Physical Properties of Erythrocyte Ghosts That Determine Susceptibility to Secretory Phospholipase A2

Abstract

Artificial membranes may be resistant or susceptible to catalytic attack by secretory phospholipase A 2 (sPLA 2 ) depending on the physical properties of the membrane. Living cells are normally resistant but become susceptible during trauma, apoptosis, and/or a significant elevation of intracellular calcium. Intact erythrocytes and ghosts were studied to determine whether the principles learned from artificial systems apply to biological membranes. Membrane properties such as phospholipid and/or protein composition, morphology, and microscopic characteristics (e.g. fluidity) were manipulated by preparing ghosts under different experimental conditions such as in the presence or absence of divalent cations with or without ATP. The properties of each membrane preparation were assessed by biochemical and physical means (fluorescence spectroscopy and electron and two-photon microscopy using the membrane probes bis-pyrene and laurdan) and compared with sPLA 2 activity. The properties that appeared most relevant were the degree of phosphatidylserine exposure on the outer face of the membrane and changes to the membrane physical state detected by bis-pyrene and laurdan. Specifically, vulnerability to hydrolysis by sPLA 2 was associated with an increase in bilayer order apparently reflective of expansion of membrane regions of diminished fluidity. These results argue that the general principles identified from studies with artificial membranes apply to biological systems.

Relationship between Erythrocyte Membrane Phase Properties and Susceptibility to Secretory Phospholipase A2

“…The two-photon excitation images were collected on an Axiovert 35 inverted microscope (Zeiss, Thornwood, NY) at the Laboratory for Fluorescence Dynamics (Urbana, Il) as described previously (12,13,21). The excitation source was a titanium-sapphire laser (Coherent, Palo Alto, CA) tuned to 780 nm and pumped by a FIGURE 1: Effects of temperature on fluorescence emission of laurdan in erythrocytes.…”

“…In previous studies, it was observed that erythrocytes normally resist the action of sPLA 2 , but that upon the addition of a calcium ionophore such as ionomycin, they become susceptible (12). This increase in susceptibility appears to be linked to an accompanying increase in the order of membrane lipids (12,13). Nevertheless, one question that remained unresolved was whether the critical issue for increased susceptibility was the average level of membrane order or the spatial distribution of membrane regions of differential order.…”

Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes

“…Two-photon laurdan images were obtained at the Laboratory for Fluorescence Dynamics (University of California, Irvine) as described previously (20,21). Laser emission was 780 or 790 nm (depending on laser stability and performance each day).…”

“…10, 11) could be the fact that intracellular hemoglobin may quench the fluorescence of DPH and laurdan in the inner leaflet through energy transfer (49). Moreover, optical absorbance of emitted light by hemoglobin also alters the measured value of laurdan GP (20). Means to correct for these phenomena have been published (20,50).…”

Thermo-sensitive response based on the membrane fluidity adaptation inParamecium multimicronucleatum

“…Laurdan in the lipid phase shows spectral sensitivity with a red shift of the emission maximum when passing from a gel to a liquid crystalline phase (Chong and Wong, 1993;Parasassi et al, 1990;Parasassi et al, 1991;Sheffield et al, 1995). Based on this property, laurdan has been used to estimate the fluidity of the membrane in various living cells (Chapman et al, 1995;Harris et al, 2001;Mamdouh et al, 1998;Palleschi and Silvestroni, 1996;Sasaki et al, 2006;Vest et al, 2004;Yu et al, 1996).…”