ATP stimulation of Ca²⁺-dependent plasminogen release from cultured microglia

1. ATP (10-100 μM), but not glutamate (100 μM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 μM) nor glutamate (100 μM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 μM), which is metabolized in the cytosol to BAPTA (an intracellular Ca²⁺ chelator), completely inhibited the plasminogen release evoked by ATP (100 μM). The Ca²⁺ ionophore A23187 induced plasminogen release in a concentration-dependent manner (0.3 μM to 10 μM). 2. ATP induced a transient increase in the intracellular calcium concentration ([Ca²⁺](i)) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 μM) had no effect on [Ca²⁺](i) (70 out of 70 cells) in microglial cells. A second application of ATP (100 μM) stimulated an increase in [Ca²⁺](i) similar to that of the first application (21 out of 21 cells). 3. The ATP-evoked increase in [Ca²⁺](i) was totally dependent on extracellular Ca²⁺, 2-Methylthio ATP was active (7 out of 7 cells), but α,β-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca²⁺](i). Suramin (100 μM) was shown not to inhibit the ATP-evoked increase in [Ca²⁺](i) (20 out of 20 cells). 2'- and 3'-O-(4-Benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a selective agonist of P2X₇ receptors, evoked a long-lasting increase in [Ca²⁺](i) even at 1 μM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5'-triphosphate-2', 3'-dialdehyde (oxidized ATP, 100 μM), a selective antagonist of P2X₇ receptors, blocked the increase in [Ca²⁺](i) induced by ATP (10 and 100 μM). 4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca²⁺](i) via the ionotropic P2X₇ receptor which stimulates the release of plasminogen from the microglia.