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Maitotoxin induces calpain activation in SH-SY5Y neuroblastoma cells and cerebrocortical cultures
1Laboratory of Neuro-biochemistry, Department of Neuroscience Therapeutics and Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan, 48105, USA.
Abstract:
Maitotoxin (MTX) is a highly potent marine toxin that activates both voltage-sensitive and receptor-operated calcium channels in the plasma membrane. This results in calcium overload that rapidly leads to cell death. We now report that maitotoxin (0.1-1 nM) induces calpain activation in both SH-SY5Y neuroblastoma cells and fetal rat cerebrocortical cultures. MTX-induced calpain activation was confirmed by the presence of autolytic fragmentation of both subunits of calpain. Secondly, the formation of calpain-produced alpha-spectrin breakdown products (150 and 145 kDa) was observed. We were also able to detect intracellular hydrolysis of a peptide substrate (succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin) by activated calpain in MTX-treated cells. Calpain inhibitors (calpain inhibitor I, MDL28170 and PD150606) inhibited spectrin breakdown and SLLVY-AMC hydrolysis in MTX-treated SY5Y cells. Our results suggest that (i) calpain is activated as a result of the maitotoxin-induced calcium influx; and (ii) coupling with the in situ calpain assays, maitotoxin would be a useful tool in investigating the physiologic and pathophysiologic roles of calpain in neuronal cells.
Insights
Maitotoxin (MTX) activates calpain, a calcium-dependent protease, in neuronal cells. This study demonstrates MTX as a tool to investigate calpain
Area of Science:
- Neuroscience
- Toxicology
- Cell Biology
Background:
- Maitotoxin (MTX) is a potent marine toxin that triggers calcium influx into cells.
- Calcium overload can lead to cell death.
- Calpains are calcium-dependent proteases implicated in various cellular processes.
Purpose of the Study:
- To investigate whether maitotoxin (MTX) induces calpain activation in neuronal cells.
- To explore the role of calcium influx in MTX-induced calpain activation.
- To assess the utility of MTX in studying calpain in neuronal contexts.
Main Methods:
- Treatment of SH-SY5Y neuroblastoma cells and fetal rat cerebrocortical cultures with MTX (0.1-1 nM).
- Assessing calpain activation via autolytic fragmentation and alpha-spectrin breakdown product formation.
- Measuring intracellular hydrolysis of a peptide substrate (SLLVY-AMC) by activated calpain.
- Utilizing calpain inhibitors (MDL28170, PD150606) to confirm calpain's role.
Main Results:
- MTX (0.1-1 nM) induced calpain activation in both cell types.
- Confirmed MTX-induced calpain activation by observing calpain subunit fragmentation and alpha-spectrin breakdown products (150 and 145 kDa).
- Detected intracellular SLLVY-AMC hydrolysis, indicating active calpain in MTX-treated cells.
- Calpain inhibitors blocked MTX-induced spectrin breakdown and substrate hydrolysis.
Conclusions:
- Calpain activation is a consequence of maitotoxin-induced calcium influx in neuronal cells.
- Maitotoxin serves as a valuable tool for in situ investigation of calpain's physiological and pathophysiological roles in neurons.