Activation of membrane cholesterol by 63 amphipaths

Abstract

A few membrane-intercalating amphipaths have been observed to stimulate the interaction of cholesterol with cholesterol oxidase, saponin and cyclodextrin, presumably by displacing cholesterol laterally from its phospholipid complexes. We now report that this effect, referred to as cholesterol activation, occurs with dozens of other amphipaths, including alkanols, saturated and cis- and trans-unsaturated fatty acids, fatty acid methyl esters, sphingosine derivatives, terpenes, alkyl ethers, ketones, aromatics and cyclic alkyl derivatives. The apparent potency of the agents tested ranged from 3 microM to 7 mM and generally paralleled their octanol/water partition coefficients, except that relative potency declined for compounds with >10 carbons. Some small amphipaths activated cholesterol at a membrane concentration of approximately 3 mol per 100 mol of bilayer lipids, about equimolar with the cholesterol they displaced. Lysophosphatidylserine countered the effects of all these agents, consistent with its ability to reduce the pool of active membrane cholesterol. Various amphipaths stabilized red cells against the hemolysis elicited by cholesterol depletion, presumably by substituting for the extracted sterol. The number and location of cis and trans fatty acid unsaturations and the absolute stereochemistry of enantiomer pairs had only small effects on amphipath potency. Nevertheless, potency varied approximately 7-fold within a group of diverse agents with similar partition coefficients. We infer that a wide variety of amphipaths can displace membrane cholesterol by competing stoichiometrically but with only limited specificity for weak association with phospholipids. Any number of other drugs and experimental agents might do the same.

FIGURE 1

Effect of amphipaths on red cells. Panel A: cholesterol oxidase lysis assay. Washed RBCs (0.24 μl) suspended in 0.2 ml PBS (pH 7.4, final volume) were pre-treated in wells with 0 (●), 1.0 (▽), 1.3 (■), 1.5 (◇) and 2.0 (▲) mM nonanoic acid [26]for 5 min at 37 °C. Cholesterol oxidase (0.4 IU) was then added, the plate incubated at 37 °C, optical absorbance determined periodically and fractional hemolysis calculated. Panel B: The time intervals for 50% lysis (horizontal line in panel A) were replotted so as to estimate H, the concentration required to achieve 50% lysis at one hour (horizontal line in panel B). Panel C: saponin lysis assay. Red cells were pre-treated as in Panel A with oleic acid [60]at 0 μM (●), 5 μM (▽), 6 μM (■), 7 μM (◇), and 8 μM (▲). Saponin (4 μg) was added to each well; the plates were incubated at room temperature, optical absorbance determined periodically and fractional hemolysis calculated. Panel D: The time intervals for 50% lysis by saponin (horizontal line in Panel C) were replotted as a function of oleic acid concentration so as to estimate S, the concentration required to achieve 50% lysis at 15 min (horizontal line in Panel D).

FIGURE 2

Relationship of the potency of amphipaths in promoting red cell lysis by cholesterol oxidase to their partition coefficients. −log H (potency, molar) was plotted versus ClogP (computed log octanol/water partition coefficient) for 63 agents numbered as in Table 1. The linear least-squares fit to the 31 points with ClogP < 4 had a slope of 0.90 and an R² of 0.68.

FIGURE 3

Correlation of the ability of 33 amphipaths to promote red cell lysis by cholesterol oxidase (H) and saponin (S). The linear least squares fit had a slope of 0.958 and an R² = 0.992.

FIGURE 4

Effect of amphipaths on the transfer of [³H]cholesterol from red cells to cyclodextrin. Panel A: RBCs were pre-equilibrated in PBS (pH 7.4) with cyclodextrin-cholesterol mixtures in proportions that do not appreciably alter RBC cholesterol (see Methods). The mixtures were centrifuged and the pellet and supernatant saved. The equilibrated cells were pulse-labeled with [³H]cholesterol and washed. Aliquots of the pre-equilibrated cyclodextrin-cholesterol acceptor were mixed with ethanol alone (0.1% final, ▽) or containing 0.14 mM hexyl ether [40] (final, ○). 60 μl aliquots of packed [³H]cholesterol-labeled cells were then added to 5.94 ml of the pre-equilibrated acceptor and the transfer of label followed at 10 °C and plotted as percent of input radioactivity transferred. Panel B: As in panel A, except that the treatments were either ethanol alone (0.1% final, ▽) or containing 4 mM nonanoic acid [26](final, ○) and the PBS was at pH 6 to increase the membrane uptake of the fatty acid.

FIGURE 5

Inhibition of hemolysis by lysophosphatidylserine (LPS). Panel A: Washed RBCs (0.24 μl) were suspended in wells containing 0.2 ml PBS (final volume) plus 1 nmol linoleic acid [57]and 0 (○), 0.2 (▽), 0.4 (□) or 0.8 (◇) nmol LPS. Cholesterol oxidase (0.4 IU) was added and the samples incubated at 37 °C. Optical absorbance was determined periodically and fractional hemolysis calculated. Panel B: To paired aliquots of 0.24 μl packed RBCs in wells containing 200 μl PBS (final) was added (1) 70 nmol 1-octanol[9]; (2) 16 nmol hexylether [40]; (3) 2 nmol 1-dodecanol [39]and (6) 1 nmol linoleic acid [57]. LPS (0.3–0.6 nmol) was added to one of each pair (right, striped bars). Finally, 0.4 IU of cholesterol oxidase was added and the plate incubated at 37 °C for 2 h before optical absorbance was determined and fractional hemolysis calculated.

FIGURE 6

Amphipaths protect of red cells from hemolysis by cholesterol depletion. Panel A: To deplete RBC cholesterol by roughly half, replicate aliquots of 1.2 μl packed cells were incubated in wells containing 1.33 mg methyl-β-cyclodextrin in 200 μl PBS (final) for 8 min at room temperature. Amphipaths were added immediately and optical absorbance determined 15 min later. Fractional hemolysis was calculated relative to undepleted controls. The n-alcohols were: heptanol [2]( ○), octanol [9]( ▽), decanol [31]( ▲), tridecanol [43]( ◇), tetradecanol [48]( ■), hexadecanol [54](●). Panel B: As in Panel A, but with these fatty acids: lauric [41]( ■), elaidic [59]( ▲), linoleic [57]( ◇), palmitic [55]( ▽), and oleic [60](●).

FIGURE 7

Effect of amphipaths on HMG-CoA reductase activity. Fibroblasts were preincubated overnight in growth medium containing 5% lipoprotein-deficient serum. The medium was replaced by 2 ml PBS containing 0.5–0.8% ethanol ± agents, the concentrations of which were scaled to their relative potency, given in Table 1. The flasks were incubated at 37 °C for 1 h, following which HMGR activity and cell protein were determined in duplicate and HMGR activities (pmol mevalonate/min/mg cell protein) plotted relative to controls lacking agents. Bar 1, ethanol control; bar 2, 160 μM 1-decanol [31]; bar 3, 160 μM hexyl ether [40]; bar 4, 20 μM linoleic acid [57]; bar 5, 80 μM 1-dodecanol [39] Composite of representative experiments.

FIGURE 8

Variation of alcohol efficacy with chain length. The mole fractions of 1-hexanol through 1-decanol in red cell membranes needed to promote their susceptibility to cholesterol oxidase were calculated from H values and RBC partition coefficients. R² = 0.53. See section 2 of Discussion.