Multiple signaling pathways of V1-vascular vasopressin receptors of A7r5 cells.
Multiple signaling pathways of V1-vascular vasopressin receptors of A7r5 cells.
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DOI：
10.1210/endo-129-6-2845
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发表时间：
1991-12
期刊：
影响因子：
4.8
通讯作者：
M. Thibonnier;A. L. Bayer;M. Simonson;M. Kester
中科院分区：
文献类型：
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作者：
M. Thibonnier;A. L. Bayer;M. Simonson;M. Kester
应用放射配基结合技术、细胞内钙监测、多聚磷脂酰肌醇和磷脂分析，探讨培养的A7r5血管加压素(AVP)V1受体信号转导途径的性质和时程。A7r5细胞的V1血管AVP受体以激动型放射性配体[~3H]AVP和拮抗型放射性配体[~3H]d(CH2)5Tyr(Me)AVP为特征。氯化镁和氟化铝可调节激动剂的亲和力和结合能力，但不能调节拮抗剂的结合能力，提示受体与鸟嘌呤核苷酸调节蛋白偶联。在Fura-2负载的A7r5细胞中，AVP在数秒内引起细胞内游离钙离子([Ca++]i)呈剂量依赖性的升高，表现为短暂的快速尖峰和持续3-5分钟的升高。基础[Ca++]i为136+/-18 nM，对AVP的最大[Ca++]i反应为1,582+/-297 nM，AVP ED50为1.87+/-0.15 nM。用EGTA、1，2-双(邻氨基苯氧基)乙烷-N，N，N‘，N’-四乙酸乙酰氧甲酯、Mn++、离子霉素、叔丁基苯并对苯二酚和尼卡地平进行的不同实验表明，最初的峰值是由内质网释放的细胞内钙离子和细胞外钙离子内流引起的，而持续的相依赖于二氢吡啶不敏感的细胞外钙离子内流。用消炎痛和花生四烯酸进行的实验表明，AVP诱导的细胞外钙内流部分依赖于磷脂酶A2的激活。在[3 H]肌醇和[3 H]花生四烯酸标记的A7r5细胞中，AVP通过激活磷脂酶C刺激1，4，5-三磷酸肌醇和1，2-二酰甘油的产生；在[3 H]1-O-烷基甘油磷胆碱标记的A7r5细胞中，AVP通过激活磷脂酶D刺激转磷脂反应。因此，A7r5细胞的V1血管AVP受体的刺激触发了几条信号通路。磷脂酶A2和C的激活以及磷脂酶D的持续激活与细胞内和细胞外钙动员引起的即刻和一过性的[Ca++]i升高有关。
We explored the nature and time course of the multiple signal transduction pathways for V1-vascular vasopressin (AVP) receptors of A7r5 aortic smooth muscle cells in culture by using radioligand binding techniques, intracellular calcium monitoring, and polyphosphoinositide and phospholipid analyses. V1-vascular AVP receptors of A7r5 cells were characterized by the agonist radioligand [3H]AVP and the antagonist radioligand [3H]d(CH2)5Tyr(Me)AVP. Affinity and capacity of agonist but not antagonist binding were modulated by MgCl2 and aluminum fluoride, suggesting that the receptors are coupled to a guanine nucleotide regulatory protein. In fura-2-loaded A7r5 cells, AVP induced within seconds a dose-dependent increase of free intracellular Ca++ ([Ca++]i) consisting of a rapid transient spike and a sustained increase lasting for 3-5 min. The baseline [Ca++]i was 136 +/- 18 nM, the maximum [Ca++]i response to AVP was 1,582 +/- 297 nM, and AVP ED50 was 1.87 +/- 0.15 nM. Diverse experiments performed with EGTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester, Mn++, ionomycin, terbutylbenzo hydroquinone, and nicardipine suggested that the initial spike resulted from both intracellular Ca++ release from the endoplasmic reticulum and extracellular Ca++ influx, whereas the sustained phase depended on dihydropyridine-insensitive extracellular Ca++ influx. Experiments done with indomethacin and arachidonic acid indicated that AVP-induced extracellular Ca++ influx was in part dependent on phospholipase A2 activation. In [3H]myoinositol and [3H]arachidonate-labeled A7r5 cells, AVP stimulated inositol 1,4,5 trisphosphate and 1,2 diacylglycerol production via activation of phospholipase C. Also, AVP stimulated a transphosphatidylation reaction through activation of phospholipase D in A7r5 cells labeled with [3H]1-O-alkyl lysoglycerophosphocholine. Thus, the stimulation of V1-vascular AVP receptors of A7r5 cells triggers several signaling pathways. The immediate and transient [Ca++]i rise due to mobilization of intracellular and extracellular Ca++ is associated with the activation of phospholipases A2 and C, and the sustained activation of phospholipase D.