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ATP-evoked increases in [Ca²⁺]_i and peptide release from rat isolated neurohypophysial terminals via a P_2X2 purinoceptor

Jean-Denis Troadec *, Sylvie Thirion *, Ghislain Nicaise *, José R. Lemos *² and Govindan Dayanithi ¹

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--S > ,-methylene-ATP > ADP. The compounds adenosine, AMP, ,-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.

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