calyculin-a and 1-norokadaone

1. Phosphorylation by kinases and dephosphorylation by phosphatases markedly affect the biological activity of proteins involved in stimulus-response coupling. In this study, we have characterized the effects of okadaic acid, an inhibitor of protein phosphatases 1 and 2A, on insulin secretion. Mouse pancreatic islets were preincubated for 60 min in the presence of okadaic acid before their function was studied. 2. Okadaic acid dose-dependently (IC50 approximately 200 nM) inhibited insulin secretion induced by 15 mM glucose. At 0.5 microM, okadaic acid also inhibited insulin secretion induced by tolbutamide, ketoisocaproate and high K+, and its effects were not reversed by activation of protein kinases A or C. 3. The inhibition of insulin secretion did not result from an alteration of glucose metabolism (estimated by the fluorescence of endogenous pyridine nucleotides) or a lowering of the ATP/ADP ratio in the islets. 4. Okadaic acid treatment slightly inhibited voltage-dependent Ca2+ currents in beta cells (perforated patch technique), which diminished the rise in cytoplasmic Ca2+ (fura-2 method) that glucose and high K+ produce in islets. However, this decrease (25%), was insufficient to explain the corresponding inhibition of insulin secretion (90%). Moreover, mobilization of intracellular Ca2+ by acetylcholine was barely affected by okadaic acid, whereas the concomitant insulin response was decreased by 85%. 5. Calyculin A, another inhibitor of protein phosphatases 1 and 2A largely mimicked the effects of okadaic acid, whereas 1-norokadaone, an inactive analogue of okadaic acid on phosphatases, did not alter beta cell function. 6. In conclusion, okadaic acid inhibits insulin secretion by decreasing the magnitude of the Ca2+ signal in beta cells and its efficacy on exocytosis. The results suggest that, contrary to current concepts, both phosphorylation and dephosphorylation of certain beta cell proteins may be involved in the regulation of insulin secretion.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Calcium Channels; Cytoplasm; Energy Metabolism; Enzyme Inhibitors; Ethers, Cyclic; Exocytosis; Female; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Marine Toxins; Mice; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases

Glucocorticoids induce apoptosis in murine T cell hybridomas. It was inhibited by okadaic acid and calyculin A, potent inhibitors of protein phosphatase 1 and 2A, but not by 1-norokadaone, a structural analog of okadaic acid without phosphatase inhibitory activity. The inhibitory effect of okadaic acid was significant even when it was added 9 h after the start of the culture. Okadaic acid did not prevent either the translocation of glucocorticoid receptor from the cytoplasm to the nucleus or the induction of luciferase activity in the T cell hybridoma transfected with a plasmid containing the luciferase gene under the control of glucocorticoid response elements. These results indicate that protein dephosphorylation is an essential step for glucocorticoid-induced apoptosis in T cell hybridomas, and that the step is at the late stage of the apoptotic process.

Topics: Animals; Apoptosis; Binding Sites; Dexamethasone; DNA; Electrophoresis, Agar Gel; Ethers, Cyclic; Hybridomas; Luciferases; Marine Toxins; Mice; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Plasmids; Protein Phosphatase 1; Receptors, Glucocorticoid; T-Lymphocytes; Transfection