Grazzini E, Durroux T, Payet M D, Bilodeau L, Gallo-Payet N, Guillon G
Centre National de la Recherche Scientifique, Institute National de la Santé et de la Recherche Médicale de Pharmacologie/Endocrinologie U 401, Montpellier, France.
Mol Pharmacol. 1996 Nov;50(5):1273-83.
In rat glomerulosa cells, vasopressin stimulates intracellular calcium mobilization via at least two distinct mechanisms: the release of calcium from inositol-1,4,5-P3-sensitive stores and the activation of transmembrane calcium influx. In this study, we focused on the second mechanism through three experimental approaches. By videomicroscopically examining Fura-2-loaded cells, we demonstrate that vasopressin induces a dose-dependent and receptor-mediated calcium influx fully inhibited by either 1 microM nifedipine or a pertussis toxin pretreatment and potentiated by 1 microM BAY K 8644. Patch-clamp experiments also indicate that vasopressin stimulates L-type calcium current by 87% and only weakly inhibits T-type calcium current. To further characterize the coupling between the vasopressin receptor and the dihydropyridine calcium channel, we performed binding studies using tritiated nitrendipine. With this technique, we showed that on intact cells, vasopressin is able to increase the specific binding of tritiated nitrendipine in a dose-dependent manner (Kact = 2 nM). Pharmacological studies using a series of vasopressin analogs revealed that this effect is mediated via a V1a vasopressin receptor subtype. Furthermore, the vasopressin-stimulated nitrendipine binding was sensitive to pertussis toxin pretreatment, which affected only the maximum binding capacity of nitrendipine-binding sites. More interestingly, we demonstrate that vasopressin still increases nitrendipine binding to plasma membrane preparation and that GTP is absolutely necessary for such a hormonal effect. Altogether, these data confirm the existence of a tight and direct coupling between the V1a vasopressin receptor and a dihydropyridine calcium channel via a pertussis toxin-sensitive G protein.
在大鼠肾小球旁细胞中，血管加压素通过至少两种不同机制刺激细胞内钙动员：从肌醇 - 1,4,5 - 三磷酸敏感储存库释放钙以及激活跨膜钙内流。在本研究中，我们通过三种实验方法聚焦于第二种机制。通过视频显微镜检查负载Fura - 2的细胞，我们证明血管加压素诱导剂量依赖性且受体介导的钙内流，1 microM硝苯地平或百日咳毒素预处理可完全抑制该内流，而1 microM BAY K 8644可增强该内流。膜片钳实验还表明，血管加压素使L型钙电流增加87%，而对T型钙电流仅有微弱抑制。为了进一步表征血管加压素受体与二氢吡啶钙通道之间的偶联，我们使用氚化尼群地平进行结合研究。通过该技术，我们表明在完整细胞上，血管加压素能够以剂量依赖性方式增加氚化尼群地平的特异性结合（Kact = 2 nM）。使用一系列血管加压素类似物的药理学研究表明，这种效应是通过V1a血管加压素受体亚型介导的。此外，血管加压素刺激的尼群地平结合对百日咳毒素预处理敏感，百日咳毒素仅影响尼群地平结合位点的最大结合能力。更有趣的是，我们证明血管加压素仍然增加尼群地平与质膜制剂的结合，并且GTP对于这种激素效应是绝对必需的。总之，这些数据证实了V1a血管加压素受体与二氢吡啶钙通道之间通过百日咳毒素敏感的G蛋白存在紧密而直接的偶联。
