Differential activation of subtype purinergic receptors modulates Ca(2+) mobilization and COX-2 in human microglia.
We have studied modulation of purinergic receptors (P(2Y) and P(2X) subtypes) on changes in intracellular Ca(2+) [Ca(2+)](i) and expression and production of COX-2 in human microglia. Measurements using Ca(2+)-sensitive spectrofluorometry showed adenosine triphosphate (ATP) to cause rapid transient increases in [Ca(2+)](i). Application of ATP plus the P(2X) antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), or treatment with adenosine diphosphate-beta-S (ADP-beta-S), a selective P(2Y) agonist, led to a considerable prolongation in [Ca(2+)](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 microM for 8 h). However, treatment using ATP plus PPADS or with ADP-beta-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(2+)-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 SOC-mediated influx of Ca(2+).
Choi
Hyun B
HB
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Hong
Seok H
SH
Ryu
Jae K
JK
Kim
Seung U
SU
McLarnon
James G
JG
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001921:Brain / Q000166:cytology / Q000196:embryology; D002118:Calcium / Q000378:metabolism*; D002452:Cell Count / Q000379:methods; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D004347:Drug Interactions; D004622:Embryo, Mammalian; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D017628:Microglia / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D058913:Purinergic Agonists; D058914:Purinergic Antagonists; D012333:RNA, Messenger / Q000096:biosynthesis; D011983:Receptors, Purinergic / Q000145:classification / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000295:instrumentation; D013873:Thionucleotides / Q000494:pharmacology
D005456:Fluorescent Dyes; D007211:Indoles; D007527:Isoenzymes; D008565:Membrane Proteins; D010975:Platelet Aggregation Inhibitors; D058913:Purinergic Agonists; D058914:Purinergic Antagonists; D012333:RNA, Messenger; D011983:Receptors, Purinergic; D013873:Thionucleotides; C030812:adenosine 5'-O-(2-thiodiphosphate); C007293:DAPI; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D002118:Calcium