoles; Phosphoprotein Phosphatases

8-Cl-cAMP and 8-NH2-cAMP induced MCF-7 cell death. The type(s) of cell death were studied in more detail and compared with the cell death type (apoptosis) induced by okadaic acid, an inhibitor of serine/threonine phosphatases. By morphological criteria dying cells showed loss of cell-cell interactions and microvilli, condensation of nuclear chromatin and segregation of cytoplasmic organelles. By in situ nick end-labelling, using digoxigenin-conjugated dUTP as probe, a large fraction of 8-Cl-cAMP, 8-NH2-cAMP and 8-Cl-adenosine-exposed cells stained positively in the advanced stages of death. In the early phase of chromatin condensation the cells stained negatively. Specific (internucleosomal) DNA fragmentation was not observed. The MCF-7 cell death induced by 8-Cl-cAMP and 8-NH2-cAMP was not mediated by activation of the cAMP kinase since more stable cAMP analogues (8-CPT-cAMP and N6-benzoyl-cAMP) or forskolin failed to induce death. Furthermore, 8-Cl-cAMP action was counteracted by adenosine deaminase and 3-isobutyl-1-methylxanthine, and mimicked by 8-Cl-adenosine, a major metabolite of 8-Cl-cAMP. It is concluded that 8-Cl- and 8-NH2-cAMP can induce morphological and biochemical effects resembling apoptotic cell death in MCF-7 cells through their conversion into potent cytotoxic metabolite(s).

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Adenocarcinoma; Adenosine Deaminase; Amino Acid Sequence; Apoptosis; Biotransformation; Breast Neoplasms; Chromatin; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA Damage; Ethers, Cyclic; Female; Humans; Marine Toxins; Microvilli; Molecular Sequence Data; Necrosis; Okadaic Acid; Organelles; Oxazoles; Phosphoprotein Phosphatases