Inhibition of lipopolysaccharide-induced cyclooxygenase-2, tumor necrosis factor-α and [Ca2+]i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195

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-alpha (TNF-alpha). PK11195 (at 50 mum) significantly inhibited the LPS-induced up-regulation of both inflammatory factors; at a lower concentration of PK11195 (2 mum) expression of TNF-alpha, but not COX-2, was reduced. Production of both factors, using immunocytochemistry for COX-2 and ELISA for TNF-alpha, was markedly reduced with 50 mum 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-alpha 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-alpha and that modulation of Ca2+ -mediated signaling pathways could be involved in the anti-inflammatory actions.