A novel bioluminescence-based method to investigate uptake of bile acids in living cells

Abstract

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.

Intestinal Absorption of Bile Acids in Health and Disease

“…ASBT does not appear to have a strong preference for conjugated compared to unconjugated bile acids (92). However, CA and its conjugated derivatives appear to have a lower affinity (~ 15-35 μM) than CDCA and its derivatives (~3-10 μM) (92,244,325,327,377,422). TLCA appears to be a high-affinity substrate of ASBT (244), while DCA species have an intermediate affinity between CA and CDCA (175,422).…”

“…Because ASBT is an intestinal transporter that absorbs relatively large molecules, a great deal of work has been devoted to using this transport system to aid in the absorption of drugs via bile acid-conjugated prodrugs (151,346). Several groups have demonstrated that bile acids conjugated to synthetic molecules are substrates for ASBT and other bile acid transporters, such as NTCP (25,246,346,377). For example, studies have shown that the antiviral acyclovir conjugate to CDCA (acyclovir valylchenodeoxycholate) has a favorable affinity to ASBT and enhanced the oral availability of acyclovir by twofold in rats (385).…”

S-acylation modulates the function of the apical sodium-dependent bile acid transporter in human cells

“…Assessment of ASBT activity by CA-SS-Luc uptake was performed as recently described (34). Briefly, cells were washed twice at room temperature with uptake buffer containing 110 mM NaCl (with sodium) or choline chloride (without sodium), 4 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 45 mM mannitol, 5 mM glucose, 10 mM HEPES (pH 7.4), and 100 µM TC.…”

“…Human ASBT cDNA was previously generated in our laboratory and subcloned into pSF-CMV-Puro-COOH-TEV-V5 (Oxford Genetics) (34). To generate the ASBT C314S mutant, site-directed mutagenesis was performed with the Quikchange II XL kit (Agilent) as per the manufacturer's instructions using the primers: 5'-TCTGCCTTGTTTTTTCCATGACTTTTCTTGT ATGCCACATAAAATC-3' and 5'-GATTTTATGTGGCATACAAGAAAAGTCAT GGAAAAAACAAGGCAGA-3'.…”

Caged luciferins for bioluminescent activity-based sensing

“…FFA-SS-Luc has been used to elucidate the role of macronutrient uptake in metabolic disorders [2830]. In a similar vein, Alrefai and co-workerset al developed cholic acid-functionalized cleavable luciferins to target bile acid transport proteins and quantify bile acid transporter activity in cell culture [31].…”