ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors.
ATP increases intracellular calcium concentration ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca(2+)-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 [Ca(2+)](i) in dissociated SON neurons. However, the ATP-induced increase in [Ca(2+)](i) persisted after removal of Ca(2+) from the perifusate ([Ca(2+)](o)). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca(2+) release from intracellular stores, whereas P2XRs are Ca(2+)-permeable ion channels. Depletion of [Ca(2+)](i) stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca(2+)](i) in zero, but not in 2 mM [Ca(2+)](o), indicating that both Ca(2+) influx and release of intracellular Ca(2+) contribute to the ATP response. Ca(2+) influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca(2+) 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 [Ca(2+)](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 [Ca(2+)](i) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca(2+)](i) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero [Ca(2+)](o). Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca(2+)](i), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.
Song
Zhilin
Z
Department of Physiology and Biophysics, University of Colorado at Denver and Health Sciences Center, P.O. Box 6511, Mail Stop 8307, Aurora, CO 80045, USA. zhilin.song@uchsc.edu
Vijayaraghavan
Sukumar
S
Sladek
Celia D
CD
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003952:Diagnostic Imaging; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D066298:In Vitro Techniques; D008297:Male; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D009994:Osmolar Concentration; D058913:Purinergic Agonists; D058914:Purinergic Antagonists; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D058469:Receptors, Purinergic P2X; D013495:Supraoptic Nucleus / Q000187:drug effects / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C111914:N(6)-methyl-2'-deoxyadenosine 3',5'-diphosphate; D018377:Neurotransmitter Agents; D058913:Purinergic Agonists; D058914:Purinergic Antagonists; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D058469:Receptors, Purinergic P2X; D002104:Cadmium; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D013779:Tetrodotoxin; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2