ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors

ATP increases intracellular calcium concentration ([Ca2+](i)) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca2+-sensitive dye, fura 2-AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin release from hypothalamo-neurohypophyseal system explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca2+](i) in dissociated SON neurons. However, the ATP-induced increase in [Ca2+](i) persisted after removal of Ca2+ from the perifusate ([Ca2+](o)). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca2+ release from intracellular stores, whereas P2XRs are Ca2+-permeable ion channels. Depletion of [Ca2+](i) stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca2+](i) in zero, but not in 2 mM [Ca2+](o), indicating that both Ca2+ influx and release of intracellular Ca2+ contribute to the ATP response. Ca2+ influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca2+ channels. PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), and iso-PPADS, P2XR antagonists, attenuated, but did not abolish, the ATP-induced increase in [Ca2+](i). Combined treatment with PPADS or iso-PPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-methylthio-ADP increased [Ca2+](i) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca2+](i) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero [Ca2+](o). Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca2+](i), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.