Activation of P2Y 1 receptor triggers two calcium signaling pathways in bone marrow erythroblasts

Abstract

In this study, we describe the presence of P2 receptor subtypes and Ca 2+ signaling in erythroblasts. ATP and ADP produced a biphasic increase of intracellular Ca 2+ concentration ([Ca 2+] i), with an initial transient phase followed by a sustained phase. Reverse transcription polymerase chain reaction (RT-PCR) showed the expression of P2Y 1, P2Y 2 and P2Y 12. The selective P2Y 1 receptor antagonist 2′-deoxy- N 6-methyl-adenosine-3′,5′-diphosphate (MRS2179) and the G i protein inhibitor pertussis toxin blocked Ca 2+ increase. The initial transient [Ca 2+] i increase phase was sensitive to the 1,4,5-inositol trisphosphate (IP 3) receptor blocker 2-aminoethoxy-diphenylborate (2-APB), while the sustained phase was sensitive to the protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1 H-indol-3-yl]-3-(1 H-indol-3-yl)-maleimide (GF109203X) and calcium calmodulin kinase II (CaMKII) inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)- N-methyl- l-tyrosyl]-4-phenylpiperazine (KN-62). In addition, the PKC activator phorbol-12,13-dibutyrate (PDBu) produced increase of [Ca 2+] i. Flow cytometry analysis showed the expression of Ca 2+-dependent PKCα, βI, γ and phospho-CaMKII. These results suggest that the activation of the P2Y 1 receptor triggers two different [Ca 2+] i increase pathways, one IP 3-dependent and the other kinase-dependent.