Song Zhilin, Vijayaraghavan Sukumar, Sladek Celia D
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.
Am J Physiol Regul Integr Comp Physiol. 2007 Jan;292(1):R423-31. doi: 10.1152/ajpregu.00495.2006. Epub 2006 Sep 14.
ATP increases intracellular calcium concentration (Ca(2+)) 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+) in dissociated SON neurons. However, the ATP-induced increase in Ca(2+) persisted after removal of Ca(2+) from the perifusate (Ca(2+)). 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+) stores with thapsigargin (TG) prevented the ATP-induced increase in Ca(2+) in zero, but not in 2 mM Ca(2+), 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+). 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+) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in Ca(2+) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero Ca(2+). Thus, both P2XR and P2YR participate in the ATP-induced increase in Ca(2+), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.
在装载了钙敏染料fura 2-AM的下丘脑-神经垂体系统外植体的视上核（SON）神经元中，ATP可增加细胞内钙浓度（Ca(2+)）。由于ATP刺激下丘脑-神经垂体系统外植体释放血管加压素需要激活P2X嘌呤能受体（P2XR），并且激活P2XR可诱导解离的SON神经元中Ca(2+)增加，因此预计P2XR参与了这一反应。然而，从灌流液（Ca(2+)）中去除Ca(2+)后，ATP诱导的Ca(2+)增加仍持续存在。这提示P2Y嘌呤能受体（P2YR）参与其中，因为P2YR可诱导细胞内钙库释放Ca(2+)，而P2XR是Ca(2+)可通透的离子通道。用毒胡萝卜素（TG）耗尽Ca(2+)库可在零钙（Ca(2+)）条件下阻止ATP诱导的Ca(2+)增加，但在2 mM Ca(2+)条件下则不能，这表明Ca(2+)内流和细胞内Ca(2+)释放均对ATP反应有贡献。镉可部分阻断Ca(2+)内流，表明电压门控Ca(2+)通道发挥了作用。P2XR拮抗剂PPADS（磷酸吡哆醛-6-偶氮苯基-2',4'-二磺酸）和异-PPADS可减弱但不能消除ATP诱导的Ca(2+)增加。PPADS或异-PPADS与TG联合处理可阻止该反应。由UTP、UDP和2-甲硫基-ADP组成的P2YR激动剂混合物可增加Ca(2+)（无论有无河豚毒素），且该增加被TG显著减弱。单独的2-甲硫基-ADP诱导的Ca(2+)增加比UTP或UDP更持续且更大。特异性P2Y(1)R拮抗剂MRS2179可消除在零Ca(2+)条件下对ATP的反应。因此，P2XR和P2YR均参与了ATP诱导的Ca(2+)增加，且在SON中P2Y(1)R亚型比P2Y(2)R、P2Y(4)R或P2Y(6)R更突出。
