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(From GHAFOORI P et al. , ONCOLOGY. Vol. 22 No. 1, 2008.)

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(From GHAFOORI P et al. , ONCOLOGY. Vol. 22 No. 1, 2008.)

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  1. Calcineurin mediates enhanced high-voltage-activated calcium currents in rat primary cortical neurons after acute hypoxia K. Xiang, E.I. Tietz, L.J.Greenfield JrDept. of Internal Medicine, Neurology and Physiology/Pharmacology, Univ. of Toledo College of Medicine, Toledo, OH.Resident symposium April 2010

  2. (From GHAFOORI P et al., ONCOLOGY. Vol. 22 No. 1, 2008.)

  3. Acute oxygen-sensing mechanisms.Weir EK, López-Barneo J, Buckler KJ, Archer SL. N Engl J Med. 2005 Nov 10;353(19):2042-55. • The response of the smooth-muscle cells in the pulmonary arteries to acute hypoxia begins within seconds and involves inhibition of potassium current, membrane depolarization, and calcium entry through L-type calcium channels; it also involves calcium release from the sarcoplasmic reticulum and calcium repletion through store-operated channels.

  4. Voltage-Gated Calcium Channels

  5. Primary cultures of rat cortical neurons • Primary cortical neuron culture: 13-15 days in vitro culture from E18 fetal rats. • Hypoxic exposure with 1% O2 , 94%N2 and 5%CO2 for 4h; normoxic exposure (controls) with 95% air and 5% CO2. • Recordings were conducted within 2h of termination of hypoxia exposure or within ±2 hours after 48h recovery.

  6. Whole-cell Electrophysiology from Purves et al., 1997

  7. Fig. 1. HVA Ca2+ currents increased immediately after hypoxia

  8. Inactivation of VGCC Point mutations in the IQ motif of 77WT affect Ca2+-dependent inactivation. Nature 399, 159 - 162 (13 May 1999); doi:10.1038/20200 Neuron. 1999 Mar;22(3):549-58.

  9. Fig. 2. Inactivation of HVA Ca2+ currents unchanged after hypoxia

  10. Fig. 3. HVA Ca2+currents unchanged after 48 h normoxic recovery

  11. Hypoxia and Calcineurin • Calcineurin (CaN, also termed protein phosphatase 2B) is a phosphatase broadly distributed throughout the body. • Calcineurin promotes hypoxia-inducible factor 1alpha expression by dephosphorylating RACK1 and blocking RACK1 dimerization. (Liu et al., 282(51):37064-73. J Biol Chem. 2007) • Full activation of phosphatase activity requires the binding of Ca2+ /calmodulin (CaM) to the catalytic A subunit of CaN with concurrent binding of Ca2+ to the regulatory CaN B subunit.

  12. Calcineurin regulation of neuronal plasticity.Rachel D. Groth, Robert L. Dunbar and Paul G. MermelsteinBiochemical and Biophysical Research Communications 311-4, 2003, P1159-1171 • Through direct dephosphorylation or disinhibition of PP1, CaN influences a diverse array of cellular proteins. • Green arrows indicate activating/enhancing responses; red arrows indicate inhibitory modulation.

  13. Evaluation of calcineurin in VGCC regulation after hypoxia • FK-506 (Tacrolimus)and Cyclosporin A (CsA) are structurally distinct immunosuppressive agents that specifically inhibit calcineurin activity by binding to separate, endogenously expressed immunophilins. FK-506 binds to FKBP-12, while CsA binds to cyclophilin A. • Okadaic acid is a relatively specific inhibitor of protein phosphatases 1 and 2A and exhibits little potency toward calcineurin at drug concentrations of ≤1 μM. • Rapamycin (Sirolimus) is an immunosuppressant that is similar in structure to FK-506 and competes for binding to FKBP-12. However, unlike the FK-506/FKBP-12 complex, the rapamycin/FKBP-12 complex does not bind to and inhibit calcineurin. Thus, rapamycin is an advantageous agent for separating FK-506’s actions on immunophilins from its actions on calcineurin. From Norris et al. (2002)Neuroscience.

  14. Fig. 4. FK506 and CsA reversed the transient HVA Ca2+ current enhancement after hypoxia

  15. Fig. 5. Okadaic acid rapamycin and did not reverse the post-hypoxic enhancement of HVA Ca2+ currents

  16. Summary & Conclusions • High-voltage activated (HVA) Ca2+ currents were increased ~1.5-fold immediately after 4 h exposure to 1% O2 but returned to baseline after 48 h normoxic recovery. • The half-maximal potentials of activation and steady-state inactivation were unchanged. • The calcineurin inhibitor FK506 (5 mM in the recording pipette) reversed the post-hypoxic increase in VGCC current. • Exposure to a structurally different calcineurin inhibitor, cyclosporine A (20 mM), during hypoxia blocked the increase in VGCC current. • Rapamycin, a FK506 analog that does not block calcineurin activity, failed to reverse the post-hypoxic increase in VGCC current. • Okadaic acid, an inhibitor of PP1 and PP2A, failed to prevent the post-hypoxic increase in VGCC current, suggesting that VGCC regulation is calcineurin-specifc. • In summary, hypoxia transiently upregulated HVA VGCC currents in primary cortical neurons via a calcium dependent process involving calcineurin, suggesting a positive feedback loop to amplify neuronal calcium signaling after hypoxia.

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