Milano Serena, Gerbino Andrea, Schena Giorgia, Carmosino Monica, Svelto Maria, Procino Giuseppe
Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.
Department of Sciences, University of Basilicata, Potenza, Italy.
Cell Physiol Biochem. 2018;48(2):847-862. doi: 10.1159/000491916. Epub 2018 Jul 20.
BACKGROUND/AIMS: We recently showed that the β3-adrenoreceptor (β3AR) is expressed in mouse kidney collecting ducts (CD) cells along with the type-2 vasopressin receptor (AVPR2). Interestingly, a single injection of a β3AR selective agonist promotes a potent antidiuretic effect in mice. Before considering the feasibility of chronic β3AR agonism to induce antidiuresis in vivo, we aimed to evaluate in vitro the signaling and desensitization profiles of human β3AR.
Human β3AR desensitization was compared with that of human AVPR2 in cultured renal cells. Video imaging and FRET experiments were performed to dissect β3AR signaling under acute and chronic stimulation. Plasma membrane localization of β3AR, AVPR2 and AQP2 after agonist stimulation was studied by confocal microscopy. Receptors degradation was evaluated by Western blotting.
In renal cells acute stimulation with the selective β3AR agonist mirabegron, induced a dose-dependent increase in cAMP. Interestingly, chronic exposure to mirabegron promoted a significant increase of intracellular cAMP up to 12 hours. In addition, a slow and slight agonist-induced internalization and a delayed downregulation of β3AR was observed under chronic stimulation. Furthermore, chronic exposure to mirabegron promoted apical expression of AQP2 also up to 12 hours. Conversely, long-term stimulation of AVPR2 with dDAVP showed short-lasting receptor signaling, rapid internalization and downregulation and apical AQP2 expression for no longer than 3 h.
Overall, we conclude that β3AR is less prone than AVPR2 to agonist-induced desensitization in renal collecting duct epithelial cells, showing sustained cAMP production, preserved membrane localization and delayed degradation after 12 hours agonist exposure. These results may be important for the potential use of chronic pharmacological stimulation of β3AR to promote antidiuresis overcoming in vivo renal concentrating defects caused by inactivating mutations of the AVPR2.
背景/目的：我们最近发现β3 - 肾上腺素能受体（β3AR）与2型血管加压素受体（AVPR2）一起在小鼠肾集合管（CD）细胞中表达。有趣的是，单次注射β3AR选择性激动剂可在小鼠中产生强效抗利尿作用。在考虑慢性β3AR激动作用在体内诱导抗利尿的可行性之前，我们旨在体外评估人β3AR的信号传导和脱敏特征。
在培养的肾细胞中比较人β3AR与AVPR2的脱敏情况。进行视频成像和FRET实验以剖析急性和慢性刺激下的β3AR信号传导。通过共聚焦显微镜研究激动剂刺激后β3AR、AVPR2和水通道蛋白2（AQP2）的质膜定位。通过蛋白质印迹法评估受体降解。
在肾细胞中，用选择性β3AR激动剂米拉贝隆急性刺激可诱导cAMP剂量依赖性增加。有趣的是，长期暴露于米拉贝隆可使细胞内cAMP显著增加，持续长达12小时。此外，在慢性刺激下观察到β3AR缓慢且轻微的激动剂诱导内化以及延迟下调。此外，长期暴露于米拉贝隆也可使AQP2的顶端表达增加，持续长达12小时。相反，用去氨加压素（dDAVP）长期刺激AVPR2显示受体信号传导持续时间短、快速内化和下调，且顶端AQP2表达不超过3小时。
总体而言，我们得出结论，在肾集合管上皮细胞中，β3AR比AVPR2更不易发生激动剂诱导的脱敏，在激动剂暴露12小时后显示出持续的cAMP产生、保留的膜定位和延迟降解。这些结果对于慢性药理学刺激β3AR以促进抗利尿、克服由AVPR2失活突变引起的体内肾浓缩缺陷的潜在用途可能很重要。
