Cyclo-oxygenase-2 mediates P2Y receptor-induced reactive astrogliosis

Abstract

Excessive cyclo-oxygenase-2 (COX-2) induction may play a role in chronic neurological diseases characterized by inflammation and astrogliosis. We have previously identified an astroglial receptor for extracellular nucleotides, a P2Y receptor, whose stimulation leads to arachidonic acid (AA) release, followed, 3 days later, by morphological changes resembling reactive astrogliosis. Since COX-2 may be upregulated by AA metabolites, we assessed a possible role for COX-2 in P2Y receptor-mediated astrogliosis. A brief challenge of rat astrocytes with the ATP analogue alpha,beta-methylene ATP (alpha,beta(me)ATP) resulted, 24 h later, in significantly increased COX-2 expression. The selective COX-2 inhibitor NS-398 completely abolished alpha,beta(me)ATP-induced astrocytic activation. Constitutive astroglial COX-1 or COX-2 did not play any role in purine-induced reactive astrogliosis. PGE2, a main metabolite of COX-2, also induced astrocytic activation. These data suggest that a P2Y receptor mediates reactive astrogliosis via induction of COX-2. Antagonists selective for this receptor may counteract excessive COX-2 activation in both acute and chronic neurological diseases.

Figure 1

The experimental protocol used to assess a role for COXs in purine-mediated reactive astrogliosis. Rat striatal astrocytic cells were initially established in complete medium, placed in serum-free medium at day 1 to favour morphological differentiation resembling reactive astrogliosis, and challenged at day 2 with either purine analogues (e.g. α,βmeATP) or other agents (see text). After challenge, cells were grown for a further 3 days in drug-free medium, fixed, stained with an anti-GFAP antibody for determination of the elongation of astrocytic processes. Left panel: micrograph depicting control astrocytes with typical bipolar shape. Right panel: activated astrocytes showing increased length of astrocytic processes. In selected experiments, samples for evaluation of COX-2 by Western blot were taken at various periods after challenge with α,βmeATP, as indicated. To evaluate a role of constitutive COXs, ASA and NS-398 were added to the cultures 30 min before challenge with the purine analogue and maintained for the 2 h challenge period (short protocol). To evaluate the role of inducible COX-2, NS-398 was also maintained in the culture medium for the entire duration of the experiment (long protocol).

Figure 2

Involvement of inducible COX-2 in P2Y receptor-mediated reactive astrogliosis. (a) Cells were challenged with α,βmeATP (10 μM) for 2 h, placed in agonist-free medium, and the presence of COX-2 assessed at 6, 24, 48 and 72 h by immunoblot analysis with a specific ant-COX-2 antibody. Results (area×optical density of the 72 kDa MW COX-2 band) are expressed as mean per cent±s.e.mean of the corresponding control value from four independent experiments run in triplicate. COX-2 was significantly increased at 24 and 48 h (^*P<0.05, 1-way ANOVA, Scheffé's F-test). (b) Under the experimental conditions, a marked astrocytic activation was found, as demonstrated by increased elongation of GFAP-positive astrocytic processes (expressed as percentage of control mean μm cell⁻¹±s.e.mean; see solid column; ^*P<0.05, 1-way ANOVA, F-test). If, after challenge with the purine analogue, cells were maintained in the presence of NS-398 to selectively inhibit the inducible enzyme (long protocol, Figure 1), a concentration-dependent inhibition of α,βmeATP-induced astrocytic elongation was found (hatched columns; ^**P<0.05 vs both control and α,βmeATP alone, ^#P<0.05 vs α,βmeATP alone and not statistically different from control, 1-way ANOVA, F-test).

Figure 3

Exogenously applied PGE₂, but not PGD₂, can mimic P2Y receptor-mediated reactive astrogliosis. Cultures were challenged for 2 h with the concentrations of prostaglandins indicated, washed, grown for a further 3 days in drug-free medium, fixed, stained and assessed for elongation of the astrocytic processes. Results are the mean±s.e.mean of three independent experiments run in quadruplicate. ^*P<0.05, 1-way ANOVA, F-test.