title: Involvement of nitric oxide synthase and ROS-mediated activation of L-type voltage-gated Ca2+ channels in NMDA-induced DPYSL3 degradation.
abstract: Dihydropyrimidinase-like 3 (DPYSL3), a member of TUC (TOAD-64/Ulip/CRMP), is believed to play a role in neuronal differentiation, axonal outgrowth and possibly in neuronal regeneration. Recently, we have shown that in primary cortical neurons (PCN) NMDA and oxidative stress (H(2)O(2)) caused a calpain-dependent cleavage of DPYSL3 (62 kDa) resulting in the appearance of a lower molecular weight form (60 kDa) of DPYSL3. Our preliminary results had shown that antioxidants significantly reduced NMDA-induced DPYSL3 degradation, indicating involvement of ROS in calpain activation. The aim of this study was to investigate the possible involvement of NOS in NMDA-induced DPYSL3 degradation. We found that NOS inhibitor (L-NAME) significantly prevented NMDA-induced ROS formation, as well as intracellular Ca(2+) increase [Ca(2+)](i), DPYSL3 degradation and cell death. Further, exposure of PCN to NO donor (SNP) resulted in significant [Ca(2+)](i) increase, ROS generation and probable calpain-mediated DPYSL3 truncation. The NMDA- and oxidative stress (ROS)-induced DPYSL3 truncation was totally dependent on extracellular [Ca(2+)](i). While NMDA-induced DPYSL3 truncation was blocked by both NMDA receptor antagonist (MK801) [Kowara, R., Chen, Q., Milliken, M., Chakravarthy, B., 2005. Calpain-mediated degradation of dihydropyrimidinase-like 3 protein (DPYSL3) in response to NMDA and H(2)O(2) toxicity. J. Neurochem. 95 (2), 466-474] and L-VGCC (nimodipine) inhibitors, H(2)O(2)-induced increase in [Ca(2+)](i), ROS generation and DPYSL3 truncation was blocked only by nimodipine. These results indicate that changes in Ca(2+) homeostasis resulting from ROS-dependent activation of L-VGCC are sufficient to induce probable calpain-mediated DPYSL3 truncation and demonstrate for the first time the role of ROS in the mechanism leading to glutamate-induced calpain activation and DPYSL3 protein degradation. The probable calpain-mediated DPYSL3 truncation may have significant impact on its interaction with actin and its assembly, and in turn on growth cone integrity.
authors.affiliation: National Research Council, Institute for Biological Sciences, M-54, Ottawa, Ontario, Canada K1A 0R6. Renata.Kowara@nrc-cnrc.gc.ca
authors.forename: Renata
authors.initials: R
authors.lastname: Kowara
authors.forename: Kristoffer Laser
authors.initials: KL
authors.lastname: Moraleja
authors.forename: Balu
authors.initials: B
authors.lastname: Chakravarthy
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002154:Calpain / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000196:embryology / Q000378:metabolism; D019610:Cytoprotection / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D016202:N-Methylaspartate / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D020746:Calcium Channels, L-Type; C449760:Dpysl3 protein, rat; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; D016194:Receptors, N-Methyl-D-Aspartate; D016202:N-Methylaspartate; D019001:Nitric Oxide Synthase; D002154:Calpain; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester