KEY WORDShuman microglia; adenosine triphosphate; P 2Y and P 2X receptors; COX-2, store-operated channel Ca 2ϩ entry
ABSTRACTWe have studied modulation of purinergic receptors (P 2Y and P 2X subtypes) on changes in intracellular Ca 2ϩ [Ca 2ϩ ] i and expression and production of COX-2 in human microglia. Measurements using Ca 2ϩ -sensitive spectrofluorometry showed adenosine triphosphate (ATP) to cause rapid transient increases in [Ca 2ϩ ] i . Application of ATP plus the P 2X antagonist, pyridoxal-phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS), or treatment with adenosine diphosphate--S (ADP--S), a selective P 2Y agonist, led to a considerable prolongation in [Ca 2ϩ ] i responses compared with ATP. The prolonged time courses were consistent with sustained activation of store-operated channels (SOC) since SKF96365, an inhibitor of SOC, blocked this component of the response. RT-PCR data showed that microglia expressed no COX-2 either constitutively or following treatment of cells with ATP (100 M for 8 h). However, treatment using ATP plus PPADS or with ADP--S led to marked expression of COX-2. The enhanced COX-2 with ATP plus PPADS treatment was absent in the presence of SKF96365 or using Ca 2ϩ -free solution. Immunocytochemistry, using a specific anti-COX-2 antibody, also revealed a pattern of purinergic modulation whereby lack of P 2X activation enhanced the production of COX-2 protein. These results suggest that modulation of subtypes of purinergic receptors regulates COX-2 in human microglia with a link involving SOCmediated influx of Ca 2ϩ .