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Comparative Study
. 2004 Feb;141(3):497-507.
doi: 10.1038/sj.bjp.0705639. Epub 2004 Jan 12.

Differential effects of P2Y1 and P2Y12 nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3-kinase signalling and cell proliferation in serum-deprived and nonstarved glioma C6 cells

Rafal Czajkowski  1 , Wiktor Banachewicz, Olga Ilnytska, Ludmila B Drobot, Jolanta Baranska
Affiliations
Comparative Study

Differential effects of P2Y1 and P2Y12 nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3-kinase signalling and cell proliferation in serum-deprived and nonstarved glioma C6 cells

Rafal Czajkowski et al. Br J Pharmacol. 2004 Feb.

Abstract

We have previously shown that, in glioma C6 cells, two nucleotide ADP-sensitive receptors coexist: P2Y1, coupled to PLC and responsible for Ca2+ release, and P2Y12, negatively coupled to adenylate cyclase. In the present study, we examined the effects of the stimulation of these two receptors on ERK1/2 and PI3-K activation, and cell proliferation in either serum-deprived or nonstarved C6 cells. In response to ADP and its analogues, in serum-starved cells, both p44 ERK1 and p42 ERK2 were activated in a time-dependent manner, as monitored by Western blot analysis using an antiphospho-p42/p44 MAPK antibody. The phosphorylation was reduced both by removal of the extracellular Ca2+ and partially or almost completely by MRS2179 or AR-C69931MX, specific antagonists of the P2Y1 and P2Y12 receptors, respectively. The inhibitory effect of antagonists was additive. These data indicate the involvement of both receptors, P2Y1 and P2Y12, in the ERK1/2 activation, but the P2Y12 receptor contribution predominates. ERK1/2 activity was positively correlated with cell proliferation of cultured glioma C6 cells. In nonstarved cells, ADP markedly decreased the PI3-K activity. In contrast, in serum-starved cells, ADP evoked an increase in the PI3-K activity. Blocking of the P2Y1 receptor by MRS2179 additionally increased this ADP response. These results suggest that the P2Y1 receptor has an inhibitory and the P2Y12 receptor a stimulatory effect on PI3-K signalling pathway. RT-PCR analysis revealed different mRNA expression of both receptors in starved and nonstarved cells. In nonstarved cells, the P2Y1 receptor mRNA predominates, whereas in serum-deprived cells the expression of P2Y12 mRNA becomes more pronounced. British Journal of Pharmacology (2004) 141, 497-507. doi:10.1038/sj.bjp.0705639

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Figures

Figure 1
Figure 1
Time-dependent activation of p44/p42 ERK1/ERK2. C6 cells were grown as described in Methods, and experiments were performed in the standard buffer after 48 h of serum deprivation (a, c–f), or in control, nonstarved cells (b). The cells were stimulated with 10 μM ADP (a, b), 10 μM 2MeSADP (c), 10 μM 2ClATP (d), 100 μM ATP (e) and 100 μM UTP (f) for 1–60 min. The cells were lysed in SDS sample buffer and subjected to standard Western blot analysis using antiphospho-p44/p42 MAPK or total p44 MAPK (as a control) polyclonal antibody. Bands were visualized by ECL method and quantified using ImageQuant and MS Excel programs. The basal level represents ERK1/2 phosphorylation in the absence of receptor agonists. The results are expressed as the means±s.d. from three independent experiments.
Figure 2
Figure 2
Effect of P2Y1 and P2Y12 receptor antagonists on ADP-evoked ERK1/ERK2 activation in glioma C6 cells. The 48 h starved cells were preincubated for 2 min with 30 μM MRS2179 (P2Y1 receptor antagonist) or with 10 μM AR-C69931MX (P2Y12 receptor antagonist), and, still in their presence, stimulated for 5 min with 10 μM ADP. UTP (100 μM) was used as a negative control for MRS2179 action and intracellular Ca2+ concentration was measured to demonstrate the effect of this antagonist on Ca2+ signals. (a) Effect of antagonists on ADP-evoked p44/p42 ERK1/2 activation. (b) Effect of MRS2179 on UTP-evoked p44/p42 ERK1/2 activation. The cells (a, b) were stimulated for 5 min, lysed in SDS sample buffer and subjected to Western blot procedure. Bands were visualized by the ECL method and quantified using ImageQuant and MS Excel programs. The results are expressed as the means±s.d. from three independent experiments. (c) Effect of MRS2179 on ADP-induced changes in [Ca2+]i. (d) Effect of MRS2179 on UTP-induced changes in [Ca2+]i. (c, d) Each bar represents the mean value of the initial calcium response peak corresponding to the emptying of intracellular calcium stores. Data are means±s.d. from 24 cells recorded on three separate occasions. Each trace in this figure represents the mean ratio value of the Ca2+ response of 16 cells recorded in two experiments.
Figure 3
Figure 3
Effect of extracellular calcium on ADP- and UTP-stimulated ERK1/ERK2 activation. The 48 h serum-starved cells were stimulated for 5 min in the standard buffer containing 2 mM CaCl2 or 500 μM EGTA (calcium free buffer) with 10 μM ADP (a) or with 100 μM UTP (b), lysed in SDS sample buffer and subjected to Western blot procedure. Bands were visualized by ECL method and quantified using ImageQuant and MS Excel programs. Mean values±s.d. of three independent experiments are shown.
Figure 4
Figure 4
Time course of ADP-evoked PI3-kinase activity. C6 cells were grown as described in Methods, and experiments were performed on nonstarved cells (a) or on the cells after 48 h of serum deprivation (b). The cells were stimulated with 10 μM ADP for 1–45 min, lysed and subjected to immunoprecipitation and kinase assay. Phosphatidylinositol 3-phosphate (PIP) bands were visualized with PhosphoImager using the Quantity One software. In each sample, the amount of immunoprecipitated p85 subunit is presented. The results are expressed as the means±s.d. from three independent experiments.
Figure 5
Figure 5
Effect of MRS2179 and GF109203X on ADP-stimulated PI3-K activity in serum-starved glioma C6 cells. The cells were grown as described in Methods, and experiments were performed in the standard buffer after 48 h of serum deprivation. MRS2179 (30 μM) and GF109203X (1 μM) were added 2 min prior to addition of ADP. After 15 min of stimulation with 10 μM ADP, the cells were lysed and subjected to immunoprecipitation and kinase assay. PI3-K activity was measured in vitro as described. Bands were visualized with PhosphoImager, using the Quantity One software. The results are expressed as the means±s.d. from three experiments.
Figure 6
Figure 6
Effect of agonists and antagonists of P2Y receptors on proliferation of serum-deprived glioma C6 cells, estimated by changes in DNA synthesis. Cells were grown in 12-well plates in serum-free medium for 48 h, as described in Methods. Agonists, 2MeSADP and 2ClATP, and antagonists, MRS2179 and AR-C69931MX, were added at concentrations used in experiments shown in Figures 1 and 2. Cells were labelled with 1 μCi ml−1 [3H]thymidine and incubated in the presence of agonists or antagonists for 24 h. For other details, see Methods. The number of cells grown without any addition (Control) was taken as the baseline. The results are expressed as the means±s.d. from 3–5 experiments.
Figure 7
Figure 7
Effect of serum deprivation on the relative expression levels of P2Y1 and P2Y12 receptors in glioma C6 cells. (a) 590 bp RT–PCR product (corresponding to P2Y1 mRNA) and (b) 489 bp RT–PCR product (corresponding to P2Y12 mRNA) were analysed on 1% agarose gel and visualized by ethidium bromide staining. All parallel PCR reactions contained the same amount of cDNA. Lane 1, 123 bp DNA ladder; lanes 2, 3, cells grown in medium supplemented with 10% serum; lanes 4, 5, cells after 48 h serum deprivation; lanes 6, 7, cells grown 4 days in the serum-free medium. (c) Summary of a relative expression of P2Y1 versus P2Y12 receptors in nonstarved, 48 h serum-starved cells and cells cultured in serum-free medium for 4 days. Each bar represents the mean ratio±s.d. from three independent experiments.
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