Conrad Kirk P, Tuna Kubra M, Mestre Cathleen T, Banwatt Esha S, Alli Abdel A
Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL, USA.
Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL, USA.
Physiol Rep. 2020 Oct;8(20):e14592. doi: 10.14814/phy2.14592.
Reports of the stimulated release of extracellular vesicles (EVs) are few, and the mechanisms incompletely understood. To our knowledge, the possibility that the activation of any one of the multitudes of G-protein-coupled receptors (GPCRs) expressed by a single cell-type might increase EV release has not been explored. Recently, we identified the expression of cholecystokinin (CCK), gastrin, gastrin/cholecystokinin types A and/or B receptors (CCKAR and/or -BR), and the bitter taste receptor, TAS2R14 in the human and mouse placenta. specifically, trophoblast. These GPCR(s) were also expressed in four different human trophoblast cell lines. The current objective was to employ two of these cell lines-JAR choriocarcinoma cells and HTR-8/SVneo cells derived from first-trimester human villous trophoblast-to investigate whether CCK, TAS2R14 agonists, and other GPCR ligands would each augment EV release. EVs were isolated from the cell-culture medium by filtration and ultracentrifugation. The preparations were enriched in small EVs (<200 nm) as determined by syntenin western blot before and after sucrose gradient purification, phycoerythrin (PE)-ADAM10 antibody labeling, and electron microscopy. Activation of TAS2R14, CCKBR, cholinergic muscarinic 1 & 3, and angiotensin II receptors, each increased EV release by 4.91-, 2.79-, 1.87-, and 3.11-fold, respectively (all p < .05 versus vehicle controls), without significantly changing EV diameter. A progressive increase of EV concentration in conditioned medium was observed over 24 hr consistent with the release of preformed EVs and de novo biogenesis. Compared to receptor-mediated stimulation, EV release by the calcium ionophore, A23187, was less robust (1.63-fold, p = .08). Diphenhydramine, a TAS2R14 agonist, enhanced EV release in JAR cells at a concentration 10-fold below that required to increase intracellular calcium. CCK activation of HTR-8/SVneo cells, which did not raise intracellular calcium, increased EV release by 2.06-fold (p < .05). Taken together, these results suggested that other signaling pathways may underlie receptor-stimulated EV release besides, or in addition to, calcium. To our knowledge, the finding that the activation of multiple GPCRs can stimulate EV release from a single cell-type is unprecedented and engenders a novel thesis that each receptor may orchestrate intercellular communication through the release of EVs containing a subset of unique cargo, thus mobilizing a specific integrated physiological response by a network of neighboring and distant cells.
关于细胞外囊泡（EVs）受刺激释放的报道较少，其机制也尚未完全明确。据我们所知，单一细胞类型所表达的众多G蛋白偶联受体（GPCRs）中，任何一种受体的激活是否会增加EVs释放的可能性尚未得到研究。最近，我们在人和小鼠胎盘中发现了胆囊收缩素（CCK）、胃泌素、胃泌素/胆囊收缩素A和/或B受体（CCKAR和/或 -BR）以及苦味受体TAS2R14的表达。具体而言，滋养层细胞中存在这些受体。这些GPCRs在四种不同的人滋养层细胞系中也有表达。当前的目标是利用其中两种细胞系——JAR绒毛膜癌细胞和源自孕早期人绒毛滋养层的HTR-8/SVneo细胞——来研究CCK、TAS2R14激动剂以及其他GPCR配体是否会各自增强EVs的释放。通过过滤和超速离心从细胞培养基中分离出EVs。在蔗糖梯度纯化前后，通过syntenin免疫印迹、藻红蛋白（PE）-ADAM10抗体标记以及电子显微镜检测，确定制备物富含小EVs（<200 nm）。TAS2R14、CCKBR、胆碱能毒蕈碱1和3以及血管紧张素II受体的激活，分别使EVs释放增加了4.91倍、2.79倍、1.87倍和3.11倍（与载体对照相比，所有p < 0.05），且未显著改变EVs直径。在长达24小时的时间里，条件培养基中EVs浓度呈逐渐增加趋势，这与预先形成的EVs释放和从头生物合成一致。与受体介导的刺激相比，钙离子载体A23187诱导的EVs释放作用较弱（1.63倍，p = 0.08）。TAS2R14激动剂苯海拉明在浓度比增加细胞内钙所需浓度低10倍的情况下，增强了JAR细胞中的EVs释放。HTR-8/SVneo细胞中CCK的激活并未增加细胞内钙，但使EVs释放增加了2.06倍（p < 0.05）。综上所述，这些结果表明，除了钙之外，其他信号通路可能是受体刺激EVs释放的基础，或者是其补充。据我们所知，多种GPCRs的激活可刺激单一细胞类型释放EVs这一发现是前所未有的，并提出了一个新的观点，即每个受体可能通过释放包含特定独特货物子集的EVs来协调细胞间通讯，从而通过邻近和远处细胞网络调动特定的综合生理反应。
