The mechanisms behind the membrane-mediated effects of ethanol were examined via the interaction of ethanol with phospholipid bilayers at hydration levels of 10-12 water molecules per lipid. 2H and 31P nuclear magnetic resonance (NMR) spectroscopy was used to monitor deuterated water and ethanol and the headgroups and acyl chains of neutral phospholipids. Ethanol was found to interact strongly with both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) bilayers, giving 2H NMR quadrupolar splittings for CH3CD2OH between 6.3 and 9.4 kHz. The quadrupolar splittings for ethanol in gel-phase lipids remained well resolved and were not significantly larger than those in the L alpha phase, suggesting that little or no ethanol was bound in the hydrocarbon interior of the bilayer. Ethanol binding significantly altered the orientation of the lipid headgroups, as shown with headgroup-deuterated PC bilayers. The entire lengths of the acyl chains were significantly disordered by the ethanol interaction, evidenced by significant reductions in the 2H NMR order parameters of the chains. The disordering corresponds to an increase in the area per lipid by an estimated 6% with one ethanol molecule per lipid, and a total of 18% with a second ethanol per lipid. This pronounced area increase is presumably caused by the disruption of lipid packing in the rigid region of the glycerol backbone rather than in the acyl chains, since the order of hydrocarbon chains is not affected to a significant degree by incorporation of alkanes and long-chain alcohols into the hydrocarbon interior.(ABSTRACT TRUNCATED AT 250 WORDS)
The stability of two-component liposomes composed of the polymerizable 1,2-bis-[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphati dylcholine (SorbPC) and either a phosphatidylethanolamine (PE) or a phosphatidylcholine (PC) were examined via fluorescence leakage assays. Ultraviolet light exposure of SorbPC-containing liposomes forms poly-SorbPC, which phase separates from the remaining monomeric lipids. If the nonpolymerizable lipids are PE's, then the photoinduced polymerization destabilizes the liposome with loss of aqueous contents. The permeability of the control dioleoylPC/SorbPC membranes was not affected by photopolymerization of SorbPC. The photodestabilization of dioleoylPE/SorbPC (3:1) liposomes required the presence of oligolamellar liposomes. NMR spectroscopy of extended bilayers of dioleoylPE/SorbPC (3:1) showed that the photopolymerization lowers the temperature for the appearance of 31P NMR signals due to the formation of isotropically symmetric lipid structures. These observations suggest the following model for the photoinduced destabilization of liposomes composed of PE/SorbPC; photopolymerization induced phase separation with the formation of enriched domains of PE, which allows the close approach of apposed regions of enriched PE lamellae and permits the formation of an isotropically symmetric structure between the lamellae. The formation of such an interlamellar attachment (ILA) between the lamellae of an oligolamellar liposome provides a permeability pathway for the light-stimulated leakage of entrapped water-soluble reagents.
In vivo magnetic resonance spectroscopy (MRS) has revealed that phosphomonoesters (PME) such as phosphocholine (PCho) and phosphoethanolamine (PEth) are elevated in tumors and rapidly proliferating tissues. The regulation of PME levels and their relationship to proliferation are not well known. In the present study, we investigated the regulation of PCho and PEth levels in rat glioma cells grown in vivo and in vitro using 31P and 13C MRS. However, the ability of cells to produce choline endogenously is variable. To fully understand regulation of PCho levels, it is necessary to characterize the activity of the endogenous pathway, if it exists. This was first investigated by following the metabolic fate of 13C-labeled methionine of 9L glioma tumors in vivo. Our results indicate that there is a significant amount of de novo choline synthesis in vivo. However, similar experiments performed in vitro using cells cultured in bioreactors indicated that glioma cells themselves are unable to synthesize choline de novo, suggesting that the in vivo results were due to the involvement of extra-tumoral organs, e.g., liver. Further in vitro experiments demonstrated that the uptake and phosphorylation of physiologically relevant concentrations of exogenous choline is very active in these systems. Thus, it appears that the exogenous pathway for PCho biosynthesis predominates and regulates PCho levels in glioma cells. Our results also demonstrate that PCho levels are lowest, and PEth levels are highest, in non-proliferating cells. These observations indicate that there is a decrease in the biosynthesis of PCho concomitant with a reduction in culture growth. The source of the increased PEth is, as yet, undefined.
Theoretical models of phospholipid systems have indicated that both intramolecular and intermolecular forces are important in governing their acyl chain order. Knowledge of the nature and magnitude of these interactions is central to understanding the balance of forces present in lipid lamellar phases, which in turn is related to their microscopic and macroscopic behavior. It is possible to explore the contribution of intermolecular interactions using lipid systems with the same headgroup and acyl chain identity by variation of the ratio of the headgroups to acyl chains. In this paper, deuterium (2H) NMR spectroscopy has been used to gain information on the orientational order of an acyl chain perdeuterated lipid, l-perdeuteriopalmitoyl-sn-Bly~3-phosphocholine (PaLpc-d31), in various molecular environments. The orientational order of PaLpGd31 was studied in four different lamellar phases, including pure PaLPC-d31 (containing 10 wt % H20), diplmitoylphosphatidyl~holine/PaLPC-d3~ (3:1), palmitic a~id/PaLPC-d~~ (l:l), and ch~lesterol/PaLPC-d~~ (1:l) (each containing 50 wt % HzO). ' H NMR spectra were obtained for the low-temperature and liquid-crystalline (L,) states of each of these mixtures. In the low-temperature state, the f i t three systems yielded ZH NMR spectra characteristic of all-trans chains undergoing axial diffusion, with the methyl groups rotating about their C3 axes. The molecular order, as judged by the presenoe of spectral discontinuities and moment analysis, was found to be almost identical in the low-tempratwe phases. A different behavior was observed for the ch~lesterol/PaLPC-d~~ (1:l) sample in that the maximum splitting was close to the all-trans rotating value, with a profile of quadrupolar splittings due to inmased disorder near the chain ends. The f i t three systems underwent orderdisorder phase transitions near the same midpoint temperature (range of T, values 40-48 "C), whereas the ch~lesterol/PaLPC-d~~ (1:l) sample did not display a transition over the temperature range studied. In the L, phase, where order profdm were determined as a function of acyl chain segmglt position, the segmental ordering d i f f e r e d significantly among the samples. The differences were interpreted using a simple diamond lattice model for the acyl chain configurational statistics, as a means of comparing the effective lengths, (L), projected along the bilayer normal and estimated chain cross-sectional areas, (A), of PaLPC-d31 in the various mixtures. The derived values of (L) and (A) can be understood qualitatively in terms of average packing parameters related to the balance of forces in the headgroup and acyl chain regions, or alternatively the curvature free energy of the membrane lipid-water interface. In lamellar phases of pure P~L P C -C~~~ the curvature stnap is potentially large, and interdigitation of the acyl chains of the appoaed monolayers may occur. However, in mixtures of PaLPC-d31 with 1 , 2 -d i p a l m i t o y l -s n -B l y~~3 -p h o s p~~~e(DPPC), the curvature elaatic stress is apparently...
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