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 mu M range (EC50 = 4.8 mu M). This effect was observed in only similar to 40% of the terminals. 3. Interestingly, ATP, in the same range (EC50 = 8.6 mu M), 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 P-2 purinergic receptor in the ATP-induced responses. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), another P-2 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-gamma-S > alpha,beta-methylene-ATP > ADP. The compounds adenosine, AMP, beta,gamma-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 P-2X 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 P-2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.