Microglia respond rapidly to injury, increasing their synthesis and release of inflammatory mediators, many of which contribute to the maintenance of persistent
pain following CNS or PNS injury. We have recently shown that the lysosomal
cysteine protease Cathepsin S (CatS) expressed by spinal microglia is vital for the full expression of
neuropathic pain. Here we evaluated the mechanisms by which CatS release occurs from primary microglia in culture. Stimulation of microglia with
lipopolysaccharide (LPS) or
adenosine tri-
phosphate (
ATP) alone was insufficient to induce release of enzymatically active CatS in extracellular media. However, following priming with LPS,
ATP at 1 mM but not 50 μM resulted in significant release of CatS in the media and maturation of CatS
protein in
cell extracts. The enzymatic activity measured in media at neutral pH was specific for CatS as it was completely prevented by the CatS inhibitor LHVS.
ATP-induced release of CatS required
potassium efflux and both extracellular
calcium influx and mobilization of intracellular
calcium. Pharmacological modulation of
ATP-induced release of CatS enzymatic activity revealed that this was dependent on activation of the
P2X7 receptor and intracellular
phospholipase C and
phospholipase A(2). In addition,
ATP-induced CatS release involved
p38 mitogen activated protein kinase (MAPK) phosphorylation, but not ERK and PI3K signalling pathways. Thus, as high concentration of extracellular
ATP promotes release of active CatS from microglia via
P2X7 receptor activation, we suggest that the inhibition of CatS release is one of the mechanisms responsible for P2X7 antagonist efficacy in
neuropathic pain.