okadaic-acid and sodium-arsenite

The mechanisms underlying cardiovascular disease induced by arsenic exposure are not completely understood. The objectives of this study were to investigate whether arsenic-fed mice have an increased vascular leakage response to vasoactive agents and whether enhanced type-2 protein phosphatase (PP2A) activity is involved in mustard oil-induced leakage. ICR mice were fed water or sodium arsenite (20 mg/kg) for 4 or 8 weeks. The leakage response to vasoactive agents was quantified using the Evans blue (EB) technique or vascular labeling with carbon particles. Increased EB leakage and high density of carbon-labeled microvessels were detected in arsenic-fed mice treated with mustard oil. Histamine induced significantly higher vascular leakage in arsenic-fed mice than in water-fed mice. Pretreatment with the PP2A inhibitor okadaic acid or the neurokinin 1 receptor (NK1R) blocker RP67580 significantly reduced mustard oil-induced vascular leakage in arsenic-fed mice. The protein levels of PP2Ac and NK1R were similar in both groups. PP2A activity was significantly higher in the arsenic-fed mice compared with the control group. These findings indicate that microvessels generally respond to vasoactive agents, and that the increased PP2A activity is involved in mustard oil-induced vascular leakage in arsenic-fed mice. Arsenic may initiate endothelial dysfunction, resulting in vascular leakage in response to vasoactive agents.

Topics: Animals; Arsenites; Blotting, Western; Capillary Permeability; Ear; Endothelium, Vascular; Enzyme Inhibitors; Evans Blue; Histamine; Isoindoles; Male; Mice; Mice, Inbred ICR; Mustard Plant; Okadaic Acid; Plant Oils; Protein Phosphatase 2; Receptors, Neurokinin-1; Sodium Compounds; Tandem Mass Spectrometry; Vascular Diseases; Vasoconstrictor Agents

The expression of the p38 subfamily of mitogen-activated protein kinases (MAPKs) was examined in rat islets of Langerhans and pancreatic beta-cell lines, and its involvement in the regulation of insulin secretion was investigated. Rat islets and several rodent beta-cell lines were shown to express p38 MAPK by Western blotting. The cellular stress agents sodium arsenite and hyperosmotic sorbitol significantly stimulated p38 MAPK activity, as did the tyrosine phosphatase inhibitor sodium pervanadate and the serine/threonine phosphatase inhibitor okadaic acid. Increases in p38 MAPK activity were not consistently correlated with increases in insulin secretion, and the dissociation between p38 MAPK activity and the regulation of insulin secretion was further demonstrated in studies using the specific p38 MAPK inhibitor SB203580, which was without significant effect on the stimulation of insulin secretion by glucose, 4beta phorbol myristate acetate and forskolin. These studies indicate that although p38 MAPK is expressed in pancreatic beta-cells and can be activated pharmacologically, its activity can be dissociated from the exocytotic release of insulin from rat islets of Langerhans.

Topics: Animals; Arsenites; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cells, Cultured; Colforsin; Enzyme Inhibitors; Glucose; Homeostasis; Imidazoles; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Male; Mitogen-Activated Protein Kinases; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Pyridines; Rats; Rats, Sprague-Dawley; Signal Transduction; Sodium Compounds; Sorbitol; Tetradecanoylphorbol Acetate; Vanadates

The phosphorylation of the C-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II has been investigated in HeLa cells exposed to heat shock. In control cells, the phosphorylated subunit, IIo, and the dephosphorylated subunit, IIa, were found in similar amounts. During heat shock, however, the phosphorylated subunit, IIo, accumulated, whereas the amount of IIa subunit decreased. Since phosphorylation of the CTD had been suggested to play a role in the initiation of transcription and since heat shock was known to perturb gene expression at the level of transcription, the phosphorylation state of RNA polymerase II was examined in cells that had been treated with various inhibitors of transcription. Under normal growth temperature, actinomycin D (over 0.1 microgram/ml) and okadaic acid, a phosphatase inhibitor, were found to inhibit polymerase dephosphorylation. Whereas 5,6-dichlorobenzimidazole riboside (DRB), N-(2-[Methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), and actinomycin D (over 5 micrograms/ml) were found to inhibit polymerase phosphorylation. Actinomycin D concentrations, which inhibited the dephosphorylation process, were lower than those required to inhibit the phosphorylation process. In contrast, during heat shock or exposure to sodium arsenite, a chemical inducer of the heat-shock response, the phosphorylated subunit, IIo, accumulated even in the presence of inhibitors of transcription such as DRB, H-8, and actinomycin D. These experiments demonstrated the existence of a heat-shock-induced CTD-phosphorylation process that might contribute to the regulation of transcription during stress.

Topics: Arsenites; Dactinomycin; Dichlororibofuranosylbenzimidazole; Ethers, Cyclic; HeLa Cells; Hot Temperature; Humans; Isoquinolines; Okadaic Acid; Phosphorylation; Protein Kinase C; RNA Polymerase II; Sodium Compounds; Transcription, Genetic

High intracellular levels of heat shock proteins and enhanced protein glycosylation are two phenomena closely associated with the cellular stress response. GP50 is the major heat-induced glycoprotein in Chinese hamster ovary (CHO) cells; however, GP50 is not well characterized, and its function is unknown. J6 is a gene originally identified in F9 murine teratocarcinoma cells after exposure to retinoic acid. In this study we show that J6 is heat-inducible and codes for a protein that shares characteristics with GP50. Western blotting of CHO cell homogenates, using a J6 polyclonal antibody, showed a single band with a molecular weight identical to that of GP50. Thermotolerant cells showed increased levels of the J6/GP50 protein. Heat-shocked CHO cells also accumulated transiently high levels of J6 mRNA between 2 and 7 h following 10 min at 45 degrees C. These induction kinetics are similar to those for GP50 labeling with D-[3H]mannose and to the activation of major heat shock genes, e.g., hsp70. Hybrid selection of J6 mRNA from CHO cells, followed by in vitro translation, produced a single band on SDS-PAGE with a molecular weight identical to that of deglycosylated GP50. Neither cellular proliferation (exponential growth versus plateau phase) nor the specific heat shock temperature (41.5 degrees C versus 45 degrees C) had significant effects on J6 induction by heat stress. Stress conditions other than hyperthermia, including ethanol, arsenite, and hypoxia, increased J6 mRNA levels. Conversely, J6 mRNA was reduced by quercetin, brefeldin A, okadaic acid, uv, and hydrogen peroxide. Our data support the hypothesis that J6 is a heat shock gene with a gene product identical to the polypeptide moiety of GP50.

Topics: Animals; Arsenites; Brefeldin A; Cell Division; Cell Hypoxia; CHO Cells; Cricetinae; Cyclopentanes; Ethers, Cyclic; Gene Expression Regulation; Heat-Shock Proteins; Hot Temperature; Hydrogen Peroxide; Kinetics; Mice; Molecular Weight; Nerve Tissue Proteins; Okadaic Acid; Protein Synthesis Inhibitors; Protein Tyrosine Phosphatases; Quercetin; RNA, Messenger; Sodium Compounds; Teratoma; Transcription, Genetic; Tretinoin; Ultraviolet Rays; X-Rays