ADP‐evoked phospholipase C stimulation and adenylyl cyclase inhibition in glioma C6 cells occur through two distinct nucleotide receptors, P2Y₁ and P2Y₁₂

Abstract

In this study we characterized the subtypes of nucleotide P2Y receptors that respond to ADP in glioma C6 cells. Direct visualization of phosphatidylinositol 4,5-bisphosphate at the cell surface revealed that extracellular ADP activates phospholipase C (PLC). Knock-down of P2Y 1 receptor with antisense oligonucleotide, as well as treatment with MRS2179 and pyridoxal-phosphate-6-azophenyl-2P P,4P P-disulfonic acid (P2Y 1 antagonists), attenuates receptor-mediated PLC activity. Adenylyl cyclase inhibition by ADP remains unchanged under these conditions. Reverse transcription-PCR analysis showed that P2Y 12 receptor is expressed in C6 cells. We therefore conclude that, in glioma C6 cells, two P2Y receptor subtypes are present: P2Y 1 , coupled to PLC, and P2Y 12 , negatively coupled to adenylyl cyclase. ß

2′, 3′-O-(4-benzoylbenzoyl)–ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y2 nucleotide receptor

“…2MeSADP is a specific agonist of ADP-sensitive receptors: P2Y 1 (coupled to G q ), P2Y 12 (coupled to G i ) and P2Y 13 (coupled to G i ) [15,22,23]. As it is shown in Fig.…”

“…We have previously shown that both the ADP-or 2MeSADP-induced stimulation of P2Y 1 , and the ATP-or UTP-induced stimulation of P2Y 2 , initiate typical biphasic Ca 2+ responses [8,10,[13][14][15]. In contrast, the presence and activity of P2X receptors in glioma C6 cells is weakly documented [14].…”

“…Recently, Wei et al [16] suggested functional expression of the P2X 7 receptor in this type of cells, as activation by BzATP led to an increase in [Ca 2+ ] i . On the other hand, Carrasquero et al [17] proposed activation of not only the P2X 7 but also the P2Y 13 receptors by this agonist in rat cerebellar astrocytes.…”

P2Y 1 receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2

“…Most P2YRs are coupled to G␣ q proteins and thus to the activation of phospholipase C␤ (PLC␤) and generation of diacylglycerol and inositol-3,4,5-trisphosphate, ultimately leading to the activation of PKC and release of Ca 2ϩ from internal stores (1). Some P2YRs, including P2Y 1 , P2Y 2 , P2Y 12 , and P2Y 13 , are also known to couple to G␣ i and the inhibition of adenylyl cyclase (2)(3)(4)(5). The purinergic P2YR type 1 (P2Y 1 R) subtype is abundantly expressed in a number of tissues, including the CNS (6,7), where it plays a key role in the transmission of astrocytic Ca 2ϩ waves (8), activation of mitogenic responses in astrocytes to brain trauma (9), inhibition of neuronal N-type voltage-activated Ca 2ϩ channels (10), and embryonic brain development (11).…”

Neuroprotective roles of the P2Y2 receptor

“…Our studies using in situ hybridization and reverse transcriptase polymerase chain reaction with rodent brain slices have shown high levels of P2Y 2 R expression in the hippocampus and cerebellum [20], and P2Y 2 R expression can be significantly upregulated in mouse cortical neurons by the proinflammatory cytokine interleukin-1β (IL-1β) [48]. P2YRs have been shown to be coupled either directly or indirectly to G q , G i , G o , and G 12 protein activation and downstream signaling pathways associated with alterations in PLC or adenylyl cyclase activities [1,20,[69][70][71]. The agonist selectivity of P2YR subtypes varies widely, in contrast to P2X receptors [1]; for example, the P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 14 receptor subtypes can be activated by uridine nucleotides or UDP-glucose that are ineffective agonists of all P2X and four P2Y receptor subtypes [1,4,19,20,56,70,[72][73][74].…”