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

Authors


Address correspondence and reprint requests to Dr James G. McLarnon, Department of Pharmacology and Therapeutics, Faculty of Medicine, 2176 Health Sciences Mall, The University of British Columbia, Vancouver, BC Canada, V6T 1Z3. E-mail: Mclarnon@interchange.ubc.ca

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.

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