ATP stimulated the release of plasminogen in a concentration-dependent manner from 10 μM to 100 μM, with a peak response at 5–10 min after the stimulation (Inoue et al., 1998). A 1-h 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 (0.3–10 μM). ATP induced a transient increase in the [Ca2+]i in a concentration-dependent manner, which was very similar to the ATP-evoked plasminogen release. 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). The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+. 2-Methylthio-ATP was effective (7 out of 7 cells), but α,β-methylene ATP was ineffective (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. A 1-h 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). These data suggest that ATP may transit information from neurons to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor, which stimulates the release of plasminogen from the microglia. It has been found that UTP also stimulates plasminogen release from a subpopulation of microglia (about 20% of total cells), presumably through store-operated Ca2+ entry (SOC) activated by ATP stimulation of G protein-coupled receptors, since the release evoked by UTP was also dependent on extracellular Ca2+ (K. Inoue and S. Kohsaka, unpublished data).
We found that ATP evokes the release of IL-6 at 24 h in a concentration-dependent manner (10–1,000 μM) in MG-5 (Ohsawa et al., 1997; Shigemoto-Mogami et al., 2001) and that the release was observed from 6 h after stimulation with ATP. Neither ADP (1,000 μM), UTP (1,000 μM) nor adenosine (1,000 μM) stimulated the release of IL-6. There is a possibility that ATP might evoke the release of IL-6 secondarily by releasing TNF-α and IL-1β because TNF-α and IL-1β are reported to stimulate IL-6 production in other glial cells (Norris et al., 1994). In MG-5, a significant amount of TNF-α appeared at 1 h and peaked at 3 h after the stimulation by 1,000 μM ATP, but TNF-α (≤10,000 pg/ml) never evoked the release of IL-6 in MG-5. This result was in accord with a previous report that TNF-α failed to stimulate IL-6 production in microglia (Sawada et al., 1992). Moreover, the release of IL-1β, a potent inducer of IL-6 gene (Lee et al., 1993), was not evoked by 1,000 μM ATP in MG-5 (K. Inoue et al., unpublished data).
ATP induced an approximately 7-fold increase in the expression of IL-6 mRNA, which was inhibited by 1 mM suramin to 5.6 ± 6.9% of ATP alone, indicating that ATP stimulates the de novo synthesis of IL-6 via a P2 receptor–mediated pathway and the subsequent production of IL-6. Previous studies with IL-1β-stimulated synoviocytes indicated that p38 activation leads to IL-6 gene expression through enhancement of IL-6 mRNA stability (Miyazawa et al., 1998). However, in our experiment, p38 appeared to act on the initial ATP signal transduction pathway affecting IL-6 gene expression, since no significant decrease of IL-6 mRNA expression was observed when SB203580, an inhibitor of p38, was added 3 or 5 h after the ATP application. The finding that the ATP-evoked phosphorylation of p38 was rapid, i.e., peaking at 1 min and almost disappearing at 60 min after ATP stimulation, may also exclude the possibility that p38 activation leads to increased IL-6 expression via its effects on IL-6 mRNA stability.
ATP could activate two distinct MAP kinases, i.e., ERK1/2 and p38, in MG-5. ADP activated ERK1/2 strongly but p38 only slightly. ATP stimulated IL-6 release but ADP did not. The ATP-stimulated IL-6 release was inhibited by SB203580 but not by an inhibitor of ERK1/2. These results strongly suggest that p38 but not ERK1/2 MAP kinase is responsible for the IL-6 release.
Phosphorylation by ATP of p38 was dependent on the extracellular Ca2+. ATP produced a phospholipase C (PLC)-dependent transient Ca2+ release by inositol-1,4,5-trisphosphate (InsP3), which was followed by sustained Ca2+ entry via both SOC and P2X7 receptors. Several groups have reported that P2X7 receptors have a central role in the production of cytokines in microglia (Ferrari et al., 1996, 1997b; Hide et al., 2000). In fact, BzATP evoked sustained Ca2+ entry via P2X7 receptors, leading to the phosphorylation of p38 in MG-5. However, BzATP induced only a very small amount of IL-6 production in the cells. Brilliant Blue G (≤10 μM), a specific P2X7 antagonist (Jiang et al., 2000), did not inhibit the release of IL-6 induced by ATP from MG-5. As opposed to the release of TNF-α from microglia, P2Y, rather than P2X7 receptors, seem to have a major role in the IL-6 production by the cells. This idea is supported by the most recent observation that ATP may evoke IL-6 production in bacterial endotoxin lipopolysaccharide (LPS)-primed P2X7-deficient mice (P2X7R−/−) (Solle et al., 2001). However, the fact that high ATP concentrations are required for the IL-6 production may still support the idea that P2X7, rather than P2Y receptors, might be involved in the responses. Very recently, it has been reported that ATP acting on P2Y receptors stimulated the release of IL-12 in human dendritic cells (Wilkin et al., 2001). The ATP concentrations required for this IL-12 induction were similar to those required for IL-6 production in MG-5. Thus, there seems to be much variety among the subclass of P2Y receptors in their sensitivity to ATP. The deduced P2Y receptors in MG-5 would therefore require high ATP concentrations for their activation. In addition, when cells were incubated with either NEM, an inhibitor of some G-proteins (Rhee et al., 2000), or U73122, the ATP-evoked IL-6 production was abolished. Thus, activation of PLC-linked P2Y receptors and their receptor-mediated signals including p38 would trigger the IL-6 synthesis in MG-5.
The activation of p38 is not sufficient for the IL-6 induction because BzATP activated p38, but it did not evoke the release of IL-6. This result would predict the existence of additional signals for the IL-6 production. A Ca2+-dependent PKC may be an additional signal, since the ATP-evoked IL-6 production was attenuated by Gö6976, an inhibitor of Ca2+-dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to PTX and was linked to phospholipase C. Some transcriptional factors, such as NF-κB p65 (RelA) (Ferrari et al., 1997c), Jun, and Fos (Neary et al., 1996), are known to be activated by ATP. Berghe et al. (1998) described the involvement of MAP kinase pathways in NF-κB transactivation, which leads to the induction of IL-6 gene expression. Although such transcriptional factors may work as the downstream signals of either p38 or Ca2+-dependent PKC, the detailed mechanism underlying such a cooperative regulation of IL-6 production remains to be clarified.
Finally, we tried to determine the subclass of P2Y receptors involved in the ATP-evoked responses. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that P2Y1, P2Y2, and P2Y6, but not P2Y4 mRNAs are present in the cells. However, the pharmacological profile for recombinant P2Y1, P2Y2, and P2Y6 receptors is inconsistent with our findings on the IL-6 gene expression by P2 agonists, as neither ADP (a potent agonist to P2Y1) nor UTP (an activator of P2Y2 and P2Y6 receptors) stimulated significant IL-6 gene expression in MG-5. This finding suggests that the properties of the P2Y receptors coupled to IL-6 gene expression in MG-5 differ from those of the recombinant P2Y receptors or that MG-5 express novel ATP-preferring receptors coupled to IL-6 production.
Although the definitive classification of P2Y receptors for the ATP-evoked IL-6 release remains to be determined, it is suggested that ATP, acting on the PTX-insensitive P2Y receptors linked to PLC, stimulates the synthesis of IL-6 through pathways involving p38 and Ca2+-dependent PKC. This is a novel pathway for the induction of cytokines in microglia.