Papain was purchased from Worthington (Lake Wood, NJ, United States), FCS from Invitrogen and Fura-2 from Molecular Probes (Life Technologies, Barcelona, Spain). Culture flasks, Plastic Petri dishes and transwell chambers with 8 μm pore transparent PET membrane inserts were supplied by Falcon Becton Dickinson Labware (Franklin Lakes, NJ, United States). Antibiotics, DMEM, UTP, and AG1478 were purchased from Sigma Aldrich (St. Louis, MO, United States). PGE₂, and the EP agonists and antagonists, were from Cayman Chemical (Ann Arbor, MI, United States), while LY294002 was from Calbiochem Co. (San Diego, CA, United States). Specific antibodies against phosphor-ERK1/2 (Tyr²⁰⁴), ERK2, phosphor-Akt (Thr³⁰⁸) and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States), and anti-COX-2 was from Abcam (Cambridge, United Kingdom). Antibodies against P2Y receptors, P2Y₂ and P2Y₄ receptors, and those against the EP3 receptor were from Alomone Labs (Jerusalem BioPark, Israel), while antibodies against GAPDH were purchased from Cell Signaling Technology (Beverly, MA, United States). Secondary horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies (Dako, Denmark) were used here. All other reagents not specified were routinely supplied by Sigma, Merck (Darmstadt, Germany) or Roche Diagnostics SL (Barcelona, Spain).

All the experiments were carried out at the Complutense University of Madrid (Madrid, Spain) following the International Council for Laboratory Animal Science guidelines (ICLAS). All the procedures were approved by both the Animal Ethics Committee of the Complutense University and the Regional Authorities in Madrid (Area of Animal Protection), according to the Spanish Royal Decree RD53/2013 and the European Directive 2010/63/UE on the protection of animals used for scientific purposes. The assays were designed to minimize the number of animals used while maintaining statistical validity.

Primary cultures of astrocytes were prepared as described previously with some modifications (Jimenez et al., 1999). Briefly, the cerebellum from Wistar rat pups (P7) was removed aseptically, digested with papain and cerebellar cells were resuspended in DMEM containing 10% FCS (v/v), 25 mM glucose, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin. The cells were plated in culture flasks at density of 70,000 cells/cm² and they were maintained in culture until reaching confluence (approximately 10–12 days), replacing the medium every 3–4 days. Cultures were depleted of microglial cells by orbital shaking and finally, the astrocytes were detached from the culture flasks by trypsin treatment and seeded onto culture plates. For microfluorometry experiments, astrocytes were plated onto glass coverslips in 35 mm Petri dishes at 50,000 cells/cm² and for Western blotting, cells were plated onto Petri dishes at a density of 35,000 cells/cm². Astrocytes were routinely used 48 h after plating.

Calcium imaging experiments were carried out essentially as described previously (Carrasquero et al., 2005). Astrocytes attached to coverslips were incubated in Locke’s solution (composition in mM: 140 NaCl, 4.5 KCl, 2.5 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 5.5 glucose, 10 HEPES pH 7.4) supplemented with 1 mg/mL bovine serum albumin (BSA) and loaded with 5–7 μM fura-2/AM for 45 min at 37°C. Once washed in fresh Locke’s solution, a coverslip was placed in a small superfusion chamber and the cells were imaged on a NIKON TE-200 microscope equipped with a Plan Fluor 20X/0.5 objective. Cells were continuously superfused with Locke’s medium at a rate of 1.5 mL/min, and agonists were usually applied for 20 s by switching the superfusion solution with the aid of WC-8 valve controller (Warner Instruments). Cells were illuminated at 340 and 380 nm, and the emitted light was isolated with a dichroic mirror (430 nm) and a 510 nm band-pass filter (Omega Optical). Images were obtained with an ORCA-ER C 47 42-80 camera (Hamamatsu City, Japan) controlled by MetaFluor 6.2r and PC software (Universal Imaging, Corp., Cambridge, United Kingdom). The exposure time was 100 ms and the wavelength of incoming light was changed from 340 to 380 nm in less than 5 ms. Fluorescence images were acquired at a sampling frequency of 2 Hz and processed by averaging signals from small elliptical regions within individual cells. Background signals were subtracted from each wavelength and the F₃₄₀/F₃₈₀ fluorescence ratio was calculated. Fluorescence ratio increases represent [Ca²⁺]_i changes. The quantification of the responses was made by measuring the magnitude of the initial transients. Data are represented as the percentage of the control responses elicited by an UTP challenge in the same experimental conditions (each individual cell).

Astrocytes were detached from the culture flasks and they were suspended in DMEM supplemented with 1% FCS. The lower chambers of Transwell 6-well culture plates were filled with 3 mL DMEM containing UTP (as indicated), and with an 8 μm membrane insert placed into the wells, a 1 mL cell suspension containing 300,000 cells was added to the upper chamber. After 18 h at 37°C in a 5% CO₂ incubator, the cells in the upper chambers were removed by scraping the membranes with a cotton swab, while the cells attached to the bottom of the membranes were fixed with 4% paraformaldehyde for 15 min, washed with PBS and stained with the nuclear marker, DAPI. The cells were visualized under a microscope and counted in five random fields. They cells attached to the bottom of the membrane represented those that had migrated from the upper to lower chamber in response to nucleotide stimulation.

Astrocytes detached from culture flasks were seeded onto Petri dishes in complete culture medium and they were maintained in the incubator to form a confluent monolayer (48 h after plating). A wound was made by scraping a conventional yellow pipette tip across the monolayer and the cells were then washed with culture medium, replacing the medium with fresh DMEM plus 1% FCS and with the addition of the nucleotide or another effector to induce migration. After 24 h at 37°C in a 5% CO₂ incubator, the cells were fixed with paraformaldehyde as described above and their nucleus was stained with DAPI.

Cells were stimulated in Locke’s solution and where indicated, the astrocytes were preincubated in the presence or absence of effectors, antagonists or inhibitors prior to stimulation with the nucleotide UTP. Controls were systematically used with the corresponding vehicle alone (DMSO always at a concentration <2%), in which cell viability was not significantly affected. Incubations were stopped by removing the incubation medium and lysing the cells in cold lysis buffer (20 mM MOPS pH 7.2, 50 mM NaF, 40 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM EDTA, 2 mM EGTA, 0.5% Triton X-100, 1 mM PMSF, and a protease inhibitor cocktail).

Total cellular lysates (15–30 μg protein) were resolved in SDS–PAGE gels and transferred to PVDF membranes. The membranes were probed overnight at 4°C with primary antibodies in either TBS 0.1% with Tween-20 containing low-fat milk powder (5%, w/v), and then for 1 h at room temperature with the secondary antibodies. Antibodies were used at following dilutions: anti-phosphor-ERK1/2, anti-ERK2 (1:1000), anti-phosphor Akt and Akt (1:1000), anti-COX-2 (1:500), anti-P2Y₂ and anti-P2Y₄ (1:200), anti-EP3 receptors (1:200), and anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:2000 and 1:1000, respectively). Specific protein bands were visualized by ECL (Western Lighting ECL PRO kit, Perkin Elmer, Madrid, Spain), and chemiluminescence images were taken with the ImageQuant LAS 500^® image system and quantified by densitometry using ImageQuantTL software.

The results are expressed as the means ± SEM calculated from at least three experiments performed on cells from different cultures. When multiple comparisons were made, one-way analysis of variance was used and Dunnett’s post-test analysis was applied only when a significant (p < 0.05) effect was indicated by the one-way analysis of variance (GraphPad Prism 5; GraphPad Software, Inc., San Diego, CA, United States).

Cerebellar astrocytes co-express several subtypes of P2Y metabotropic nucleotide receptors (Jimenez et al., 2000), and metabotropic calcium responses to ATP mediated by P2Y₂/P2Y₄ nucleotide receptors were evident in the entire astrocyte population, in which ATP and UTP were equipotent. To examine the modulatory role of PGE₂ on P2Y signaling in cerebellar astrocytes, we investigated the effect of this prostaglandin on UTP induced calcium responses. We did not use ATP, because cerebellar astrocytes also express ATP sensitive P2X7 receptors (Carrasquero et al., 2009). As expected all the cells tested responded to the UTP challenge (100 μM) by increasing their intracellular calcium [Ca²⁺]_i, which peaked (ΔF₃₄₀/F₃₈₀ of 0.73 ± 0.02 relative to the basal levels between 0.3 and 0.5; n = 327 cells) and then returned to the basal level within the first 20 s of stimulation (Figure 1A). These cells also responded to a second UTP challenge with similar calcium transients, the magnitude of which was reduced by 10%. It is known that EP1 receptors couple to Gq proteins and EP2 receptor stimulation promotes calcium responses in rat cortical astrocytes (Di Cesare et al., 2006). Thus, before analyzing the modulatory role of PGE₂ on UTP calcium responses, cells were challenged with PGE₂ (1 μM) alone, which did not evoke any increase in [Ca²⁺]_i. Cells were also insensitive to stimulation with 6,16-Dimethylprostaglandin E₂, a synthetic analog that resists metabolism and that has a prolonged half-life in vivo (results not shown). These results indicated that cerebellar astrocytes did not express functional EP1 receptors, although they did not rule out the presence of functional EP2/EP4 receptors coupled to adenylate cyclase activation. PGE₂ inhibited UTP calcium responses (Figure 1B) and preincubation with PGE₂ (1 μM, 5 min) dampened the calcium responses elicited by 100 μM UTP, the maximal effective concentration. This inhibition was observed in all the cells tested and the magnitude of the initial Ca²⁺ transient was reduced by a 40%. Nevertheless, the effect of preincubation with PGE₂ was not necessary due to the simultaneous co-stimulation of astrocytes with PGE₂ and UTP reduced the calcium responses triggered by the nucleotide alone by 35%. These results were consistent with the regulatory influence of PGE₂ on P2Y signaling evident in macrophages and fibroblasts (Ito and Matsuoka, 2008; Traves et al., 2013; Pimentel-Santillana et al., 2014).

The presence of EP2/EP4 receptors coupled to Gs proteins in cerebellar astrocytes was analyzed indirectly, assessing whether UTP-mediated responses could be enhanced by PGE₂ (Figure 2). We previously reported an important cross-talk between Gs-coupled receptors and P2Y nucleotide receptors in these glial cells (Jimenez et al., 1999). Indeed, the activation of A_2B adenosine receptors, or other signals coupled to adenylate cyclase stimulation, strongly potentiated the metabotropic calcium responses to nucleotides. Co-stimulation of astrocytes with adenosine (10 μM) and ATP (1 μM, ineffective when administered alone) elicited [Ca²⁺]_i transients that represented 60% of the maximal calcium response elicited by ATP/UTP (100 μM). This potentiation was parallel to but independent of cAMP accumulation, suggesting the involvement of βγ subunits released upon Gs stimulation. Co-stimulation of astrocytes with PGE₂ and ineffective concentrations of UTP (1 μM) did not evoke any signals (Figure 2), and no signal was observed after preincubation with PGE₂ and simultaneous co-stimulation with UTP (1 μM). Hence, cerebellar astrocytes do not appear to express functional EP2/EP4 receptors and if they are present, they were unable to modulate UTP calcium responses in the same way as other signals coupled to adenylate cyclase activation.

Having excluded the participation of EP1 and EP2/EP4 receptors in the inhibitory effect of PGE₂ on the calcium responses to UTP, we assessed the involvement of EP3 receptors in this effect (Figure 3). The EP3 agonist sulprostone is a synthetic analog of PGE₂ that is resistant to metabolism and it reproduced the effect of PGE₂ (Figure 4A). Stimulation with UTP (100 μM) after preincubation with sulprostone (30 nM, 5 min) dampened the calcium responses, which reached 57.44% of the control responses. Likewise, co-stimulation with sulprostone and UTP resulted in calcium responses that reached 61.71% of the control UTP responses. These findings supported the involvement of EP3 receptors in this phenomenon. In fact, EP3 receptors were detected in Western blots, although assays carried out with an EP3 receptor antagonist, L798106 ((2E)-N-[(5-bromo-2-methoxyphenyl) sulfonyl]-3-[2-(2-naphthalenylmethyl) phenyl]-2-propenamide), proved inconclusive. This antagonist did not modify the basal calcium levels but it did depress the calcium responses to UTP by 40% (Figure 4B). Moreover, pretreatment of astrocytes with L798106 prior to their stimulation with PGE₂ or sulprostone did not prevent their inhibitory effects. Thus, despite the detection of EP3 receptors in Western blots (Figure 8), their involvement in the regulatory effect of PGE₂ remained unclear. Indeed, we suggest that L798106 might be interacting with P2Y receptors given its similar structure to non-selective antagonists of nucleotide receptors, such as PPADS or suramin (Ho et al., 1995; Communi et al., 1996; Jimenez et al., 2000).

We next examined whether PGE₂ modulated the migration of cerebellar astrocytes induced by UTP, as reported in macrophages and fibroblasts (Pimentel-Santillana et al., 2014). In both assays used, transwell migration and wound-healing, PGE₂ attenuated the migration of astrocytes induced by UTP (Figure 5). When UTP (100 μM) was added to the lower transwell chamber of cell culture dishes containing membrane inserts, transmembrane chemotactic migration of astrocytes was enhanced (Figure 5A). However, preincubation of cells with PGE₂ (1 μM, 5 min) prior to their introduction into the upper chamber diminished their migration under control conditions and that induced by the nucleotide by 45%. UTP-induced chemotaxis was also prevented by U0126 (Figure 5A), an inhibitor of MEK, which indicated that ERK activation was required for cell migration. In wound-healing migration assays, PGE₂ completely prevented cell migration in control conditions (without additional exogenous nucleotide), as well as that induced by the nucleotide (Figure 5B). In this case, prostaglandin was included in the fresh medium added after the cells were washed to remove cell debris.

The impact of PGE₂ on the intracellular signaling cascades triggered by UTP was analyzed to further study the mechanism through which PGE₂ modulates UTP responses. The ERK1/2 and PI3-kinase/Akt proteins are known to play relevant roles in nucleotide signaling and cell migration (Brambilla et al., 2002; Jimenez et al., 2002; Neary et al., 2004). Indeed, stimulation of astrocytes with UTP (100 μM) induced a remarkable increase in the phosphorylation of ERK1/2 (a fourfold to eightfold increase over basal levels) and of the Akt protein (a twofold increase over basal levels: Figure 6). Pretreatment of astrocytes with PGE₂ (1 μM) reduced the ERK and Akt phosphorylation induced by UTP 35 and 40%, respectively. Stimulating astrocytes with PGE₂ alone had no significant effect on ERK phosphorylation but it did dampen the phosphorylation of Akt. The inhibitory effects of PGE₂ were also reproduced by sulprostone. Preincubation with the prostanoids was not necessary, and similar effects were observed after co-stimulation with PGE₂/sulprostone and the nucleotide (results not shown). The extent of inhibition is consistent with the changes in the calcium responses observed.

The EGF receptor plays a key role in the intracellular signaling cascades triggered by GPCRs, including P2Y₂ receptors (Liu et al., 2004). To determine whether EGFR activation was required for the activation of ERK and/or Akt by the nucleotide, or for the modulatory effect of PGE₂, the influence of an EGFR tyrosine kinase inhibitor was tested, AG1478. Interestingly, pretreatment of cells with AG1478 (1 μM) for 20 min reduced the basal phosphorylation of the two kinases, although it did not affect the UTP-induced ERK and Akt phosphorylation (Figure 7). Nevertheless, the EGFR inhibitor impaired the regulatory effect of PGE₂, indicating that EGFR transactivation was required for the effect of the prostaglandin. Moreover, inhibition of PI3K with LY294002 (50 μM, 20 min) not only reduced the basal phosphorylation of ERK but it also prevented the regulatory effect of PGE₂, although like the EGFR inhibitor it did not affect UTP-induced ERK phosphorylation. Thus, PGE₂ could regulate ERK and Akt activation by a mechanism dependent on the transactivation of the EGFR and PI3K kinase, which would also support the involvement of EP3 receptors. To clearly demonstrate the possible implication of these receptors, more comprehensive studies will be necessary that take into account the different splice variants of these receptors described.

To determine whether the modulation of PGE₂ might be relevant to neuroinflammation and pathological conditions, we investigated the cross-talk between PGE₂ and nucleotides in astrocytes treated with LPS, a toll-like receptor (TLR) ligand. Pretreatment with LPS (10 ng/mL, 24 h) did not affect either UTP-induced ERK phosphorylation or the modulatory effect of PGE₂ (Figure 8A). UTP-induced ERK activation was higher (a 7.98-fold increase over unstimulated cells) than that observed in untreated cells (4.5-fold increase over basal levels) in the same experimental conditions, while PGE₂ decreased UTP-induced ERK activation by 55%. Pretreatment of astrocytes with LPS notably increased the basal levels of the Akt phosphorylation and it diminished UTP-induced Akt phosphorylation by 55% (Figure 8B). In addition, PGE₂ prevented Akt activation induced by the nucleotide. LPS induced strong expression of COX-2 (twofold over basal) but it did not significantly modify the levels of P2Y₂/P2Y₄ receptors in cerebellar astrocytes. Note that as reported previously, the P2Y₂ receptor is more abundant in these cells than the P2Y₄ receptor. Nevertheless, LPS also increased the levels of EP3 receptors (twofold to threefold increase over basal levels) (Figure 8C).

This study aimed to explore the cross-talk between PGE₂ and UTP-sensitive P2Y receptors in cerebellar astrocytes. We found that PGE₂ negatively modulated UTP responses, as reported previously in macrophages and fibroblasts. However, there were two differences inasmuch as the effect of PGE₂ appeared to be dependent on EP3 receptors and also, it was observed in LPS treated cells. In macrophages the interaction took place in thioglycollate-elicited and alternative macrophages (M2), but not in macrophages activated with the endotoxin (M1). While the metabolic changes that occur during macrophage activation and the transition from M1 to M2 phenotype have been largely explored (Rodriguez-Prados et al., 2010; Traves et al., 2012), this is not the case for astrocytes activated with LPS. Nevertheless, macrophages and astrocytes share some metabolic features and they are constitutively glycolytic (Bolanos, 2016). Hence, it will be very interesting to investigate the metabolic implications of the cross-talk reported in cerebellar astrocytes, both in control conditions and in LPS-treated astrocytes, and the intracellular targets of the endotoxin. The negative effects of PGE₂ on UTP signaling could contribute to limit excessive astrogliosis, which could represent part of the adaptive mechanism to neuroinflammation. Several studies have indicated that both P2Y and P2X receptors are upregulated in the astrogliosis process involving ERK and Akt activation (Franke et al., 2001; Neary et al., 2005; Franke and Illes, 2014). We are developing studies to determine whether P2Y₂/P2Y₄ receptors are upregulated after prolonged treatment with the endotoxin and whether they can be down-regulated by PGE₂.