The role of membrane cholesterol in determining bile acid cytotoxicity and cytoprotection of ursodeoxycholic acid

Abstract

In cholestatic liver diseases, the ability of hydrophobic bile acids to damage membranes of hepatocytes/ductal cells contributes to their cytotoxicity. However, ursodeoxycholic acid (UDC), a hydrophilic bile acid, is used to treat cholestasis because it protects membranes. It has been well established that bile acids associate with and solubilize free cholesterol (CHOL) contained within the lumen of the gallbladder because of their structural similarities. However, there is a lack of understanding of how membrane CHOL, which is a well-established membrane stabilizing agent, is involved in cytotoxicity of hydrophobic bile acids and the cytoprotective effect of UDC. We utilized phospholipid liposomes to examine the ability of membrane CHOL to influence toxicity of individual bile acids, such as UDC and the highly toxic sodium deoxycholate (SDC), as well as the cytoprotective mechanism of UDC against SDC-induced cytotoxicity by measuring membrane permeation and intramembrane dipole potential. The kinetics of bile acid solubilization of phosphatidylcholine liposomes containing various levels of CHOL was also characterized. It was found that the presence of CHOL in membranes significantly reduced the ability of bile acids to damage synthetic membranes. UDC effectively prevented damaging effects of SDC on synthetic membranes only in the presence of membrane CHOL, while UDC enhances the damaging effects of SDC in the absence of CHOL. This further demonstrates that the cytoprotective effects of UDC depend upon the level of CHOL in the lipid membrane. Thus, changes in cell membrane composition, such as CHOL content, potentially influence the efficacy of UDC as the primary drug used to treat cholestasis.

Fig. 1

A schematic drawing of the correlation between intramembrane lipid dipole moments and molecular orientation/packing density. When lipids are packed tightly, all membrane internal dipole moments (indicated by the black arrows) are oriented in an orderly fashion, thus giving a large dipole potential (top). When lipid packing is disrupted, lipid dipoles are aligned at various angles, effectively canceling each other out (bottom). This would give a small dipole potential. Note that this intramembrane dipole originates from lipid carbonyl dipole and the molecular dipoles of interfacial water molecules and is different from the headgroup dipole arising from phosphate-choline dipolar interactions.

Fig. 2

DLPC membrane permeability to calcein was determined as a function of deoxycholic acid (SDC) or ursodeoxycholic acid (UDC) concentration with or without 30% CHOL When exposed SDC, the value of C₅₀ was ∼ 0.56 mM with no CHOL (○) and shifted to ∼0.9 mM with 30 mol% CHOL (●). In UDC, the value of C₅₀ was ∼2.2 mM with no CHOL (Δ) and shifted to ∼3.9 mM with 30 mol% CHOL (▲). The slope of the concentration dependence of increase in calcein permeability in SDC was calculated to be ∼267, while the corresponding slope for UDC is ∼50, indicating more gradual increase in the case of UDC.

Fig. 3

Changes in the DLPC membrane dipole potential as a function of bile acid concentrations. Both bile acids lead to dose-dependent decrease in the dipole potential of DLPC liposomes (p < 0.05). UDC (◊) was found to yield less profound decrease in the dipole potential than SDC (■).

Fig. 4

Effects of 30 mol% of CHOL incorporated in DLPC membranes on the dipole potential. When compared with the dipole potential values in liposomes with no CHOL in Fig. 3, CHOL was found to have minimal effect when the liposomes were exposed to SDC (■) (ANOVA two-factor p>0.05). In contrast, the addition of 30% CHOL to DLPC membranes made liposomes more resistant to the effects of UDC on dipole potential (Δ) (ANOVA two-factor p<0.05).

Fig. 5

A comparative plot illustrating the effect of DLPC membranes with or without 30 mol% CHOL on the cytoprotective effect of UDC against the membrane-destabilizing action of a combination of UDC and SDC (1:1). The cytoprotective effect of UDC against the membrane disruptive action of SDC is more effective with 30 mol% CHOL in the membrane (◊).

Fig. 6

Effects of 30 or 50 mol% CHOL in the membrane on the cytoprotective effect of UDC against SDC in various bile acid combinations. Pure DLPC (white bars), DLPC liposomes containing 30 mol% CHOL in the membrane (gray bars) or DLPC liposomes containing 50 mol% CHOL (black bars) were exposed to PBS (first group), 5 mM SDC (second group), 5 mM UDC (third group), or SDC/UDC mixtures at 1:1, 1:2, 1:3, 1:4 and 1:5. The total concentrations of all SDC/UDC mixtures were fixed at 5 mM. Data is presented as mean+/−SD of 3 individual experiments.

Fig. 7

(A) The levels of undissolved lipids indicated by the turbidity measurements at 350 nm were monitored over time. It shows that without CHOL in the membrane, 5 mM SDC alone was highly damaging and completely solubilized DLPC lipids within 10 s. The presence of CHOL in the membrane at 30 mol% was unable to protect the DLPC membrane while 50 mol% CHOL display some level of protection. (B) The levels of undissolved lipids indicated by the turbidity measurements at 350 nm were monitored over time. It shows that without CHOL in the membrane, a SDC/UDC combination was mostly as damaging as SDC alone (both at 5 mM). The presence of CHOL in the membrane at 30 or 50 mol% prevented lipids from being solubilized by bile acids, demonstrating the ability of CHOL in the membrane to influence the cytoprotective effect of UDC against SDC attack.

Fig. 8

(A) Values of C₅₀, which is the bile acid concentration at which 50% of maximal permeability to calcein was obtained, was plotted against D₅₀, which is the bile acid concentration at which 50% of the maximal dipole potential was obtained. A linear correlation shown in the plot indicates that the change in membrane integrity and permeability is likely caused by the ability of bile acids to influence lipid orient and packing density within the membrane (r²=0.947). (B) The values of the membrane dipole potential of liposomes treated with various bile acid combinations were plotted against Ln (k), the rate of solubilization, for corresponding treatments. A linear correlation (r²=0.78) was found between the two parameters, further suggesting that the ability of bile acids to alter membrane integrity is tied to their ability to change lipid packing density.