An ultrastructural study of the effects of acidic phospholipid substitutions on calcium phosphate precipitation in anionic liposomes

Publisher Website
Google Scholar
Abstract
A model membrane system was used to investigate the ability of specific membrane constituents to modulate the precipitation of calcium phosphate. Intraliposomal precipitation was induced in phosphate-encapsulated liposomes composed of 7:2:1 molar mixtures of phosphatidylcholine (PC), dicetyl phosphate (DCP), and cholesterol (Chol) by ionophore-supported (X-537A) Ca2+ uptake. Extraliposomal precipitation occurred when these reactions were initiated in metastable external solutions. In this case, the endogenously formed crystals penetrated through the enclosing lipid bilayers and seeded the external solution phase. Transmission electron microscopy (TEM) was used to monitor the effect of acidic phospholipids [phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG)] on the precipitation reactions when these molecular species were incorporated into the liposome membranes. Compared with the precipitation reactions in 7PC:2DCP:1Chol liposomes containing no acidic phospholipids, calcium phosphate formation in the presence of monoester phosphate (PA) and amino-(PS) phospholipids was inhibited. Analyses of the lipid-mineral interactions in PA-containing (10 mol%) liposomes revealed close physical contact between the small crystals of apatite and the inner lipid bilayers; there was only minimal extraliposomal precipitation. A few small crystals adhered to the external surfaces of the liposomes. In PS-containing liposomes, lipid-mineral interactions were dependent upon the DCP content of the lipid membrane. Discrete clusters of crystals formed within the interior aqueous compartment when intraliposomal precipitation was initiated in 7PC:2DCP:1Chol liposomes doped with up to 10 mol% PS. There was no evidence for specific associations between these crystals and the enclosing lipid bilayers. In contrast, the liposomes clustered around extraliposomally formed crystals, with the lipid membranes adhering tightly to the exposed crystal surfaces. These crystallipid interactions were reversed when the DCP component was omitted from the liposome membrane (7PC:1PS:1Chol liposomes). These results suggest that PS may be localized preferentially on the outer membrane surface in the presence of DCP but concentrated on the inner aspect in its absence. No such interactions were observed in PI or PG-containing liposomes. The liposome-mediated precipitation events were not affected in these preparations. The data suggest that the inhibition of calcium phosphate formation resulted from specific interactions between the nascent crystals and lipid species present in the liposome membrane. The molecular conformation of the head group, the molecular geometry of the phospholipids in the membrane, and the relative affinity of the incorporated species for Ca2+ were key determinats of these interactions.