Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago , Chicago, Illinois.
Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois at Chicago , Chicago, Illinois.
Am J Physiol Gastrointest Liver Physiol. 2018 Oct 1;315(4):G529-G537. doi: 10.1152/ajpgi.00133.2018. Epub 2018 Jun 21.
Bile acid transporters, including the ileal apical sodium-dependent bile acid transporter (ASBT) and the hepatic sodium-taurocholate cotransporting polypeptide (NTCP), are crucial for the enterohepatic circulation of bile acids. Our objective was to develop a method for measuring bile acid transporter activity in real time to precisely evaluate rapid changes in their function. We designed a reporter system relying on a novel probe: cholic acid attached to luciferin via a disulfide-containing, self-immolating linker (CA-SS-Luc). Incubation of human embryonic kidney-293 cells coexpressing luciferase and ASBT with different concentrations of CA-SS-Luc (0.01-1 μM) resulted in bioluminescence with an intensity that was concentration- and time-dependent. The bioluminescence measured during incubation with 1 μM CA-SS-Luc was dependent on the levels of ASBT or NTCP expressed in the cells. Coincubation of CA-SS-Luc with natural bile acids enhanced the bioluminescence in a concentration-dependent manner with kinetic parameters for ASBT similar to those previously reported using conventional methods. These findings suggest that this method faithfully assesses ASBT function. Further, incubation with tyrosine phosphatase inhibitor III (PTPIII) led to significantly increased bioluminescence in cells expressing ASBT, consistent with previous studies showing an increase in ASBT function by PTPIII. We then investigated CA-SS-Luc in isolated mouse intestinal epithelial cells. Ileal enterocytes displayed significantly higher luminescence compared with jejunal enterocytes, indicating a transport process mediated by ileal ASBT. In conclusion, we have developed a novel method to monitor the activity of bile acid transporters in real time that has potential applications both for in vitro and in vivo studies. NEW & NOTEWORTHY This article reports the development of a real-time method for measuring the uptake of bile acids using a bioluminescent bile acid-based probe. This method has been validated for measuring uptake via the apical sodium-dependent bile acid transporter and the sodium-taurocholate cotransporting polypeptide in cell culture and ex vivo intestinal models.
胆酸转运蛋白，包括回肠顶端钠依赖性胆酸转运蛋白（ASBT）和肝脏钠牛磺胆酸共转运蛋白（NTCP），对于胆酸的肠肝循环至关重要。我们的目标是开发一种实时测量胆酸转运蛋白活性的方法，以精确评估其功能的快速变化。我们设计了一种依赖于新型探针的报告系统：通过含有二硫键的自毁连接子将胆酸连接到荧光素上（CA-SS-Luc）。将共表达荧光素和 ASBT 的人胚肾 293 细胞与不同浓度的 CA-SS-Luc（0.01-1 μM）孵育会产生强度与浓度和时间相关的生物发光。在用 1 μM CA-SS-Luc 孵育过程中测量的生物发光取决于细胞中表达的 ASBT 或 NTCP 的水平。CA-SS-Luc 与天然胆酸共同孵育以浓度依赖的方式增强生物发光，其 ASBT 的动力学参数与使用传统方法先前报道的相似。这些发现表明该方法忠实地评估了 ASBT 的功能。此外，用酪氨酸磷酸酶抑制剂 III（PTPIII）孵育可导致表达 ASBT 的细胞中的生物发光显著增加，这与先前的研究一致，表明 PTPIII 可增加 ASBT 的功能。然后，我们在分离的小鼠肠上皮细胞中研究了 CA-SS-Luc。回肠肠上皮细胞的发光明显高于空肠肠上皮细胞，表明这是一种由回肠 ASBT 介导的转运过程。总之，我们开发了一种实时监测胆酸转运蛋白活性的新方法，该方法在体外和体内研究中都具有潜在的应用。
