The following chemicals were used. RPMI-1640 powder (Cytiva, Tokyo, Japan); fetal bovine serum (FBS), goat anti-rabbit immunoglobin (Ig) G (H+L) cross-adsorbed secondary antibody, Alexa Fluor™ 594 (Thermo Fisher Scientific, Waltham, MA, USA); thapsigargin (Tg), Tween20 (Fujifilm Wako Chemicals, Osaka, Osaka, Japan); A23187 (Cayman Chemicals, Ann Arbor, MI, USA); rabbit polyclonal anti-Orai1 antibody against 18 amino acid peptide from near the amino terminus (ANA SPEC, Fremont, CA, USA), mouse monoclonal anti-STIM1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-Orai1 antibody against extracellular loop between transmembrane (TM)3 and TM4, C-KKQPGQPRPTSKP (Abnova, Neihu District., Taipei, Taiwan); anti-rabbit secondary antibody conjugated to horse radish peroxidase (HRP) (MBL, Tokyo, Japan); ImmunoStar LD, MagCapture™ TamavidinR2-Rev (Fuji Film Wako Chemicals, Osaka, Osaka, Japan); Fluo8/acetoxy methyl (AM) (AAT Bioquest, Pleasanton, CA, USA); cholic acid (CA; 3$\alpha$,7$\alpha$,12$\alpha$-trihydroxy-5$\beta$-cholan-24-oic acid), deoxycholic acid (DCA; 3$\alpha$,12$\alpha$-dihydroxy-5$\beta$-cholan-24-oic acid), dinitrophenyl (DNP)-bovine serum albumin (BSA), anti-DNP IgE, TritonX-100, ethylene glycol-bis (2-aminoethylether)-n,n,n^′,n^′-tetraacetic acid (EGTA), p-nitrophenyl-N-acetyl-$\beta$-D-glucosaminide, Fura2/AM, (+)Biotin N-hydroxysuccinimide ester, HCl, bisbenzimide H33342 trihydrochloride (Hoechst33342) (Sigma-Aldrich, St. Louis, MO, USA); thin-layer chromatography (TLC) aluminum sheets Silica gel 60 F254, thickness 0.2 mm (Merck, St. Louis, MO, USA); and ULTRARIPA® Kit for Lipid Raft (Biodynamics Laboratory Inc., Tokyo, Japan); tetrahydrofuran, triethylamine, ethyl chlorocarbonate, NaBH₄, NaHCO₃, Na₂SO₄, benzene, isopropanol, acetic acid, dioxan, tributylamine, ammonia solution, N, N-Dimethylformamide (Kanto Chemicals, Tokyo, Japan).

CDCA (3$\alpha$,7$\beta$-dihydroxy-5$\beta$-cholan-24-oic acid) and UDCA (3$\alpha$,7$\beta$-dihydroxy-5$\beta$-cholan-24-oic acid) were purchased from Mitsubishi Tanabe Pharma (Tokyo, Japan).

Taurine-conjugated CA (TC), taurine-conjugated UDCA (TUDC), taurine-conjugated CDCA (TCDC), taurine-conjugated DCA (TDC), glycine-conjugated CA (GC), glycine-conjugated UDCA (GUDC), glycine-conjugated CDCA (GCDC), and glycine-conjugated DCA (GDC) were provided by Dr. T. Hoshita (Hiroshima University, Hiroshima, Japan).

All bile acids and alcohols are presented in Fig. 1. Bile alcohols were synthesized according to a previously reported method [22, 23].

Fig. 1.

Chemical structure of bile acids and bile alcohols.

The bile acids presented in Fig. 1 (5 g) were dissolved in 100 mL anhydrous tetrahydrofuran; 3 mL triethylamine was added, and 2 mL ethyl chlorocarbonate was added dropwise. After stirring at room temperature (~20 °C) for 2 h, 6 g NaBH₄ in 35 mL water was added gradually. The reaction mixture was stirred at room temperature for 12 h, after which 35 mL of water was added, and the mixture was stirred at room temperature for 5 min. Next, 100 mL of 1 M HCl was added to the reaction mixture; the acidified reaction mixture was extracted using ethyl acetate ester, and washed with water to neutral pH. Subsequently, 5% NaHCO₃ was added to remove unreacted bile acids, followed by washing with water, which was removed using anhydrous Na₂SO₄. The solvent was evaporated, and ethyl acetate ester was added to the residue to obtain crystals or precipitates. The purity of each sample was determined using thin-layer chromatography (TLC). (development solvent: benzene: isopropanol: acetic acid = 3:1:0.1).

Rf values: 5$\beta$-cholan-3$\alpha$,7$\alpha$,12$\alpha$,24-tetrol (C-OH) 0.218, 5$\beta$-cholan-3$\alpha$,7$\beta$,24-triol (UDC-OH) 0.443, 5$\beta$-cholan-3$\alpha$,7$\alpha$,24-triol (CDC-OH) 0.406, 5$\beta$-cholan-3$\alpha$,12$\alpha$,24-triol (DC-OH) 0.412

Purity: C-OH 98%, UDC-OH 90%, CDC-OH 98%, DC-OH 98%

The synthesis route is illustrated in Fig. 2. Briefly, bile acid, CA, CDCA, UDCA, and DCA (1.0 g) [1] were dissolved in anhydrous dioxan (20 mL), 0.28 mL tributylamine was added, and 0.25 mL ethyl chlorocarbonate was added dropwise [22, 23] [2]. After stirring the reaction mixture at room temperature for 2 h, 0.25 mL 30% ammonia solution was added dropwise. After extraction with ethyl acetate ester and evaporation of the solvent, each 0.7–0.8 g 24-amido 5$\beta$-cholan residue was collected [3]. Each 24-amido 5$\beta$-cholan residue 100 mg/20 mL 0.1 M NaHCO₃ (pH 8.0) was added to 85 mg D-biotin N-succinimidyl ester in 4 mL N, N-Dimethylformamide and incubated overnight at room temperature [24, 25]. Each biotin derivative was extracted using ethyl acetate ester, and 20–60 mg of each biotin derivative [4] was collected after evaporating the ethyl acetate ester.

Fig. 2.

The synthesis of bile acid-biotin derivatives. Synthesis route of biotinylated bile acid derivatives. Briefly, bile acids (CA, chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), and deoxycholic acid (DCA)) [1] were reacted with ethyl chlorocarbonate in the presence of tributylamine ([1]$\to$[2]), followed by treatment with ammonia to yield 24-amido 5$\beta$-cholan residues ([2]$\to$[3]). Each residue was then conjugated with D-biotin N-succinimidyl ester to produce the corresponding biotin derivatives ([3]$\to$[4]). The resulting products were extracted and collected. Detailed experimental procedures are provided in the Methods and Materials section.

All compounds (CA biotin derivative (C-biotin), UDCA biotin derivative (UDC-biotin), CDCA biotin derivative (CDC-biotin), DCA biotin derivative (DC-biotin)) were confirmed to be biotinylated based on the presence of a peak at ppm specific to biotin in the Nuclear Magnetic Resonance apparatus (ECA600, Akishima, Tokyo, JEOL).

RBL-2H3 cells were cultured in RPMI-1640 medium supplemented with 15% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin. The RBL-2H3 cells were donated to Prof. Ozawa by Dr. Beven, who was studying overseas. A mycoplasma test using a fluorescence staining method was performed for the RBL-2H3 cells, and the cells were found to be mycoplasma-free. We confirmed their identity by functional characterization. Specifically, the cells released $\beta$-hexosaminidase, a specific marker enzyme of mast cells, in response to stimulation, which is consistent with their established use as a mast cell model. For Antigen–Antibody (Ag-Ab) stimulation, the cells were treated with 0.5 µg/mL anti-dinitrophenol (anti-DNP) IgE overnight. The concentrations are presented as final.

We measured the activity of released $\beta$-hexosaminidase as an indicator of mast cell degranulation according to Ozawa et al. [26]. RBL-2H3 cells were seeded at a density of 4 $\times$ 10⁴ cells/well in a 96-well plate. For sensitization, 0.5 µg/mL anti-DNP IgE was added. After overnight culture, the cells were washed with Siraganian buffer (below prepared Siraganian buffer; 10 mM HEPES, 125.4 mM NaCl, 11.5 mM glucose, 5.9 mM KCl, 1.2 mM MgCl₂; pH 7.4) prepared immediately prior; 1 mM CaCl₂ and 0.1% BSA were added, and the cells were treated with 40 µL bile acids or bile alcohols in prepared Siraganian buffer at each concentration for 10 min. Then, 10 µL dinitrophenol-bovine serum albumin (DNP-BSA, 20 ng/mL) was added as antigen (Ag) and incubated at 37 °C for 15 min. Non-sensitized cells were stimulated for 15 min with either 2.0 µM Tg or 0.5 µM A23187. As a positive control, Triton X-100 solution (0.2%) was added to lyse the cell membrane. After centrifugation at 90 $\times$g for 5 min at 4 °C, each supernatant (10 µL) was transferred to a new 96-well plate, and 10 µL 1.3 mg/mL p-nitrophenyl-N-acetyl-$\beta$-D-glucosaminide in sodium citrate buffer (0.04 M, pH 4.5) was added as the substrate. The plate was incubated at 37 °C for 1 h, and the reaction was stopped by adding 250 µL carbonate–bicarbonate buffer (0.2 M, pH 10.0). The absorbance was measured at 415 nm with a reference filter at 600 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Waltham, MA, USA). The percentage of degranulation was calculated after subtracting spontaneous release using the following formula:

$$\% \text{degranulation}={\mathrm{Abs}}_{(415-\mathrm{600 }\mathrm{n}\mathrm{m})}/$$ $${\text{Abs}}_{\text{Triton }X-\mathrm{100 }(415-\mathrm{600 }\mathrm{n}\mathrm{m})}\times 100$$

The intracellular calcium concentration [Ca²⁺]_i was measured using Fura2/AM as an intracellular calcium indicator in the presence of Ca²⁺. RBL-2H3 cells sensitized on glass-bottom dishes as mentioned earlier were loaded with 1 µM Fura2/AM and bile acid or bile alcohol in prepared Siraganian buffer containing 1 mM CaCl₂ and incubated for 30 min at 37 °C and 95% CO₂. After washing twice with the prepared Siraganian buffer, 900 µL prepared Siraganian buffer was added. The cells were excited every 10 s at 340/380 nm and fluorescence images were captured at 510 nm using an ARGUS HiSCA calcium imaging system (Hamamatsu Photonics, Iwata, Shizuoka, Japan). Three minutes after the start of capture, the RBL-2H3 cells were stimulated with 20 ng/mL DNP–BSA [27].

[Ca²⁺]_i was measured using Fluo8/AM under Ca²⁺-free conditions. RBL-2H3 cells in glass-bottom dishes overnight at 37 °C and 95% CO₂ were loaded with 1 µM Fluo8/AM as an intracellular calcium indicator and bile acid or bile alcohol in Siraganian buffer containing 1 mM CaCl₂ for 30 min at 37 °C and 95% CO₂. After washing twice with Siraganian buffer without CaCl₂, 900 µL of Ca-free Siraganian buffer with 0.1 mM EGTA was replaced. Every 10 s, the fluorescence image was captured using a fluorescence microscope equipped with excitation (490 nm) and emission (520 nm) filters (BIOREVO 9000; Keyence, Osaka, Osaka, Japan). Then, 2.0 µM Tg was added to RBL-2H3 cells 1 min after capture, and 1 mM CaCl₂ was added 4 min later.

RBL-2H3 cells grown on glass coverslips were used for fluorescence staining. RBL-2H3 cells sensitized as mentioned earlier were washed twice with the prepared Siraganian buffer, pretreated with CA, UDCA, CDCA, DCA, C-OH, UDC-OH, CDC-OH or DC-OH for 10 min, and stimulated with Ag for 2.5 min. The cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature followed by washed three times with 10 mM Glycine-PBS for 3 min, permeabilized with 0.1% Triton X-100 for 5 min, washed three times again, blocked with 3% BSA–PBS for 10 min, and incubated with anti-STIM1 antibody (1:100) in 1% BSA–PBS overnight at 4 °C. After washing with 0.1% BSA–PBS four times for 3 min each, the cells were incubated with anti-rabbit IgG (H+L) Secondary Antibody, Alexa Fluor™ 594 (1:2000, Thermo Fisher Scientific, Waltham, MA, USA) and Hoechst33342 for 1 h at room temperature. After washing four times for 3 min each, the cells were mounted in mounting medium Moiwol 4-88 Reagent and prepared as a slide.

A fluorescence microscope (BZ-X810; Keyence, Osaka, Osaka, Japan) equipped with excitation/emission (360/460, 560/630 nm) filters was used to capture the images.

Fig. 3 indicates Bile Acid Biotin Derivative-binding Protein Extraction and Analysis. The ULTRARIPA® Kit for Lipid Raft extracts cell membrane lipid rafts under non-denaturing conditions [28]. After washing, the RBL-2H3 cells were lysed using RIPA A buffer, sonicated for 10 min, and centrifuged at 18,000 $\times$g for 5 min at 4 °C. The supernatant was removed, and the remaining pellet was dissolved in ultra-RIPA B buffer and centrifuged at 18,000 $\times$g for 5 min at 4 °C. The supernatant was used as the lipid raft fraction including Orai1 protein.

Fig. 3.

Purification of binding protein to BA-biotin derivative by Tamavidin® 2-REV magnetic beads. Isolation of lipid raft fractions and analysis of proteins bound to biotinylated bile acid derivatives. Lipid rafts from rat basophilic leukemia (RBL-2H3) cell membranes were extracted under non-denaturing conditions using the ULTRARIPA® Kit. Biotinylated bile acid derivatives were incubated with MagCapture Tamavidin2-REV for 30 min. After washing three times with PBS containing 0.025% Tween-20, the MagCapture Tamavidin2-REV–bound bile acid derivatives were incubated with lipid raft fractions. Proteins bound to the steroidal nucleus were then analyzed by SDS-PAGE and western blotting. Detailed experimental procedures are provided in the Methods and Materials section.

MagCapture™ TamavidinR2-Rev was used to purify the biotinylated molecules using a competitive biotin elution method [29].

The bead suspension (40 µL) was washed with washing buffer, and each bile acid biotin derivative which were used at approximate amounts: C-biotin and CDC-biotin ~24 nmol, UDC-biotin and DC-biotin ~12 nmol was added to the washed beads, rotated for 30 min, and washed three times with 0.05% Tween20 in PBS after adding the lipid raft, including the collected supernatant. The solution was resuspended in a rotator for 1 h at 4 °C, the supernatant was removed, and the beads were washed three times with washing buffer.

The beads obtained in the first round, 2$\times$ sample buffer, were added and boiled for 5 min, and the supernatant was loaded and separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for silver staining analysis of proteins bound to the steroidal nucleus of each bile acid-biotin derivative.

The beads obtained in the second round were mixed well with 15 µL of excess biotin solution (2 mM biotin in PBS) and incubated for 15 min at 4 °C. The tube was placed on a magnetic stand, and the supernatant was recovered. The supernatant was subjected to western blot analysis. Laemmli sample buffer was added to the supernatant, boiled for 3 min, loaded, and separated using 10% SDS-PAGE for analysis of proteins bound to the steroidal nucleus of each bile acid biotin derivative. Proteins were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skimmed milk, the membrane was treated with a 1:200-diluted anti-Orai1 antibody and a 1:20,000-diluted secondary rabbit antibody. The membranes were analyzed by chemiluminescence using an Immuno Star LD system with a chemiluminescence detector (LAS 4000 Mini; Cytiva, Tokyo, Japan).

The cells were treated overnight with or without 0.5 µg/mL anti-Orai1 antibody against the extracellular loop between transmembrane domains (TM)3 and TM4 of the Orai1 subunit before stimulation with Ag-Ab, Tg or A23187 to investigate the interaction between bile alcohols and the Orai1 extracellular loop. For Ag-Ab stimulation, the cells were sensitized to 0.5 µg/mL anti-DNP IgE and 0.5 µg/mL anti-Orai1 antibody overnight. For Tg and A23187 stimulation, the cells were used without prior sensitization. These cells were then subjected to degranulation.

The experimental results were expressed as mean $\pm$ standard error (S.E.). Analysis of variance (ANOVA) followed by Dunnett’s test were used to compare two or more groups (R version 4.0.3 (2020-10-10)/R Core Team: A language and environment for statistical computing, Open-source software, available at https://www.r-project.org/). Statistical significance was set at p 0.05.

We investigated whether bile acids and their C₂₄ bile alcohols suppress mast cell activation across different activation pathways, Ag–Ab crosslinking, Tg, or the calcium ionophore A23187. All tested bile acids and C₂₄ bile alcohols inhibited the degranulation of RBL-2H3 cells by up to 10% stimulated with the Ag–Ab, Tg, and A23187 (Fig. 4). Bile acids and alcohols significantly inhibited the degranulation induced by all stimuli. The inhibitory effects of bile acids paralleled the hydrophobicity of each bile acid. The order of hydrophobicity, determined via chromatography using a reverse-phase column, was CA UDCA CDCA DCA [30]. The more hydrophobic CDCA and DCA were active at relatively low concentrations. The correlation with hydrophobicity suggests that membrane interactions or binding to the hydrophobic domains of target proteins contribute to the inhibitory effect. Both bile acids and alcohols are amphipathic; however, alcohols possess stronger surfactant properties, and the number of hydroxyl groups influences bile acids more than that of alcohols. Higher concentrations of tested bile acids promoted mast cell degranulation without stimulators (Supplementary Fig. 1).

Fig. 4.

Bile acids and bile alcohols inhibit degranulation. Degranulation of RBL-2H3 cells treated with bile acids or bile alcohols. Treatment: Cells sensitized with anti-DNP IgE were incubated with the indicated bile acids (CA, UDCA, CDCA, DCA; (A)) or bile alcohols (5$\beta$-cholan-3$\alpha$,7$\alpha$,12$\alpha$,24-tetrol (C-OH), 5$\beta$-cholan-3$\alpha$,7$\beta$,24-triol (UDC-OH), 5$\beta$-cholan-3$\alpha$,7$\alpha$,24-triol (CDC-OH), 5$\beta$-cholan-3$\alpha$,12$\alpha$,24-triol (DC-OH); (B)). Stimulation: Cells were stimulated with DNP-BSA (20 ng/mL), thapsigargin (Tg) (2 µM), or A23187 (0.5 µM). Triton X-100 served as a positive control. Assay: $\beta$-Hexosaminidase activity in the supernatant was measured as an indicator of degranulation. Data presentation: Mean $\pm$ SEM (n = 3). Statistical significance: ****p 0.0001, ***p 0.001, **p 0.01, *p 0.05 vs. 0 µM. Detailed experimental procedures are provided in the Methods and Materials section.

To examine the inhibitory mechanism of bile acids and alcohols on degranulation, we investigated whether the inhibition of degranulation was due to reduced intracellular Ca²⁺ ([Ca²⁺]_i) elevation after Ag–Ab stimulation with or without bile acid/alcohol pretreatment. The increase in [Ca²⁺]_i is essential for mast cell degranulation, and the immediate increase in [Ca²⁺]_i after stimulation decreased rapidly following treatment with all bile acids or alcohols at concentrations that inhibited degranulation (Fig. 5). The increase in [Ca²⁺]_i associated with Ag–Ab stimulation involves Ca²⁺ efflux from the ER, followed by extracellular Ca²⁺ influx. Bile acids and alcohols inhibit the extracellular Ca²⁺ influx.

Fig. 5.

Pre-treatment with bile acid and bile alcohol inhibited the sustained increase in [Ca²⁺]_𝐢 induced by Ag-Ab stimulation. Calcium imaging of RBL-2H3 cells treated with bile acids or bile alcohols. Treatment: Cells sensitized with anti-DNP IgE were loaded with 1 µM Fura2/AM and pretreated with the indicated bile acids (a) or bile alcohols (b) in Ca²⁺ (+, 1 mM) Siraganian buffer. After washing, cells were maintained in Ca²⁺ (+, 1 mM) buffer. Stimulation: Three minutes after image capture, cells were stimulated with DNP-BSA (20 ng/mL). Detection: Changes in intracellular calcium were monitored using a Calcium Imaging System ARGUS HiSCA (Hamamatsu Photonics, Japan). Data representation: (a) Bile acids: black, green, azure, pink, and mazarine lines represent control, CA 1000 µM, UDCA 500 µM, CDCA 100 µM, and DCA 100 µM, respectively. (b) Bile alcohols: black, mazarine, pink, green, and azure lines represent control, C-OH 100 µM, UDC-OH 100 µM, CDC-OH 70 µM, and DC-OH 70 µM, respectively. Detailed experimental procedures are provided in the Methods and Materials section.

These results indicated that bile acids and alcohols primarily interfered with the influx phase, possibly by disrupting STIM1 aggregation, STIM1–Orai1/TRPC coupling, or SOC channel function.

We evaluated whether bile acid and bile alcohols could open SOC channels in 2.0 µM Tg-stimulated RBL-2H3 cells in Ca²⁺-free Siraganian buffer, followed by Ca²⁺ re-addition (Fig. 6). Upon Ca²⁺ loading, bile acid-pretreated cells showed increased intracellular Ca²⁺ levels, comparable to those in control cells. However, [Ca²⁺]_i in bile alcohol-pretreated cells after Ca²⁺ loading decreased by 25–37% compared to [Ca²⁺]_i in control cells (Area under the curve: Control 1.0, CA 1.18, UDCA, 1.11, CDCA 0.91, DCA 0.81, C-OH 0.75, UDC-OH 0.63, CDC-OH 0.66, DC-OH 0.66). These results indicated that bile acids cannot inhibit SOC channel opening, whereas bile alcohols can partially inhibit SOC channels. However, in the presence of Ca²⁺, bile acids and alcohols completely inhibited Ca²⁺ entry into the cells (Fig. 5).

Fig. 6.

1.0 mM Ca²⁺ Addition to RBL-2H3 cells stimulated with 2 µM Tg in Ca²⁺-free buffer increased [Ca²⁺]_𝐢 with or without bile acid or bile alcohol. Time course of cytosolic Ca²⁺ concentration changes in RBL-2H3 cells treated with bile acids or bile alcohols and stimulated with thapsigargin (Tg). Treatment and Stimulation: Cells were treated with the indicated bile acids (a) or bile alcohols (b). In Ca²⁺-free extracellular solution, 2 µM Tg was added at 1 min to induce Ca²⁺ release from the endoplasmic reticulum, followed by addition of 1.0 mM Ca²⁺ at 5 min. Detection: Changes in fluorescence over time were monitored using a fluorescence microscope (BIOREVO 9000, Keyence, Osaka, Osaka, Japan). Data representation: (a) Bile acids: black, green, azure, pink, and mazarine lines represent control, CA 1000 µM, UDCA 500 µM, CDCA 100 µM, and DCA 100 µM, respectively. (b) Bile alcohols: black, green, azure, pink, and mazarine lines represent control, C-OH 100 µM, UDC-OH 100 µM, CDC-OH 70 µM, and DC-OH 70 µM, respectively. Detailed experimental procedures are provided in the Methods and Materials section.

In an antigen-antibody reaction, the following events occur in sequence: STIM1 aggregation, interaction with Orai1, and then interaction with TRPCs [9, 10, 11]. However, in the case of Tg stimulation in the calcium-free state shown in Fig. 6, both interactions, STIM1 with Orai1 or TRPCs, occur, then calcium is added to the buffer, which may result in calcium influx from Orai1 and TRPCs. Further the differences between the effects of bile acids and alcohols were probably caused by differences in the side chains. SOC entry involves the CRAC and TRPCs channels. However, TRPC1, which acts as an SOC channel, does not induce SOCE on its own [6, 31]. More hydrophobic bile alcohols may have a higher affinity for CRAC channels than bile acids, and may block CRAC channels.

We investigated the effects of UDCA and UDC-OH on STIM1 aggregation in Ag-Ab-stimulated RBL-2H3 cells (Fig. 7). We compared STIM1 aggregation in Ag–Ab-stimulated RBL-2H3 cells pretreated with 250 µM UDCA, 30 µM UDC-OH, or the control. Other tested bile acids and bile alcohols results are shown in Supplementary Fig. 2. We did not observe any apparent effects of tested bile acids or alcohols on STIM1 aggregation in the Ag-Ab-stimulated RBL-2H3 cells.

Fig. 7.

UDCA and UDC-OH did not inhibit antigen antibody-induced stromal interaction molecule 1 (STIM1) aggregation. Immunofluorescence analysis of STIM1 in RBL-2H3 cells treated with UDCA or UDC-OH. Treatment and Staining: Sensitized cells were pretreated with UDCA (250 µM) or UDC-OH (30 µM) and then stimulated with DNP-BSA. Cells were fixed, permeabilized, blocked, and immunostained with anti-STIM1 antibody and Alexa Fluor™ 594-conjugated secondary antibody. Imaging: Images were captured using a fluorescence imaging microscope (BZ-X810, KEYENCE, Osaka, Osaka, Japan). Scale bar, 20 µm. Detailed experimental procedures are provided in the Methods and Materials section.

The inhibitory mechanism likely acts downstream of STIM1 activation, consistent with interference at the Orai1/TRPC channel level and SOC function.

Initially, bile acids had hydrophilic and hydrophobic portions; therefore, they were amphipathic in nature. Taurine- and glycine-conjugated bile salts (TC, TCDC, TDC, GC, GCDC, and GDC) were more hydrophilic than unconjugated bile acids. However, they also had weak but significant inhibitory effects (10–37%) on the degranulation of stimulated RBL-2H3 cells with Ag-Ab and Tg (Supplementary Fig. 3).

These results support the hypothesis that bile acid and alcohol steroid nuclei inhibit the degranulation of activated RBL-2H3 cells.

We synthesized bile acid-biotin derivatives because the inhibitory effect of bile acid and bile alcohol on degranulation is likely derived from the hydroxylated steroid nucleus. Fig. 2 shows the synthetic route in which the carboxyl group [1] is converted to an amido group [3] after adding biotin [4].

C-biotin, UDC-biotin, CDC-biotin, DC-biotin, biotin derivatives of the four bile acids, CA, UDCA, CDCA, and DCA, exerted similar inhibitory effects on degranulation following stimulation (Ag–Ab, Tg, and A23187) (Fig. 8). These results indicate the significance of the structure of the steroid nucleus in the inhibition of degranulation.

Fig. 8.

Bile acid-biotin derivatives also inhibit histamine release. Degranulation assay of RBL-2H3 cells treated with biotinylated bile acid derivatives. Treatment and Stimulation: Sensitized cells were pretreated with C-biotin, UDC-biotin, CDC-biotin, or DC-biotin and stimulated with DNP-BSA (20 ng/mL). Non-sensitized cells were treated similarly and stimulated with Tg (2 µM) or A23187 (0.5 µM). Assay: $\beta$-D-glucosaminidase activity in the supernatant was measured as an indicator of degranulation (Using the same procedure as in Fig. 4). Data presentation: Mean $\pm$ SEM (n = 3). Statistical significance: ****p 0.0001, **p 0.01, *p 0.05 vs. 0 µM. Detailed experimental procedures are provided in the Methods and Materials section.

Orai1 protein exists in lipid rafts that form the CRAC channel. Fig. 3 shows a scheme for lipid raft fractionation and bile acid-binding lipid raft protein purification. The lipid raft and detergent-resistant membranes were solubilized with less degeneration using the ULTRARIPA B buffer from the ULTRARIPA® Kit for Lipid Raft, and the supernatant contained the lipid raft fraction after centrifugation. The supernatant was incubated with MagCapture™ Tamavidin®2-Rev, which binds to the bile acid biotin derivative via biotin overnight at 4 °C. Protein binding to the bile acid biotin derivative was eluted with a 0.2 M biotin solution (Fig. 3). Samples treated with bile acid derivatives showed a band of approximately 50 kDa. Therefore, an anti-Orai1 antibody was used for western blotting, and each derivative bound to Orai1, although the amount varied (Fig. 9). The binding of Orai1 to each bile acid derivative may induce bile acid and bile alcohol inhibitory effects on degranulation.

Fig. 9.

Bile acid-biotin derivative binds to Orai1. Analysis of proteins bound to bile acid biotin derivatives in lipid raft fractions. Lipid raft fractions were incubated with MagCapture™ Tamavidin®2-Rev beads bound to the indicated bile acid biotin derivatives. Proteins bound to the derivatives were eluted with biotin and analyzed by SDS-PAGE and western blotting (the procedure shown in Fig. 3). Detailed experimental procedures are provided in the Methods and Materials section.

As bile acid biotin derivatives can bind to the Orai1 protein, this binding site is probably involved in Ca²⁺ entry. To investigate the inhibitory site, RBL-2H3 cells were pretreated overnight with an anti-Orai1 antibody against the extracellular loop between TM3 and TM4 (C-KKQPGQPRPTSKP). The results showed that CDC- and DC-OH induced the inhibition of Ag–Ab- and Tg-induced degranulation was not observed in anti Orai1 antibody-sensitized cells. Moreover, C- and UDC-OH-induced inhibition of Ag–Ab- and Tg-induced degranulation were observed in anti Orai1 antibody-sensitized cells (Fig. 10). This result indicates that the CDC- and DC-OH steroidal structure binding site of Orai1 is within the TM3–TM4 extracellular loop, and the C- and UDC-OH steroidal structure binding site is not an anti-Orai1 antibody binding site within TM3–TM4, but may reside in the TM1–TM2 extracellular loop.

Fig. 10.

Bile alcohol binding sites to Orai1 is not anti-Orai1 antibody against the extracellular loop between transmembrane (TM)3 and TM4 binding sites. Effect of anti-Orai1 antibody on degranulation of RBL-2H3 cells treated with bile alcohols. Treatment: Cells were incubated overnight with anti-Orai1 antibody targeting the extracellular loop between TM3 and TM4. Anti-Orai1 antibody-treated (orange bars) and untreated (blue bars) cells were pretreated with the indicated bile acids or bile alcohols and then stimulated with Ag-Ab (20 ng/mL), Tg (2 µM), or A23187 (0.5 µM) for 15 min. Assay: $\beta$-D-glucosaminidase activity in the supernatant was measured to determine % degranulation. (Using the same procedure as in Fig. 4). Data presentation: Mean $\pm$ SEM (n = 3). Statistical significance: **p 0.01, *p 0.05. Detailed experimental procedures are provided in the Methods and Materials section.