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Keywords:

  • cyclooxygenase-2;
  • human microglia;
  • intracellular calcium;
  • lipopolysaccharide;
  • mitochondrial effector;
  • PK11195;
  • tumor necrosis factor-α

Abstract

The anti-inflammatory actions of the mitochondrial peripheral benzodiazepine receptor (PBR) agonist PK11195 [1-(2-chloro- phenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide] were investigated in human microglia. Application of the microglial inflammatory stimulus lipopolysaccharide (LPS, at 100 ng/mL for 3 h), induced enhancement of the expressions of the inducible enzyme, cyclooxygenase-2 (COX-2) and the pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α). PK11195 (at 50 µm) significantly inhibited the LPS-induced up-regulation of both inflammatory factors; at a lower concentration of PK11195 (2 µm) expression of TNF-α, but not COX-2, was reduced. Production of both factors, using immunocytochemistry for COX-2 and ELISA for TNF-α, was markedly reduced with 50 µm of PK11195 added to LPS solution. Acute application of LPS induced a transient increase in intracellular Ca2+[Ca2+]i exhibiting both a slow development and recovery in kinetic behavior. This increase in [Ca2+]i consisted primarily of a Ca2+ influx component accompanied by a smaller mobilization from intracellular Ca2+ stores. In the presence of PK11195, the amplitude of the [Ca2+]i response induced by LPS was reduced by 54%. Another mitochondrial agent cyclosporin A (CsA), which also acts at the permeability transition pore (PTP) of mitochondrial membrane but at a site different from the PBR, was ineffective in reducing either the LPS-induced expression of COX-2 and TNF-α or the endotoxin increase in [Ca2+]i. These results indicate that the mitochondrial effector PK11195 is a specific and effective agent for inhibiting LPS-induced microglial expressions of COX-2 and TNF-α and that modulation of Ca2+-mediated signaling pathways could be involved in the anti-inflammatory actions.