okadaic-acid and calmidazolium

Excitotoxic cell death (ECD) is characteristic of mammalian brain following min of anoxia, but is not observed in the western painted turtle following days to months without oxygen. A key event in ECD is a massive increase in intracellular Ca(2+) by over-stimulation of N-methyl-d-aspartate receptors (NMDARs). The turtle's anoxia tolerance may involve the prevention of ECD by attenuating NMDAR-induced Ca(2+) influx. The goal of this study was to determine if protein phosphatases (PPs) and intracellular calcium mediate reductions in turtle cortical neuron whole-cell NMDAR currents during anoxia, thereby preventing ECD. Whole-cell NMDAR currents did not change during 80 min of normoxia, but decreased 56% during 40 min of anoxia. Okadaic acid and calyculin A, inhibitors of serine/threonine PP1 and PP2A, potentiated NMDAR currents during normoxia and prevented anoxia-mediated attenuation of NMDAR currents. Decreases in NMDAR activity during anoxia were also abolished by inclusion of the Ca(2+) chelator -- BAPTA and the calmodulin inhibitor -- calmidazolium. However, cypermethrin, an inhibitor of the Ca(2+)/calmodulin-dependent PP2B (calcineurin), abolished the anoxic decrease in NMDAR activity at 20, but not 40 min suggesting that this phosphatase might play an early role in attenuating NMDAR activity during anoxia. Our results show that PPs, Ca(2+) and calmodulin play an important role in decreasing NMDAR activity during anoxia in the turtle cortex. We offer a novel mechanism describing this attenuation in which PP1 and 2A dephosphorylate the NMDAR (NR1 subunit) followed by calmodulin binding, a subsequent dissociation of alpha-actinin-2 from the NR1 subunit, and a decrease in NMDAR activity.

Topics: Animals; Calcium; Calmodulin; Cerebral Cortex; Egtazic Acid; Female; Hypoxia; Imidazoles; Marine Toxins; Okadaic Acid; Oxazoles; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Protein Phosphatase 1; Pyrethrins; Receptors, N-Methyl-D-Aspartate; Turtles

Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca2+/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca2+/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca2+-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca2+, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.

Topics: Animals; Antiemetics; Calcium; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Calmodulin-Binding Proteins; Calponins; Carcinogens; Carotid Arteries; Chelating Agents; Diglycerides; Egtazic Acid; Electrophoresis; Enzyme Inhibitors; Imidazoles; Microfilament Proteins; Okadaic Acid; Organ Culture Techniques; Peptide Fragments; Phorbol 12,13-Dibutyrate; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Staurosporine; Sulfonamides; Swine; Trifluoperazine; Vasodilator Agents

We have provided functional and molecular evidence for the presence of Na+/Ca2+ exchange in isolated porcine cortical thick ascending limb (CTAL) cells. The present studies were designed to show that this exchange activity may be modulated by phosphorylative processes. Control of intracellular Ca2+ concentration ([Ca2+]i) was determined in isolated CTAL cells with microfluorescence. CTAL cells were pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) from 10 to 20 mM. These cells had normal basal [Ca2+]i (79 +/- 3 nM). Substitution of extracellular NaCl (50 mM) with KCl resulted in the rapid elevation of [Ca2+]i to maximal levels of 795 +/- 60 nM (n = 17). The increments of [Ca2+]i were associated with [Na+]i. We next determined the modulation of Na+/Ca2+ exchange activity with phosphorylative inhibitors. Pretreatment of cells with calmidazolium, a Ca(2+)-calmodulin inhibitor, resulted in a shift of the [Na+]i dependence curve to the right. Pretreatment with okadaic acid, a phosphatase 1 and 2A inhibitor, increased the Na+/Ca2+ exchanger activity resulting in half-maximal [Ca2+]i increase near normal [Na+]i of 12 mM. Furthermore, in the presence of okadaic acid in normal CTAL cells, pretreatment with ouabain and the elevation of [Na+]i was not required to elicit increments in [Ca2+]i. These data indicate that Na+/Ca2+ exchange is present in CTAL cells and the exchange activity appears to be modulated, directly or indirectly, by phosphorylation events.

Topics: Animals; Carrier Proteins; Cells, Cultured; Enzyme Inhibitors; Epithelial Cells; Epithelium; Ethers, Cyclic; Imidazoles; Intracellular Membranes; Loop of Henle; Okadaic Acid; Osmolar Concentration; Sodium; Sodium-Calcium Exchanger; Swine

Cytoplasmic dynein is a microtubule-binding protein which is considered to serve as a motor for retrograde organelle movement. In cultured fibroblasts, cytoplasmic dynein localizes primarily to lysosomes, membranous organelles whose movement and distribution in the cytoplasm have been shown to be dependent on the integrity of the microtubule cytoskeleton. We have recently identified conditions which lead to an apparent dissociation of dynein from lysosomes in vivo, indicating that alterations in membrane binding may be involved in the regulation of retrograde organelle movement (Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579-588). Both brief serum withdrawal and low extracellular calcium levels induced this alteration, and the effect was reversed upon addition of serum or additional calcium. Here we demonstrate that the phosphorylation state of the dynein molecule is correlated with changes in its intracellular distribution in normal rat kidney fibroblasts. Dynein heavy chain phosphorylation level increased during serum starvation, and decreased back to control levels upon subsequent addition of serum. We found that okadaic acid, a phosphoprotein phosphatase inhibitor, mimicked the effects of serum starvation on both phosphorylation and the intracellular redistribution of dynein from a membrane-associated pool to one that was more soluble, with similar dose dependence for both phenomena. Cell fractionation by differential detergent extraction revealed that a higher proportion of dynein was present in a soluble pool after serum starvation than was found in comparable fractions from control cells. Our data indicate that cytoplasmic dynein is phosphorylated in vivo, and changes in phosphorylation state may be involved in a regulatory mechanism affecting the distribution of this protein among intracellular compartments.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphate; Alkaloids; Animals; Bucladesine; Calcimycin; Calmodulin; Cell Line; Colforsin; Culture Media; Cytoplasm; Dyneins; Edetic Acid; Ethers, Cyclic; Imidazoles; Kidney; Kinetics; Macromolecular Substances; Okadaic Acid; Phosphates; Phosphoprotein Phosphatases; Phosphorus Radioisotopes; Phosphorylation; Rats; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate

Apoptosis of murine thymocytes induced by either methylprednisolone or valinomycin was studied by flow cytometry. The apoptosis induced by methylprednisolone followed three stages: an initial decrease in cell volume, indicated by a fall in forward scatter accompanied by faint ethidium bromide staining, a second stage in which the cells became brightly stained by ethidium bromide, and a final stage when the cells were apparently less fluorescent as the nuclei disintegrated into apoptotic bodies. As the forward scatter of cells decreased there was a simultaneous depolarization of the cells and an elevation of intracellular calcium. These early changes preceded the fragmentation of the DNA which also preceded the intense staining of the cells by ethidium bromide. Methylprednisolone-induced apoptosis was inhibited by low concentrations (1 x 10(-7) M) of valinomycin and nonactin, neither of which could themselves induce apoptosis at these low concentrations. Cadmidazolium and cycloheximide arrested the program at an early stage. Okadaic acid allowed volume loss and ethidium bromide staining to proceed in the absence of DNA fragmentation. At high concentrations (1 x 10(-5) M) valinomycin induced a form of apoptosis, but nonactin only caused the cells to fragment. The valinomycin-induced apoptosis, although it involved the degradation of DNA and the disintegration of the nuclei into apoptotic bodies, differed from the methylprednisolone apoptosis as it did not involve a decrease of cell volume and was not inhibited by cycloheximide or affected by okadaic acid.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Cells, Cultured; Cycloheximide; DNA Damage; Ethers, Cyclic; Flow Cytometry; Imidazoles; In Vitro Techniques; Macrolides; Male; Methylprednisolone; Mice; Okadaic Acid; Thymus Gland; Valinomycin

We have demonstrated that the alpha 2,3 sialyltransferase (alpha 2,3 ST) from C6 cultured glioma cells was inhibited in vivo by W-7 and related Ca2+/Calmodulin (Ca/CaM) antagonists while protein kinase C effectors had no effect. Dephosphorylation of alpha 2,3 ST by the wide specificity alkaline phosphatase led to inactivation indicating that the enzyme is phosphorylated. The serine/threonine protein phosphatase inhibitors okadaic acid and Calyculin A led also to an inhibition of alpha 2,3 ST activity. In addition, Ca/CaM antagonists and phosphatase inhibitors led both to an inhibition of a alpha 2,3 sialoglycoprotein from C6 glioma cells as demonstrated with lectin affinity blotting. A concerted regulatory mechanism with phosphorylation/dephosphorylation of alpha 2,3 ST is then postulated.

Topics: Animals; beta-Galactoside alpha-2,3-Sialyltransferase; Calmodulin; Carbohydrate Sequence; Ethers, Cyclic; Glioma; Glycosylation; Homeostasis; Imidazoles; Kinetics; Marine Toxins; Molecular Sequence Data; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Sialyltransferases; Sulfonamides; Tumor Cells, Cultured

A protein phosphatase and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized bovine chromaffin cells. Addition of okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, or 1-naphthylphosphate, a more general phosphatase inhibitor, to digitonin-permeabilized chromaffin cells caused about a 100% increase in the amount of norepinephrine secreted in the absence of Ca2+ (in 5 mM EGTA) without affecting the amount of norepinephrine secreted in the presence of 10 microM free Ca2+. This stimulation of norepinephrine secretion by protein phosphatase inhibitors suggests that in the absence of Ca2+ there is a slow rate phosphorylation and that this phosphorylation triggers secretion. Addition of an exogenous type 2A protein phosphatase caused almost a 50% decrease in Ca(2+)-dependent norepinephrine secretion. Thus, the amounts of norepinephrine released both in the absence of Ca2+ and in the presence of Ca2+ appear to depend upon the level of protein phosphorylation.

Topics: Adrenal Medulla; Animals; Calcium; Cattle; Cell Membrane Permeability; Ethers, Cyclic; Imidazoles; Naphthalenes; Norepinephrine; Okadaic Acid; Organophosphorus Compounds; Phosphoprotein Phosphatases; Phosphorylation; Proteins; Trifluoperazine