Nucleotide P2Y13-stimulated phosphorylation of CREB is required for ADP-induced proliferation of late developing retinal glial progenitors in culture.

Nucleotides stimulate phosphorylation of CREB to induce cell proliferation and survival in diverse cell types. We report here that ADP induces the phosphorylation of CREB in a time- and concentration-dependent manner in chick embryo retinal progenitors in culture. ADP-induced increase in phospho-CREB is mediated by P2 receptors as it is blocked by PPADS but not by the adenosine antagonists DPCPX or ZM241385. Incubation of the cultures with the CREB inhibitor KG-501 prevents ADP-induced incorporation of [3H]-thymidine, indicating that CREB is involved in retinal cell proliferation. No effect of this compound is observed on the viability of retinal progenitors. While no significant increase in CREB phosphorylation is observed with the P2Y1 receptor agonist MRS2365, ADP-induced phosphorylation of CREB is blocked by the P2Y13 receptor selective antagonist MRS2211, but not by MRS2179 or PSB0739, two antagonists of the P2Y1 and P2Y12 receptors, respectively, suggesting that ADP-induced CREB phosphorylation is mediated by P2Y13 receptors. ADP-induced increase in phospho-CREB is attenuated by the PI3K inhibitor LY294002 and completely prevented by the MEK inhibitor U0126, suggesting that at least ERK is involved in ADP-induced CREB phosphorylation. A pharmacological profile similar to the activation and inhibition of CREB phosphorylation is observed in the phosphorylation of ERK, suggesting that P2Y13 receptors mediate ADP induced ERK/CREB pathway in the cultures. While no increase in [3H]-thymidine incorporation is observed with the P2Y1 receptor agonist MRS2365, both MRS2179 and MRS2211 prevent ADP-mediated increase in [3H]-thymidine incorporation, but not progenitor's survival, suggesting that both P2Y1 and P2Y13 receptor subtypes are involved in ADP-induced cell proliferation. P2Y1 receptor-mediated increase in [Ca2+]i is observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca2+]i is detected with MRS2365 and no inhibition of ADP-mediated calcium response is observed with MRS2179. In contrast, MRS2211 attenuates ADP-mediated increase in [Ca2+]i in glial cells from cultures at both stages, suggesting the presence of P2Y13 receptors coupled to calcium mobilization in proliferating retinal glial progenitors in culture.

On the role of ATP release, ectoATPase activity, and extracellular ADP in the regulatory volume decrease of Huh-7 human hepatoma cells.

Hypotonicity triggered in human hepatoma cells (Huh-7) the release of ATP and cell swelling, followed by volume regulatory decrease (RVD). We analyzed how the interaction between those processes modulates cell volume. Cells exposed to hypotonic medium swelled 1.5 times their basal volume. Swelling was followed by 41% RVD(40) (extent of RVD after 40 min of maximum), whereas the concentration of extracellular ATP (ATP(e)) increased 10 times to a maximum value at 15 min. Exogenous apyrase (which removes di- and trinucleotides) did not alter RVD, whereas exogenous Na(+)-K(+)-ATPase (which converts ATP to ADP in the extracellular medium) enhanced RVD(40) by 2.6 times, suggesting that hypotonic treatment alone produced a basal RVD, whereas extracellular ADP activated RVD to achieve complete volume regulation (i.e., RVD(40) ≈100%). Under hypotonicity, addition of 2-(methylthio)adenosine 5'-diphosphate (2MetSADP; ADP analog) increased RVD to the same extent as exposure to Na(+)-K(+)-ATPase and the same analog did not stimulate RVD when coincubated with MRS2211, a blocker of ADP receptor P2Y(13). RT-PCR and Western blot analysis confirmed the presence of P2Y(13). Cells exhibited significant ectoATPase activity, which according to RT-PCR analysis can be assigned to ENTPDase2. Both carbenoxolone, a blocker of conductive ATP release, and brefeldin A, an inhibitor of exocytosis, were able to partially decrease ATP(e) accumulation, pointing to the presence of at least two mechanisms for ATP release. Thus, in Huh-7 cells, hypotonic treatment triggered the release of ATP. Conversion of ATP(e) to ADP(e) by ENTPDase 2 activity facilitates the accumulated ADP(e) to activate P2Y(13) receptors, which mediate complete RVD.