Release of prostaglandin E(2) and nitric oxide from spinal microglia is dependent on activation of p38 mitogen-activated protein kinase.
BACKGROUND
The spinal release of prostaglandins (PGs), nitric oxide (NO), and cytokines has been implicated in spinal nociceptive processing. Microglia represent a possible cell of origin for these proexcitatory mediators. Spinal microglia possess Toll-like receptor 4 (TLR4) and neurokinin 1 (NK1) receptors, and both receptors play a significant role in peripheral nerve injury- and inflammation-induced spinal sensitization. Accordingly, we examined the properties of the cascades activated by the respective targets, which led to the release of PGE(2) and an increase in nitrite (NO(2)(-)) (a marker of NO) from cultured rat spinal microglia.


METHODS
Spinal microglia isolated from Sprague-Dawley neonatal rats were cultured with lipopolysaccharide (LPS) or substance P (SP) alone, with LPS in combination with SP, and with LPS in the presence of each inhibitor of cyclooxygenase (COX), NO synthase 2 (NOS2) or p38 mitogen-activated protein kinase (p38), or minocycline for 24 hours and 48 hours. Concentrations of PGE(2) and NO(2)(-) in culture supernatants were measured using an enzyme immunoassay and a colorimetric assay, respectively.


RESULTS
Application of LPS (a TLR4 ligand, 0.1 to 10 ng/mL) to cultured microglia produced a dose- and time-dependent increase in PGE(2) and NO(2)(-) production, whereas no effects were observed after incubation with SP (an NK1 agonist, up to 10(-5) M) alone or in combination with LPS. Antagonist studies with SC-560 (COX-1 inhibitor) and SC-236 (COX-2 inhibitor) showed that LPS-induced PGE(2) release was generated from both COX-1 and COX-2. LPS-induced NO release was suppressed by 1400W, an inhibitor of NOS2. Minocycline, an agent blocking microglial activation, and SB203580, an inhibitor of p38, both attenuated the LPS-induced PGE(2) and NO release. The 1400W, at the doses that suppressed NO release, also blocked increased PGE(2) release.


CONCLUSIONS
Our findings suggest that (a) activation of spinal microglia via TLR4 but not NK1 receptors produces PGE(2) and NO release from these cells; (b) the evoked PGE(2) release is generated by both COX-1 and COX-2, and (c) the COX-PGE(2) pathway is regulated by p38 and NOS2. Taken together with our previous in vivo work, the current findings emphasize that p38 in spinal microglia is a key player in regulating production of pronociceptive molecules, such as PGE(2) and NO.
Matsui
Tomohiro
T
Department of Laboratory Sciences, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
Svensson
Camilla I
CI
Hirata
Yuka
Y
Mizobata
Kanae
K
Hua
Xiao-Ying
XY
Yaksh
Tony L
TL
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D051545:Cyclooxygenase 1 / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D017628:Microglia / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013116:Spinal Cord / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D013373:Substance P / Q000378:metabolism; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D018040:Receptors, Neurokinin-1; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D009569:Nitric Oxide; D013373:Substance P; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496513:Ptgs1 protein, rat; C489691:Ptgs2 protein, rat; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone