P(2Y) purinoceptor subtypes recruit different mek activators in astrocytes

Abstract

Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P(2Y) receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P(2Y) receptors expressed in astrocytes have not been defined and it is not known whether all P(2Y) receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P(2Y) receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. Reverse transcriptase-polymerase chain reaction (RT - PCR) studies using primer pairs for cloned rat P(2Y) receptors revealed that rat cortical astrocytes express P(2Y(1)), a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P(2Y(2)) and P(2Y(4)), subtypes in rats for which ATP and UTP are equipotent. Transcripts for P(2Y(6)), a pyrimidine-preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P(2Y(11)), an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. These signal transduction and RT - PCR experiments reveal differences in the activation of cRaf-1 by P(2Y) receptor agonists that are inconsistent with properties of the P(2Y(1)), P(2Y(2)) and P(2Y(4)) receptors shown to be expressed in astrocytes, i.e. ATP=UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P(2Y) receptors coupled to the Erk cascade differ from the recombinant P(2Y) receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.

Figure 1

P_2Y receptor agonists stimulate phosphorylation and activation of Erk. Primary cultures of rat cortical astrocytes were treated for 10 min with FGF-2 (25 ng ml⁻¹), ATP (100 μM), UTP (100 μM), or 2-methylthioATP (2MeSATP, 10 μM). Cells were lysed, and lysates containing equivalent amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis. Immunoblots were probed with antibodies which recognize dual-phosphorylated Erk1/Erk2 (upper panel) or total Erk1/Erk2 (lower panel). All three P_2Y receptor agonists stimulated Erk phosphorylation to an extent similar to that obtained with FGF-2.

Figure 2

Mek activation by P_2Y agonists and FGF-2. Primary astrocyte cultures were treated for 2 min with ATP (100 μM), UTP (100 μM), 2MeSATP (10 μM) or FGF-2 (25 ng ml⁻¹); Mek activity was determined as described in Methods. Data (mean±s.e.mean) were obtained from a minimum of six experiments. ATP, UTP, 2MeSATP and FGF-2 stimulated Mek activity (P<0.05).

Figure 3

Inability of extracellular ATP to activate cRaf-1. Primary astrocyte cultures were treated with ATP (100 μM) or FGF-2 (25 ng ml⁻¹) for 5 min. cRaf-1 was immunoprecipitated from lysates, and cRaf-1 activity was determined by the coupled assay described in Methods. Erk assays were conducted using aliquots of the same samples. Data (mean±s.e.mean) were obtained from seven independent experiments. FGF-2 activated Raf-1 but ATP did not (left panel), even though both FGF-2 and ATP stimulated Erk to the same extent (right panel).

Figure 4

Time course studies: cRaf-1 activation by UTP and 2MeSATP. (A) Primary astrocyte cultures were treated with ATP (100 μM), UTP (100 μM), 2MeSATP (10 μM) or FGF-2 (25 ng ml⁻¹) for the indicated times. cRaf-1 was immunoprecipitated from lysates, and cRaf-1 activity was determined by the coupled assay described in Methods. Data (mean±s.e.mean) were obtained from at least three independent experiments. The data for the 1 min time points obtained with P_2Y agonists and all ATP time points were not significantly different from control values (P>0.05). All other data were significantly different from control values (P<0.05). UTP and 2MeSATP, but not ATP, activated cRaf-1. (B) Aliquots of samples from A were assayed for Erk activation. All P_2Y agonists tested stimulated Erk activity from 2 to 10 min; by 60 min Erk activity in ATP- and 2MeSATP-treated cells had returned to near basal levels, whereas Erk activity in UTP- and particularly in FGF-2 (data not shown)-treated cells remained elevated.

Figure 5

Effect of the P_2Y agonists and FGF-2 on different Mek activators. Primary astrocyte cultures were treated for 2 min with ATP (100 μM), UTP (100 μM), 2MeSATP (10 μM) or FGF-2 (25 ng ml⁻¹). B-Raf, MekK1 and MekK2/3 were immunoprecipitated from lysates, and B-Raf, MekK1 and MekK2/3 activities were determined by the coupled assay described in Methods. Data (mean±s.e.mean) were obtained from a minimum of three independent experiments. ATP and 2MeSATP did not activate any of the Mek activators tested (P>0.05). UTP and FGF-2 activated MekK1 and MekK2/3 (^*P<0.05).

Figure 6

RT–PCR analysis of P_2Y receptor subtype expression in rat neonatal cortical astrocytes. Primer pairs specific for the amplification of rat P_2Y1, P_2Y2, P_2Y4, and P_2Y6 receptor subtypes were used in these experiments. Amplification products were resolved by agarose gel electrophoresis. M, size markers as indicated; –, PCR reactions conducted without the reverse transcriptase step to control for amplification of genomic DNA in the RNA samples. Results are representative of experiments conducted with RNA extracted from at least three independent culture seedings. P_2Y1, P_2Y2 and P_2Y4, but not P_2Y6, receptor subtypes were expressed in rat neonatal cortical astrocytes.

Figure 7

Concentration dependence of ATP and UTP stimulation of Erk activity. Primary cultures of rat cortical astrocytes were treated for 10 min with the indicated concentrations of ATP or UTP, and Erk activity was determined. Data (mean±s.e.mean) are from four independent experiments and were fitted in a sigmoid log concentration-response curve by Prism® v2.0 (GraphPad™). The concentration-response relationships revealed that ATP and UTP have similar potencies in terms of Erk activation.

Figure 8

Effect of ATP and forskolin on cyclic AMP synthesis. Primary astrocyte cultures were treated for 10 min with ATP (100 μM), forskolin (Fsk, 10 μM) or ATP+forskolin, and cyclic AMP synthesis was determined as described in Methods. Data (mean±s.e.mean) were obtained from five experiments. ATP did not stimulate cyclic AMP formation but it significantly potentiated forskolin-induced cyclic AMP synthesis (P<0.01, ANOVA).