Independent signaling pathways in ATP-evoked secretion of plasminogen and cytokines from microglia

We investigated the action of ATP on the secretion of plasminogen, TNF-α, and IL-6 from microglia. ATP (10-100 μM) stimulated the release of plasminogen from rat cultured microglia in a concentration-dependent manner with a peak response at 5-10 min after the stimulation. The release was dependent on extracellular Ca²⁺ and was blocked by pretreatment with oxidized ATP, a blocker of P2X₇. UTP, an agonist of P2Y₂, also stimulated the release of plasminogen from a subpopulation (about 20% of total cells) of cultured microglia. The release was also dependent on extracellular Ca²⁺, suggesting a role of stocker-operated calcium entry (SOC). ATP potently stimulated TNF-α release from 2 h after the stimulation with TNF-α mRNA expression in primary cultures of rat brain microglia. The TNF-α release was maximally elicited by 1 mM ATP and 2′- and 3′-O-(4-benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP), a P2X₇ selective agonist, suggesting the involvement of P2X₇. This TNF-α release was correlated with a sustained Ca²⁺ influx. The release was inhibited by PD98059, an inhibitor of MEK1 which activates extracellular signal-regulated protein kinase (ERK), and SB203580, an inhibitor of p38 MAP kinase. However, both ERK and p38 were rapidly activated by ATP even in the absence of extracellular Ca²⁺. These results indicate that extracellular ATP triggers TNF-α release in rat microglia via P2X₇ in a manner dependent on the sustained Ca²⁺ influx and via the ERK/p38 cascade independently of Ca²⁺ influx. ATP caused the mRNA expression and release of IL-6 in a concentration-dependent manner in MG-5. The physiological meaning of these independent release mechanisms is also discussed.