calyculin-a and cypermethrin

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

In the present work we studied the effect of protein phosphatase inhibitors on the phosphorylation state and function of alpha(1b)-adrenoceptors. Okadaic acid increased receptor phosphorylation in a time- and concentration-dependent fashion (maximum at 30 min, EC(50) of 30 nM). Other inhibitors of protein phosphatases (calyculin A, tautomycin and cypermethrin) mimicked this effect. Staurosporine and Ro 31-8220, inhibitors of protein kinase C, blocked the effect of okadaic acid on receptor phosphorylation. Neither genistein nor wortmannin altered the effect of okadaic acid. The intense adrenoceptor phosphorylation induced by okadaic acid altered the adrenoceptor-G protein coupling, as evidenced by a small decreased noradrenaline-stimulated [(35)S]GTPgammaS binding. Okadaic acid did not alter the noradrenaline-stimulated increases in intracellular calcium or the production of inositol trisphosphate. Our data indicate that inhibition of protein phosphatases increases the phosphorylation state of alpha(1b)-adrenoceptors; this effect seems to involve protein kinase C. In spite of inducing an intense receptor phosphorylation, okadaic acid alters alpha(1b)-adrenergic actions to a much lesser extent than the direct activation of protein kinase C by phorbol myristate acetate.

Topics: Animals; Antifungal Agents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Indoles; Kinetics; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Pyrans; Pyrethrins; Rats; Receptors, Adrenergic, alpha-1; Spiro Compounds; Staurosporine; Tetradecanoylphorbol Acetate

The K(+)-Cl(-) cotransporters (KCCs) are members of the cation-chloride cotransporter gene family and fall into two phylogenetic subgroups: KCC2 paired with KCC4 and KCC1 paired with KCC3. We report a functional comparison in Xenopus oocytes of KCC1 and KCC4, widely expressed representatives of these two subgroups. KCC1 and KCC4 exhibit differential sensitivity to transport inhibitors, such that KCC4 is much less sensitive to bumetanide and furosemide. The efficacy of these anion inhibitors is critically dependent on the concentration of extracellular K(+), with much higher inhibition in 50 mm K(+) versus 2 mm K(+). KCC4 is also uniquely sensitive to 10 mm barium and to 2 mm trichlormethiazide. Kinetic characterization reveals divergent affinities for K(+) (K(m) values of approximately 25.5 and 17.5 mm for KCC1 and KCC4, respectively), probably due to variation within the second transmembrane segment. Although the two isoforms have equivalent affinities for Cl(-), they differ in the anion selectivity of K(+) transport (Cl(-) > SCN(-) = Br(-) > PO(4)(-3) > I(-) for KCC1 and Cl(-) > Br(-) > PO(4)(-3) = I(-) > SCN(-) for KCC4). Both KCCs express minimal K(+)-Cl(-) cotransport under isotonic conditions, with significant activation by cell swelling under hypotonic conditions. The cysteine-alkylating agent N-ethylmaleimide activates K(+)-Cl(-) cotransport in isotonic conditions but abrogates hypotonic activation, an unexpected dissociation of N-ethylmaleimide sensitivity and volume sensitivity. Although KCC4 is consistently more volume-sensitive, the hypotonic activation of both isoforms is critically dependent on protein phosphatase 1. Overall, the functional comparison of these cloned K(+)-Cl(-) cotransporters reveals important functional, pharmacological, and kinetic differences with both physiological and mechanistic implications.

Topics: Animals; Barium Compounds; Biological Transport; Bumetanide; Carrier Proteins; Chlorides; Ethylmaleimide; Furosemide; Humans; K Cl- Cotransporters; Kinetics; Marine Toxins; Mice; Okadaic Acid; Oocytes; Oxazoles; Phosphoprotein Phosphatases; Potassium; Protein Isoforms; Protein Phosphatase 1; Pyrethrins; Recombinant Proteins; Rubidium; Sharks; Symporters; Xenopus laevis