Effect of benzyl alcohol on phospholipid transverse mobility in human erythrocyte membrane

Abstract

The effect of benzyl alcohol on the transverse mobility and repartition of phospholipids in the human erythrocyte membrane was investigated using electron spin resonance and morphological modification of red blood cells. Transmembrane internalization rates and equilibrium distribution in red blood cells of short-chain spin-labeled phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were strongly modified by treatment with 10 -70 mM benzyl alcohol. A dual effect was observed: (a) at 4°C and 37°C there was an N-ethylmaleimide-sensitive, long lasting and fully reversible increase in the spin-labeled phosphatidylserine and phosphatidylethanolamine internalization rate; (b) at 37"C, an enhancement of N-ethylmaleimide-insensitive fluxes of all the labeled phospholipids through the membrane occurred. Both effects were dose-dependent. Erythrocytes submitted to benzyl alcohol incubation also showed dose-dependent shape changes : an immediate one from discocytes to echinocytes, followed by a slower N-ethylmaleimide-and ATP-dependent change to stomatocytes. Moreover, benzyl alcohol treatment was shown to lead to enhanced hydrolysis of intracellular ATP. All the effects of benzyl alcohol can be described as an accumulation of labeled phosphatidylethanolamine (and labeled phosphatidylcholine at 37 "C) in the inner leaflet. This can be interpreted as a perturbation of the erythrocyte membrane, leading to an energy-consuming specific increase in aminophospholipid translocase activity, in addition to a slow and passive bidirectional flux of all phospholipids at 37°C.Benzyl alcohol has been observed to modify the activity of numerous membrane proteins [I -41 and more complex mechanisms, for example endocytosis [5]. Its effect on membranous enzyme activity is often related to a change in membrane fluidity, as is the case for other local anaesthetics. Furthermore, it has been postulated that benzyl alcohol has a direct effect on proteins [6]. Inhibition of the fusion stage between lysosomes and endocytic vesicles was also suggested to explain the effect of benzyl alcohol on asialo-glycoprotein receptors [7]. The effect of benzyl alcohol on phospholipid reorientation kinetics and transverse repartition in the red cell membrane is also noteworthy. Transverse distribution of phospholipids in the erythrocyte membrane is asymmetric, mainly regulated by a specific aminophospholipid carrier protein, aminophospholipid translocase [8 -101. Translocase activity in erythrocytes was found to be sensitive to certain membrane perturbations, due to cholesterol/phospholipid ratio variation [ I l l or chlorpromazine incorporation [I 21, for example. Conversely, other amphiphdic drugs do not modify translocase activity [12]. There are several advantages to using benzyl alcohol as a membrane-perturbing agent: (a) this molecule is neutral and does not preferentially invade one of the two membrane leaflets, thus avoiding hemolysis even at a high concentration; (b) being a neutral compound precludes any selective interaction with charg...

Cholesterol- and Sphingolipid-rich Microdomains Are Essential for Microtubule-based Membrane Protrusions Induced by Clostridium difficile Transferase (CDT)

“…Finally, we employed BA as an agent, which is known to increase membrane fluidity and to redistribute components of the liquid ordered phase (24,25). For this purpose, cells were treated with CDT until formation of the first small protrusions was observed, and BA (40 mM) was then added.…”

“…Second, myriocin, a serine palmitoyltransferase inhibitor that blocks sphingosine biosynthesis (41), efficiently reduced protrusion formation caused by CDT. Finally, benzyl alcohol, which increases the fluidity of membranes and destroys lipid rafts (24,25), strongly inhibited protrusion formation.…”

“…For hydrolysis of sphingomyelin to ceramide and phosphorylcholine, cells were treated with 0.5 unit/ml of sphingomyelinase (Smase) from Bacillus cereus (23). For manipulation of membrane fluidity and to redistribute components of the liquid ordered phase, cells were treated with 40 mM benzyl alcohol (BA) after 45 min when CDT had already entered cells (24,25).…”

Ethanol specifically alters the synthesis, acylation and transbilayer movement of aminophospholipids in rat-liver microsomes

“…Translocase activity in erythrocytes has been found to be sensitive to certain membrane perturbations such as a variation in the cholesterol / phospholipid ratio or chlorpromazine incorporation (Rosso et al, 1988). Interestingly, some fluidizing treatments such as benzyl alchohol (Bassé et al, 1992) and ethanol (Schwichtenhovel et al, 1992) increase the rate of APL translocase activity and enhance the passive transverse diffusion of any type of phospholipid.…”

“…Moreover, ethanol (Schwichtenhovel et al, 1992) and other alcohols (Bassé et al, 1992) in vitro profoundly alter the phospholipid flip-flop in human erythrocyte membranes, accelerating the internalization rate of NBD-labeled phospholipids due to an increase in APL translocase activity. There is no experimental evidence on the other hand to describe the effect of ethanol on the transbilayer distribution of APL in biogenic membranes and for this reason we decided to investigate its effect in vitro on the microsomal synthesis and acylation of PE and PS and their translocation between the two leaflets of the membrane bilayer.…”