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 microM range (EC50 = 4.8 microM). This effect was observed in only approximately 40 % of the terminals. 3. Interestingly, ATP, in the same range (EC50 = 8.6 microM), 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-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 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.
UMR 6548-CNRS, Laboratoire de Physiologie Cellulaire et Moleculaire, Universite de Nice-Sophia Antipolis, F-06108 Nice, France. troadec@hermes.unice.fr
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Troadec
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Thirion
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Nicaise
J R
JR
Lemos
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Dayanithi
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008954:Models, Biological; D009411:Nerve Endings / Q000187:drug effects / Q000502:physiology*; D010121:Oxytocin / Q000378:metabolism*; D010904:Pituitary Gland, Posterior / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology*; D058476:Receptors, Purinergic P2X2; D013498:Suramin / Q000494:pharmacology
C546403:P2rx2 protein, rat; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D001127:Arginine Vasopressin; D010121:Oxytocin; D013498:Suramin; D000255:Adenosine Triphosphate; D002118:Calcium