Nobles M, Abbott N J
King's College London, Biomedical Sciences Division, Physiology Group, Strand, UK.
Eur J Pharmacol. 1998 Nov 13;361(1):119-27. doi: 10.1016/s0014-2999(98)00671-2.
This study examined the intracellular regulation of signal transduction initiated by activation of the P2Y2 purinoceptor in a cultured rat brain microvascular endothelial cell line (RBE4). Intracellular free Ca2+ ([Ca2+]i) was monitored in single cells, using FURA-2 fluorimetry. As previously described [Nobles, M., Revest, P.A., Couraud, P.-O., Abbott, N.J., 1995. Characteristics of nucleotide receptors that cause elevation of cytoplasmic calcium in immortalized rat brain endothelial cells, RBE4, and in primary cultures. Br. J. Pharmacol., 115, 1245-1252], extracellular ATP (100 microM, 20 s) evoked a transient increase in intracellular free calcium concentration ([Ca2+]i). The amplitude of the Ca2+ transient evoked by ATP decreased with successive applications (desensitisation), as expected for a P2 purinoceptor. The modulation of the Ca2+ signal downstream to the activation of the ATP receptor was investigated, using agents selected for their ability to interfere with the intracellular pathways activated by ATP. The amplitude of the Ca2+ transient observed on the second application of ATP was compared in the presence and absence of these agents. The Ca2+ transient triggered by ATP was decreased by the inhibitor of nitric oxide synthesis, N-omega-nitro-L-arginine methyl ester (L-NOARG). The inhibition induced by 100 microM L-NOARG was reversed by coapplication of the permeant cGMP analogue 8-brcGMP (100 microM). 8-BrcGMP caused a transient increase in [Ca2+]i when applied alone, and a dose-dependent inhibition of the increase in [Ca2+]i elicited by ATP. Indomethacin, an inhibitor of prostaglandin synthesis, inhibited the response to ATP. The inhibition caused by 10 microM indomethacin was reversed by coapplication of the permeant analogue of cAMP, 8-brcAMP (100 microM). 8-BrcAMP caused a transient rise in [Ca2+]i when applied alone, and a dose-dependent inhibition of the Ca2+ response evoked by ATP. The non-permeant cyclic nucleotides cAMP and cGMP did not affect the desensitising response to ATP, nor did they reverse the inhibitory actions of L-NOARG or indomethacin. It is concluded that cyclic nucleotides, nitric oxide, and prostaglandin synthesis pathways are able to interact with the Ca2+ second messenger pathway in rat brain endothelial cells activated by extracellular ATP.
本研究检测了培养的大鼠脑微血管内皮细胞系（RBE4）中P2Y2嘌呤受体激活引发的信号转导的细胞内调节。使用FURA-2荧光测定法监测单细胞内的游离钙离子浓度（[Ca2+]i）。如先前所述[Nobles, M., Revest, P.A., Couraud, P.-O., Abbott, N.J., 1995. 导致永生化大鼠脑内皮细胞RBE4及原代培养细胞胞质钙升高的核苷酸受体的特性。《英国药理学期刊》，115, 1245 - 1252]，细胞外ATP（100 μM，20秒）可引起细胞内游离钙浓度（[Ca2+]i）的短暂升高。如对P2嘌呤受体所预期的那样，ATP诱发的Ca2+瞬变幅度随着连续应用（脱敏）而降低。使用因其干扰ATP激活的细胞内途径的能力而选择的试剂，研究了ATP受体激活下游的Ca2+信号调节。在有或无这些试剂存在的情况下，比较第二次应用ATP时观察到的Ca2+瞬变幅度。一氧化氮合成抑制剂N-ω-硝基-L-精氨酸甲酯（L-NOARG）可降低ATP触发的Ca2+瞬变。共应用渗透性cGMP类似物8-溴环鸟苷（100 μM）可逆转100 μM L-NOARG所诱导的抑制作用。单独应用8-溴环鸟苷时可引起[Ca2+]i的短暂升高，并对ATP引起的[Ca2+]i升高产生剂量依赖性抑制。前列腺素合成抑制剂吲哚美辛可抑制对ATP的反应。10 μM吲哚美辛所引起的抑制作用可通过共应用cAMP的渗透性类似物8-溴环腺苷酸（100 μM）而逆转。单独应用8-溴环腺苷酸时可引起[Ca2+]i的短暂升高，并对ATP诱发的Ca2+反应产生剂量依赖性抑制。非渗透性环核苷酸cAMP和cGMP既不影响对ATP的脱敏反应，也不能逆转L-NOARG或吲哚美辛的抑制作用。得出的结论是，环核苷酸、一氧化氮和前列腺素合成途径能够与细胞外ATP激活的大鼠脑内皮细胞中的Ca2+第二信使途径相互作用。
