Background/Aims: In nucleated cells, bile acids may activate cation channels subsequently leading to entry of Ca2+. In erythrocytes, increase of cytosolic Ca2+ activity triggers eryptosis, the suicidal death of erythrocytes characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis is triggered by bile duct ligation, an effect partially attributed to conjugated bilirubin. The present study explored, whether bile acids may stimulate eryptosis. Methods: Phosphatidylserine exposing erythrocytes have been identified utilizing annexin V binding, cell volume estimated from forward scatter, cytosolic Ca2+ activity determined using Fluo-3 fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Results: The exposure of human erythrocytes to glycochenodesoxycholic (GCDC) and taurochenodesoxycholic (TCDC) acid was followed by a significant decrease of forward scatter and significant increase of Fluo-3 fluorescence, ceramide abundance as well as annexin V binding. The effect on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Bile acids stimulate suicidal cell death, an effect paralleled by and in part due to Ca2+ entry and ceramide. The bile acid induced eryptosis may in turn lead to accelerated clearance of circulating erythrocytes and, thus, may contribute to anemia in cholestatic patients.

Erythrocytes express Ca2+ permeable cation channels [1,2,3,4,5,6,7]. Ca2+ entry through those channels triggers phosphatidylserine exposure due to cell membrane scrambling [7,8,9,10,11] and cell shrinkage due to activation of Ca2+-sensitive K+ channels [12,13,14]. Cell membrane scrambling and cell shrinkage are hallmarks of eryptosis, the suicidal death of erythrocytes [15]. Eryptotic erythrocytes are engulfed by macrophages and are thus rapidly eliminated from circulating blood [16,17,18]. Thus, eryptosis contributes to establishment of anemia.

Triggers of eryptosis include bile duct ligation, an effect attributed in part to conjugated bilirubin [19]. Bile duct ligation further increases the blood concentration of bile acids [20], which have been shown to activate cation channels [21] and to increase cytosolic Ca2+ activity [22,23].

The present study explored whether the bile acids glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) are capable of stimulating Ca2+ entry into and thus trigger eryptosis of human erythrocytes drawn from healthy individuals. GCDC has previously been shown to trigger apoptosis [24,25,26,27], whereas TCDC-induced apoptosis [28,29] may be prevented by simultaneous activation of the phosphoinositide-3-kinase (PI3K)/Akt/NF-κB survival pathway [29]. Thus, the effect of those two bile acids was compared.

Erythrocytes, solutions and chemicals

Erythrocytes were provided by healthy volunteers. The study was approved by the ethics committee of the Medical Faculty of the Heinrich-Heine-University Düsseldorf. Erythrocytes were incubated in vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4 at 37°C for 48 hours. Where indicated, the bile acids glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (Sigma, Schnelldorf, Germany) were added at the indicated concentrations.

Annexin V-binding and forward scatter

After incubation under the respective experimental condition, cells were washed in Ringer solution containing 5 mM CaCl2 and then stained with Annexin-V-Fluos (Roche, Mannheim, Germany) in this solution for 20 min under protection from light. In the following, the forward scatter of the cells was determined and annexin V fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Canto II (BD, Heidelberg, Germany).

Hemolysis

After incubation at 37°C, the samples were centrifuged (3 min at 400 g, RT), and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatants was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis. To correct for absorption of bile acids contained in the supernatant the standard curve contained the same concentration of bile acids.

Cytosolic Ca2+

After incubation erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM (Calbiochem, Bad Soden, Germany) in Ringer solution containing 5 mM CaCl2 and 2 µM Fluo-3/AM. The cells were incubated at 37°C for 20 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µl Ringer. Then, Ca2+-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS calibur (BD, Heidelberg, Germany).

Ceramide abundance

To determine ceramide abundance at the erythrocyte surface, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 hour at 37°C with 1 ìg/ml anti-ceramide antibody (clone MID 15B4; Alexis, Grünberg, Germany) in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:5. According to the supplier, under physiological in vitro and in vivo conditions the antibody is highly specific for ceramide and does not cross-react with sphingomyelin, cholesterol or other phospholipids. After two washing steps with PBS-BSA, cells were stained for 30 minutes with polyclonal fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse IgG and IgM specific antibody (Pharmingen, Hamburg, Germany) diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. Samples were then analysed at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS calibur (BD, Heidelberg, Germany).

Statistics

Data are expressed as arithmetic means ± SEM. Statistical analysis was made using paired ANOVA with Tukey's test as post-test, as appropriate. n denotes the number of different erythrocyte specimens studied.

To investigate whether the bile acids glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) stimulate Ca2+ entry in human erythrocytes drawn from healthy individuals, Fluo3 fluorescence has been employed to estimate cytosolic Ca2+ activity ([Ca2+]i). As illustrated in Fig. 1, a 24 hour incubation of erythrocytes in the presence of GCDC was followed by an increase of [Ca2+]i, an effect reaching statistical significance at 500 µM GCDC concentration. Similarly, exposure to TCDC was followed by a marked increase of [Ca2+]i, an effect reaching statistical significance at 62.5 µM TCDC concentration.

Fig. 1

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on cytosolic Ca2+ concentration in erythrocytes. A. Histograms of Fluo3 fluorescence in representative experiments of erythrocytes exposed for indicated time points to Ringer without and with different concentrations (as indicated) of glycochenodeoxycholate (one representative of n=4 is shown). B. Arithmetic means ± SEM (n = 4) of the geo means of Fluo3 fluorescence in erythrocytes following a 24 hour incubation to Ringer without (control) or with indicated concentrations of GCDC. C. Histograms of Fluo3 fluorescence in representative experiments of erythrocytes exposed for different time points to Ringer without and with different concentrations of taurochenodeoxycholate (TCDC, one representative of n=4 is shown). D. Arithmetic means ± SEM (n = 4) of the geo means of Fluo3 fluorescence in erythrocytes following a 24 hour incubation to Ringer without (control) or with different concentrations of TCDC. *** indicates p<0.001 significant difference from the respective value in the absence of bile acids.

Fig. 1

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on cytosolic Ca2+ concentration in erythrocytes. A. Histograms of Fluo3 fluorescence in representative experiments of erythrocytes exposed for indicated time points to Ringer without and with different concentrations (as indicated) of glycochenodeoxycholate (one representative of n=4 is shown). B. Arithmetic means ± SEM (n = 4) of the geo means of Fluo3 fluorescence in erythrocytes following a 24 hour incubation to Ringer without (control) or with indicated concentrations of GCDC. C. Histograms of Fluo3 fluorescence in representative experiments of erythrocytes exposed for different time points to Ringer without and with different concentrations of taurochenodeoxycholate (TCDC, one representative of n=4 is shown). D. Arithmetic means ± SEM (n = 4) of the geo means of Fluo3 fluorescence in erythrocytes following a 24 hour incubation to Ringer without (control) or with different concentrations of TCDC. *** indicates p<0.001 significant difference from the respective value in the absence of bile acids.

Close modal

An increase of cytosolic Ca2+ activity is expected to induce cell shrinkage, which should be reflected by a decrease of forward scatter in FACS analysis. As shown in Fig. 2, exposure of erythrocytes for 24 hours to GCDC was followed by a decrease of forward scatter, an effect reaching statistical significance at 250 µM GCDC concentration. Similarly, exposure to TCDC was followed by a decrease of forward scatter, an effect reaching statistical significance at 125 µM GCDC concentration.

Fig. 2

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on erythrocyte forward scatter. A, B. Erythrocytes were incubated with and without different concentrations of GCDC. (A) Histograms of the erythrocyte forward scatter of a representative experiment (out of n=14) following exposure for 24 hours to Ringer without and with different concentrations of GCDC. (B) Arithmetic means ± SEM (n = 10-14) of the erythrocyte forward scatter following a 24 hour incubation to Ringer without (control) or with indicated concentrations of GCDC. C,D. Erythrocytes were incubated with and without different concentrations of TCDC. (C) Histograms of Fluo3 fluorescence in representative experiments of erythrocytes exposed for 24 hours to Ringer without and with different concentrations of TCDC (one representative of n=10 is shown). (D) Arithmetic means ± SEM (n = 10) of the erythrocyte forward scatter following a 24 hour incubation to Ringer without (control) or with different concentrations of TCDC. *** indicates p<0.001 significant difference from the respective value in the absence of bile acids.

Fig. 2

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on erythrocyte forward scatter. A, B. Erythrocytes were incubated with and without different concentrations of GCDC. (A) Histograms of the erythrocyte forward scatter of a representative experiment (out of n=14) following exposure for 24 hours to Ringer without and with different concentrations of GCDC. (B) Arithmetic means ± SEM (n = 10-14) of the erythrocyte forward scatter following a 24 hour incubation to Ringer without (control) or with indicated concentrations of GCDC. C,D. Erythrocytes were incubated with and without different concentrations of TCDC. (C) Histograms of Fluo3 fluorescence in representative experiments of erythrocytes exposed for 24 hours to Ringer without and with different concentrations of TCDC (one representative of n=10 is shown). (D) Arithmetic means ± SEM (n = 10) of the erythrocyte forward scatter following a 24 hour incubation to Ringer without (control) or with different concentrations of TCDC. *** indicates p<0.001 significant difference from the respective value in the absence of bile acids.

Close modal

An increase of cytosolic Ca2+ activity is further expected to trigger cell membrane scrambling. Accordingly, phosphatidylserine exposure was determined by measurement of annexin V-binding. As illustrated in Fig. 3, the percentage of phosphatidylserine expressing erythrocytes was low following a 24 hour incubation in Ringer solution without bile acids. In sharp contrast exposure of erythrocytes for 24 hours to GCDC was followed by an increase of annexin V binding, an effect reaching statistical significance at 500 µM GCDC concentration. Similarly, exposure to TCDC was followed by an increase of annexin V binding, an effect reaching statistical significance at 125 µM GCDC concentration.

Fig. 3

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on phosphatidylserine exposure of erythrocytes. A, B. Erythrocytes were incubated with and without different concentrations of GCDC. (A) Histograms of the erythrocyte annexin V-binding of a representative experiment following exposure for 24 hours to Ringer without and with GCDC. (B) Arithmetic means ± SEM (n = 14) of the erythrocyte annexin V-binding following a 24 hour incubation to Ringer without or with indicated concentrations of GCDC. C,D. Erythrocytes were incubated with and without different concentrations of TCDC. (C) Histograms of annexin V-binding in a representative experiment of erythrocytes exposed for 24 hours to Ringer without and with different concentrations of TCDC (one representative of n=10 is shown). (D) Arithmetic means ± SEM (n = 10) of the erythrocyte annexin V-binding following a 24 hour incubation to Ringer without or with different concentrations of TCDC. *** indicates p<0.001 significant difference from the respective value in the absence of bile acids.

Fig. 3

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on phosphatidylserine exposure of erythrocytes. A, B. Erythrocytes were incubated with and without different concentrations of GCDC. (A) Histograms of the erythrocyte annexin V-binding of a representative experiment following exposure for 24 hours to Ringer without and with GCDC. (B) Arithmetic means ± SEM (n = 14) of the erythrocyte annexin V-binding following a 24 hour incubation to Ringer without or with indicated concentrations of GCDC. C,D. Erythrocytes were incubated with and without different concentrations of TCDC. (C) Histograms of annexin V-binding in a representative experiment of erythrocytes exposed for 24 hours to Ringer without and with different concentrations of TCDC (one representative of n=10 is shown). (D) Arithmetic means ± SEM (n = 10) of the erythrocyte annexin V-binding following a 24 hour incubation to Ringer without or with different concentrations of TCDC. *** indicates p<0.001 significant difference from the respective value in the absence of bile acids.

Close modal

In order to test whether the effect of bile acids on phosphatidylserine translocation required entry of extracellular Ca2+, erythrocytes were incubated for 48 hours in the absence or presence of bile acids in the presence or nominal absence of extracellular Ca2+. As apparent from Fig. 4, a removal of extracellular Ca2+ significantly reduced the effect of 500 µM GCDC on annexin-V-binding. However, even in the absence of extracellular Ca2+, GCDC significantly increased the percentage of annexin-V-binding erythrocytes (Fig. 4a). Similarly, removal of extracellular Ca2+ significantly blunted the effect of 125 µM TCDC on annexin-V-binding (Fig. 4b). Similar to GCDC, TCDC significantly increased the percentage of annexin-V-binding erythrocytes in the absence of extracellular Ca2+ (Fig. 4b). Thus, the bile acids triggered erythrocyte cell membrane scrambling in part but not exclusively by stimulating entry of extracellular Ca2+.

Fig. 4

Ca2+ sensitivity of bile acid -induced phosphatidylserine exposure. A, B. Arithmetic means ± SEM (n = 12) of the percentage of annexin V-binding erythrocytes after a 24 hour treatment with Ringer solution without (mCa, open bars) or with (Ringer, closed bars) Calcium in the (A) absence (-) or presence(+) of 500 µM GCDC or (B) absence (-) or presence(+) of 125 µM TCDC. *(p<0.05),**(p<0.01), ***(p<0.001) indicates significant difference from the absence of bile acids, #(p<0.05), ##(p<0.01) indicates significant difference from the presence of Ca2+ (ANOVA).

Fig. 4

Ca2+ sensitivity of bile acid -induced phosphatidylserine exposure. A, B. Arithmetic means ± SEM (n = 12) of the percentage of annexin V-binding erythrocytes after a 24 hour treatment with Ringer solution without (mCa, open bars) or with (Ringer, closed bars) Calcium in the (A) absence (-) or presence(+) of 500 µM GCDC or (B) absence (-) or presence(+) of 125 µM TCDC. *(p<0.05),**(p<0.01), ***(p<0.001) indicates significant difference from the absence of bile acids, #(p<0.05), ##(p<0.01) indicates significant difference from the presence of Ca2+ (ANOVA).

Close modal

As cell membrane scrambling could be triggered by ceramide even without increase of [Ca2+]i, additional experiments explored whether the bile acids increase ceramide abundance in the cell membrane. As illustrated in Fig. 5, exposure of erythrocytes for 24 hours to 500 µM GCDC was followed by a significant increase of ceramide abundance. Consistently, exposure of erythrocytes for 24 hours to 125 µM TCDC also induced a significant increase of ceramide abundance.

Fig. 5

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on ceramide abundance. A, B. Arithmetic means ± SEM (n = 10) of ceramide abundance in erythrocytes following incubation for 24 hours to Ringer solution without (closed bars) or with (open bars) presence of (A) 500 µM GCDC or (B) 125 µM TCDC. *(P<0.05),**(p<0.01) indicates significant difference from the absence of bile acids (ANOVA).

Fig. 5

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on ceramide abundance. A, B. Arithmetic means ± SEM (n = 10) of ceramide abundance in erythrocytes following incubation for 24 hours to Ringer solution without (closed bars) or with (open bars) presence of (A) 500 µM GCDC or (B) 125 µM TCDC. *(P<0.05),**(p<0.01) indicates significant difference from the absence of bile acids (ANOVA).

Close modal

A final series of experiments explored whether the bile acids triggered hemolysis. As illustrated in Fig. 6, exposure of erythrocytes for 24 hours to 500 µM GCDC or to 125 µM TCDC was followed by a significant increase of the percentage of hemolysed erythrocytes. Taken together, these data indicate that the bile acids GCDC and TCDC induce Ca2+ entry into erythrocytes, ceramide production, eryptosis and hemolysis.

Fig. 6

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on hemolysis. A, B. Arithmetic means ± SEM (n = 10) of the percentage hemolysed erythrocytes following incubation for 24 hours to Ringer solution without (closed bars) or with (open bars) presence of (A) 500 µM GCDC or (B) 125 µM TCDC. **(p<0.01) indicates significant difference from the absence of bile acids (ANOVA).

Fig. 6

Effect of glycochenodeoxycholate (GCDC) and taurochenodeoxycholate (TCDC) on hemolysis. A, B. Arithmetic means ± SEM (n = 10) of the percentage hemolysed erythrocytes following incubation for 24 hours to Ringer solution without (closed bars) or with (open bars) presence of (A) 500 µM GCDC or (B) 125 µM TCDC. **(p<0.01) indicates significant difference from the absence of bile acids (ANOVA).

Close modal

The present study reveals a novel effect of bile acids, i.e. the stimulation of Ca2+ entry, cell membrane scrambling and cell shrinkage. Accordingly, bile acids are potent stimulators of suicidal erythrocyte death or eryptosis. The concentrations required for the effect are higher than those (50 - 200 µM) required for stimulation of apoptosis [24,25,29].

The effect of the bile acids on cell membrane scrambling was partially due to Ca2+ entry from the extracellular space leading to increase of cytosolic Ca2+ activity ([Ca2+]i). Accordingly, the cell membrane scrambling following exposure to the bile acids was blunted by removal of extracellular Ca2+. Presumably, Ca2+ entered through Ca2+ permeable cation channels [30].

The increase of [Ca2+]i presumably further accounts for the observed erythrocyte shrinkage following bile acid treatment. An increase of [Ca2+]i is followed by activation of Ca2+ sensitive K+ channels with subsequent cell shrinkage due to K+ exit, cell membrane hyperpolarization, Cl- exit and thus cellular loss of KCl with water [14]. The effect of GCDC on [Ca2+]i reaches statistical significance only at a concentration of 500 µM whereas statistical significance of cell shrinkage is already observed at 250 µM. Nevertheless, the data are compatible with an increase of [Ca2+]i at a GCDC concentration of 250 µM and cannot be taken as evidence for Ca2+ insensitive cell shrinkage.

The stimulation of eryptosis by bile acids presumably contributes to the enhanced eryptosis and anemia during cholestasis and hepatic failure. Stimulation of eryptosis was observed following bile duct ligation [19] and in patients with liver disease [19]. The effect has been attributed to increased plasma levels of conjugated bilirubin [19] but could have been in part due to enhanced bile acid concentration. In any case, the stimulation of eryptosis was due to a component in plasma, as exposure of erythrocytes drawn from healthy individuals to plasma isolated from patients was followed by triggering of eryptosis [19].

Eryptosis has been observed in a variety of further clinical disorders, such as chronic kidney disease (CKD) [31,32,33,34], Hemolytic Uremic Syndrome [35], dehydration [36], hyperphosphatemia [37], phosphate depletion [38], iron deficiency [16], diabetes [39], sepsis [40], malignancy [30], malaria [41,42,43], sickle-cell disease [30], beta-thalassemia [30], Hb-C and G6PD-deficiency [30], as well as Wilsons disease [44]. Moreover, a wide variety of xenobiotics and endogenous substances have been shown to stimulate eryptosis [30,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65].

Phosphatidylserine exposing erythrocytes are recognized by macrophages [66], which engulf and subsequently degrade phosphatidylserine-exposing cells [67]. As a result, eryptotic cells are rapidly cleared from circulating blood [16]. The stimulation of eryptosis thus leads to anemia, if the accelerated loss of circulating erythrocytes is not compensated by a similarly increased formation of new erythrocytes. Following bile duct ligation, anemia develops despite enhanced formation of new erythrocytes, as reflected by increased percentage of reticulocytes [19].

Phosphatidylserine exposing erythrocytes are further known to adhere to the vascular wall [68,69,70,71,72], and foster blood clotting [68,73,74]. Thus, triggering of eryptosis may compromize the microcirculation. As a matter of fact, eryptosis has been suggested to participate in the vascular injury during metabolic syndrome [75].

In conclusion, the present observations reveal that bile acids stimulate suicidal erythrocyte death or eryptosis. The effect may contribute to the anemia and may cause a derangement of microcirculation in several hepatic diseases and cholestasis.

The authors acknowledge the meticulous preparation of the manuscript by Tanja Loch. This study was supported by the Carl-Zeiss-Stiftung, the Deutsche Forschungsgemeinschaft, (SFB974, KFO217, LA2558/4-1, LA315/4-3, LA315/6-1 and RTG1949), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research), and the Alexander von Humboldt Foundation (SKA2010).

The authors of this manuscript state that they have no conflicts of interest to declare.

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E. Lang, V.I. Pozdeev; and K.S. Lang and P.A. Lang contributed equally to this work.

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