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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Blockade of chloride conductance antagonizes PMA-induced ramification in the murine microglial cell line, BV-2
Susanna Zierler1, Hubert H Kerschbaum
1Division of Animal Physiology, Department of Cellular Biology, University of Salzburg, Hellbrunnerstr. 34, 5020 Salzburg, Austria.
Abstract:
In microglial cells, activation of ion channels and ion transporters is associated with the transformation from an amoeboid to a ramified phenotype and vice versa. In the present study, we evaluated the contributions of protein kinase C (PKC) activity and ion conductance to the phorbol 12-myristate 13-acetate (PMA)-dependent ramification in the murine microglial cell line, BV-2. In a first set of experiments, we showed that PMA, a commonly used activator of PKC, but not the bioinactive analog 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), induces ramification in BV-2 cells. Surprisingly, the PKC inhibitors calphostin C, chelerythrine, or bisindolylmaleimide II did not antagonize PMA-induced ramification. In a further set of experiments, we found that 4,4'-diisocyanatostilbene-2,2' disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), which block chloride channels and K-Cl cotransporters, and SKF 96365, a non-selective ion channel blocker, consistently suppressed PMA-induced ramification in BV-2 cells. Additional ion channel blockers, including lanthanides, amiloride, Ba2+, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), and flufenamic acid did not affect PMA-induced ramification in BV-2 cells. Cs+ accentuated the PMA-dependent ramification in BV-2 cells. Thus, our results indicate (1) that a PMA-binding protein, excluding PKC isoforms, is critical in structural remodeling of microglial cells and (2) that chloride conductance plays a pivotal role in induction of ramification in microglial cells.
Insights
Phorbol 12-myristate 13-acetate (PMA) induces microglial cell ramification, but not through protein kinase C (PKC). Chloride conductance, not PKC, is critical for this structural change in BV-2 cells.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Microglial cell morphology, specifically the transformation between amoeboid and ramified phenotypes, is linked to ion channel and transporter activity.
- Understanding the molecular mechanisms driving microglial structural changes is crucial for comprehending their function in the central nervous system.
Purpose of the Study:
- To investigate the roles of protein kinase C (PKC) activity and ion conductance in phorbol 12-myristate 13-acetate (PMA)-induced microglial ramification.
- To identify specific ion channels or transporters involved in PMA-mediated structural remodeling of BV-2 microglial cells.
Main Methods:
- Utilized the murine microglial cell line BV-2 for experiments.
- Administered PMA and its inactive analog (4 alpha-PDD) to induce morphological changes.
- Applied various PKC inhibitors (calphostin C, chelerythrine, bisindolylmaleimide II) and ion channel/transporter blockers (DIDS, SITS, SKF 96365, lanthanides, amiloride, Ba2+, NPPB, flufenamic acid, Cs+).
- Observed and quantified cell morphology changes (ramification).
Main Results:
- PMA induced significant ramification in BV-2 cells, while 4 alpha-PDD did not.
- PKC inhibitors failed to block PMA-induced ramification, suggesting a PKC-independent mechanism.
- Blockers of chloride channels and K-Cl cotransporters (DIDS, SITS) and a non-selective ion channel blocker (SKF 96365) suppressed PMA-induced ramification.
- Other tested ion channel blockers had no effect, and Cs+ enhanced ramification.
Conclusions:
- A PMA-binding protein, independent of PKC isoforms, is essential for microglial structural remodeling.
- Chloride ion conductance plays a critical role in mediating PMA-induced microglial ramification.
- These findings highlight novel pathways regulating microglial morphology and function.
