ATP‐evoked increases in [Ca2+]i and peptide release from rat isolated neurohypophysial terminals via a P2X2 purinoceptor

1 The effect of externally applied ATP on cytosolic free Ca2+ concentration ([Ca2+]i) was tested in single isolated rat neurohypophysial nerve terminals by fura‐2 imaging. The release of vasopressin (AVP) and oxytocin (OT) upon ATP stimulation was also studied from a population of terminals using specific radioimmunoassays. 2 ATP evoked a sustained [Ca2+]i increase, which was dose dependent in the 1‐100 μM range (EC50= 4·8 μM). This effect was observed in only ≈40 % of the terminals. 3 Interestingly, ATP, in the same range (EC50= 8·6 μM), evoked AVP, but no significant OT, release from these terminals. 4 Both the [Ca2+]i increase and AVP release induced by ATP were highly and reversibly inhibited by suramin, suggesting the involvement of a P2 purinergic receptor in the ATP‐induced responses. Pyridoxal‐5‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), another P2 purinergic receptor antagonist, strongly reduced the ATP‐induced [Ca2+]i response. 5 To further characterize the receptor, different agonists were tested, with the following efficacy: ATP = 2‐methylthio‐ATP > ATP‐γ‐S > α,β‐methylene‐ATP > ADP. The compounds adenosine, AMP, β,γ‐methylene‐ATP and UTP were ineffective. 6 The ATP‐dependent [Ca2+]i increase was dependent on extracellular Ca2+ concentration ([Ca2+]o). Fluorescence‐quenching experiments with Mn2+ showed that externally applied ATP triggered a Mn2+ influx. The ATP‐induced [Ca2+]i increase and AVP release were independent of and additive to a K+‐induced response, in addition to being insensitive to Cd2+. The ATP‐induced [Ca2+]i increase was strongly reduced in the presence of Gd3+. These results suggest that the observed [Ca2+]i increases were elicited by Ca2+ entry through a P2X channel receptor rather than via a voltage‐dependent Ca2+ channel. 7 We propose that ATP, co‐released with neuropeptides, could act as a paracrine‐autocrine messenger, stimulating, via Ca2+ entry through a P2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.