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Thrombin induces NO release from cultured rat microglia via protein kinase C, mitogen-activated protein kinase, and
1Department of Pharmacology, Ajou University School of Medicine, Suwon, 442-721, Korea.
Abstract:
Microglia, brain resident macrophages, become activated in brains injured due to trauma, ischemia, or neurodegenerative diseases. In this study, we found that thrombin treatment of microglia induced NO release/inducible nitric-oxide synthase expression, a prominent marker of activation. The effect of thrombin on NO release increased dose-dependently within the range of 5-20 units/ml. In immunoblot analyses, inducible nitric-oxide synthase expression was detected within 9 h after thrombin treatment. This effect of thrombin was significantly reduced by protein kinase C inhibitors, such as Go6976, bisindolylmaleimide, and Ro31-8220. Within 15 min, thrombin activated three subtypes of mitogen-activated protein kinases: extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase/stress-activated protein kinase. Inhibition of the extracellular signal-regulated kinase pathway and p38 reduced the NO release of thrombin-treated microglia. Thrombin also activated nuclear factor kappaB (NF-kappaB) within 5 min, and N-acetyl cysteine, an inhibitor of NF-kappaB, reduced NO release. However, thrombin receptor agonist peptide (an agonist of protease activated receptor-1 (PAR-1)), could not mimic the effect of thrombin, and cathepsin G, a PAR-1 inhibitor, did not reduce the effect of thrombin. These results suggest that thrombin can activate microglia via protein kinase C, mitogen-activated protein kinases, and NF-kappaB but that this occurs independently of PAR-1.
Insights
Thrombin activates microglia, the brain's immune cells, leading to nitric oxide (NO) release. This activation involves protein kinase C, mitogen-activated protein kinases, and NF-kappaB signaling pathways, independent of PAR-1.
Area of Science:
- Neuroimmunology
- Cellular Signaling
Background:
- Microglia are key immune cells in the brain, becoming activated in response to injury and neurodegenerative diseases.
- Understanding microglia activation mechanisms is crucial for developing treatments for neurological disorders.
Purpose of the Study:
- To investigate the signaling pathways involved in thrombin-induced microglia activation.
- To determine the role of protease-activated receptor-1 (PAR-1) in thrombin-mediated microglial responses.
Main Methods:
- Microglia were treated with thrombin, and nitric oxide (NO) release and inducible nitric-oxide synthase (iNOS) expression were measured.
- The involvement of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and nuclear factor kappaB (NF-kappaB) was assessed using specific inhibitors and activators.
- The role of PAR-1 was evaluated using a PAR-1 agonist peptide and a PAR-1 inhibitor.
Main Results:
- Thrombin treatment dose-dependently induced NO release and iNOS expression in microglia.
- Thrombin activated extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) MAPKs, as well as NF-kappaB.
- Inhibition of ERK, p38, and NF-kappaB pathways significantly reduced thrombin-induced NO release.
- PKC inhibitors also attenuated thrombin's effect on NO release.
- Neither a PAR-1 agonist nor a PAR-1 inhibitor mimicked or blocked thrombin's effects, suggesting PAR-1 independence.
Conclusions:
- Thrombin activates microglia through PKC, MAPK, and NF-kappaB signaling pathways.
- Microglia activation by thrombin occurs independently of the PAR-1 receptor.
- These findings elucidate novel mechanisms of neuroinflammation and potential therapeutic targets.