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Pentylenetetrazole-induced chemoshock affects protein kinase C and substrate proteins in mouse brain
1Department of Pharmacology, College of Medicine, National Taiwan University, Taipei.
Abstract:
Protein kinase C (PKC) activity, western blot analysis of PKC alpha, beta, gamma, epsilon, and zeta by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentylenetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 mM KCl. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 mM KCl eluates from DE-52 columns contain more PKC alpha and beta in both cytosol and membrane than the 200 mM KCl eluates, whereas PKC gamma, epsilon, and zeta appear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 mM KCl eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC gamma isozyme. As for novel-type epsilon and zeta isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 mM KCl from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 mM KCl eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKC gamma isoform were involved in the pentylenetetrazole-induced chemoshock.
Insights
Pentylenetetrazole-induced chemoshock in mice increased Protein Kinase C (PKC) gamma activity and neuromodulin phosphorylation. These changes in PKC signaling pathways are linked to chemoshock development.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Protein Kinase C (PKC) isozymes play crucial roles in neuronal signaling.
- Pentylenetetrazole (PTZ)-induced chemoshock is a model for studying seizure activity and associated molecular changes in the brain.
Purpose of the Study:
- To investigate the changes in PKC isozyme activity and their endogenous substrates in the mouse brain following PTZ-induced chemoshock.
- To elucidate the specific PKC isoforms and substrates involved in the chemoshock response.
Main Methods:
- Western blot analysis using isozyme-specific antibodies to quantify PKC alpha, beta, gamma, epsilon, and zeta.
- Partial purification of PKC isozymes and substrates from cytosolic and membrane fractions using DE-52 column chromatography.
- In vitro phosphorylation assays to assess the activity of endogenous substrate proteins like neurogranin and neuromodulin.
- Autoradiography and SDS-PAGE to identify and quantify specific substrate proteins.
Main Results:
- PTZ-induced chemoshock led to increased kinase activity in both cytosolic and membrane fractions, primarily associated with the PKC gamma isozyme.
- The content of PKC gamma increased significantly in chemoshocked mice, while novel PKC isozymes (epsilon and zeta) remained unchanged.
- Phosphorylation of the endogenous substrate neuromodulin (43 kDa) was elevated in chemoshocked mice, with neurogranin (17 kDa) showing different elution profiles.
- PKC alpha and beta were predominantly found in lower KCl eluates, while PKC gamma, epsilon, and zeta appeared in both lower and higher KCl eluates.
Conclusions:
- Increased PKC gamma content and enhanced neuromodulin phosphorylation are key molecular events implicated in pentylenetetrazole-induced chemoshock.
- Specific PKC isoforms and their substrates exhibit differential responses to chemoshock, highlighting the complexity of PKC signaling in seizure disorders.
- DE-52 chromatography provides an effective method for separating PKC isozymes and their substrates, aiding in the study of their roles in neurological conditions.