Brevetoxin-induced phosphorylation of Pyk2 and Src in murine neocortical neurons involves distinct signaling pathways

Abstract

Brevetoxins (PbTx-1 to PbTx-10) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis. Brevetoxins bind to site 5 of the α-subunit of voltage-gated sodium channels (VGSCs) and augment Na + influx. In neocortical neurons brevetoxins elevate intracellular Ca 2+ and augment NMDA receptor signaling. In this study, we explored the effects of PbTx-2 on Pyk2 and Src activation in neocortical neurons. We found that both Pyk2 and Src were activated following PbTx-2 exposure. PbTx-2-induced Pyk2 Tyr402 phosphorylation was dependent on elevation of Ca 2+ influx through NMDA receptors. Moreover, Pyk2 Tyr402 phosphorylation was also found to require PKC activation inasmuch as RO-31-8425 and GF 109203x both attenuated the response. In contrast, PbTx-2-induced Src Tyr416 phosphorylation involved a Gq-coupled receptor inasmuch as U73122, a specific PLC inhibitor, abolished the response. This Gq-coupled receptor appears to be mGluR 5. The PKCδ inhibitor rottlerin abolished PbTx-2-induced Src activation demonstrating that this isoform of PKC is involved in the activation of Src by PbTx-2. Considered together these data suggest that although activation of neuronal Pyk2 and Src result from PbTx-2 stimulation of VGSC, engagement of these two non-receptor tyrosine kinases involves distinct signaling pathways.

Mechanisms of Pyrethroid Insecticide-Induced Stimulation of Calcium Influx in Neocortical Neurons

“…All animal use protocols were approved by the Creighton University Institutional Animal Care and Use Committee. Primary cultures of neocortical neurons were obtained from embryonic day 16 Swiss-Webster mice as described by Cao et al (2007). In brief, pregnant mice were euthanized by CO 2 asphyxiation, embryos were removed from the dam, and their neocortices were collected.…”

Sodium Channel Activation Augments NMDA Receptor Function and Promotes Neurite Outgrowth in Immature Cerebrocortical Neurons

“…Western blot analysis was performed in cells grown in 12-well plates as described previously (Cao et al, 2007). Cells were washed three times with Locke’s buffer and then allowed to equilibrate in Locke’s buffer for 30 min.…”

Early mitochondrial abnormalities in hippocampal neurons cultured from Fmr1 pre‐mutation mouse model

“…The sample preparation for western blot was performed as described previously (Cao et al 2007). Equal amounts (20 lg) of samples were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane by electroblotting.…”