Maleylated-BSA induces hydrolysis of PIP2, fluxes of Ca2+, NF-kappaB binding, and transcription of the TNF-alpha gene in murine macrophages.

The interaction of altered lipids or proteins with the several scavenger receptors (SR) on macrophages can lead to disparate results in both gene expression and cell function. However, the molecular bases of signaling induced by SR ligation have remained obscure. Here we report that maleylated-bovine serum albumin (maleyl-BSA) binds a low-affinity SR, initiating PIP2 hydrolysis, [Ca2+]i spikes, phospholipase A2 (PLA2) activation, nuclear factor-kappa(B) (NF-kappa(B)) binding to its cognate nucleotide and tumor necrosis factor alpha (TNF-alpha) gene transcription. We recently reported that oxidized low-density lipoprotein (ox-LDL), which binds another macrophage SR, induced pertussis-toxin-sensitive hydrolysis of PIP2 and elevations in [Ca2+]i [J. Biol. Chem. 270, 3475-3478, 1995]. By contrast, maleyl-BSA-initiated events were not pertussis toxin-sensitive and produced less [Ca2+]i spiking than ox-LDL. Furthermore, maleyl-BSA led to binding of NF-kappa(B) to its cognate nucleotide and TNF-alpha gene transcription, whereas ox-LDL suppressed these events. Collectively, this data suggests that maleyl-BSA and ox-LDL bind to distinct SR on murine macrophages, initiate distinct signal transduction pathways, and produce different functional effects.

Oxidized low density lipoprotein suppresses activation of NF kappa B in macrophages via a pertussis toxin-sensitive signaling mechanism.

The interaction of oxidized low density lipoprotein (ox-LDL) and macrophages is generally believed to be a significant inductive step in atherogenesis. Endocytosis of ox-LDL by scavenger receptors (SR) on macrophages is one result of this interaction, as is suppressed expression of several lipopolysaccharide (LPS)-stimulated, inflammatory genes such as tumor necrosis factor-alpha (TNF-alpha). Events subsequent to SR ligation, including intracellular signaling events if any, have not been established. We report here that ox-LDL initiates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate 2 (PIP2) and intracellular fluxes of Ca2+ in macrophages, both of which are sensitive to pertussis toxin. ox-LDL also suppresses the LPS-induced binding of macrophage extracts to an NF kappa B sequence oligonucleotide and the LPS-initiated accumulation of RNA specific for TNF-alpha. These latter two effects are pertussis toxin-sensitive. Ligation of SR by ox-LDL thus initiates a pertussis toxin-sensitive signaling pathway in macrophages, which involves hydrolysis of PIP2 and which can suppress expression of the TNF-alpha gene by modulating activation of NF kappa B.

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury.

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.

Lipid order in hepatocyte plasma membrane blebs during ATP depletion measured by digitized video fluorescence polarization microscopy.

Low-light digitized video fluorescence polarization microscopy was used to measure lipid order parameters in plasma membrane blebs of single, cultured rat hepatocytes during ATP depletion with the metabolic inhibitors cyanide and iodoacetic acid. Hepatocytes were labeled on the microscope stage with the plasma membrane probe trimethylammoniumdiphenylhexatriene at successive stages of cell injury. A pair of fluorescence polarization ratio images was obtained from a series of four fluorescence images recorded with a polarizer in the emission path oriented first parallel and then perpendicular to each of two orthogonal excitation light polarization directions. From the polarization ratio images, the lipid order parameter S was determined in individual plasma membrane blebs. Results indicate that the plasma membrane becomes uniformly rigid within a few minutes of the addition of metabolic inhibitors when small surface blebs have formed and ATP levels have fallen by greater than 95%. The measured order parameter of S approximately 0.95 in plasma membrane blebs, compared with S approximately 0.75 in normoxic cell plasma membranes, remained unchanged throughout the course of bleb development and ultimate cell death. These findings demonstrate that significant alteration in hepatocyte plasma membrane structure occurs early in hypoxic cell injury.

Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy.

Low-light digitized video fluorescence microscopy has been utilized to measure the steady-state polarized fluorescence from the membrane probe diphenylhexatriene (DPH) and its cationic and phosphatidylcholine derivatives 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 2-[3-(diphenylhexatrienyl)propanoyl]-3-palmitoyl-L-alpha-phosphati dylcholine (DPH-PC), respectively, in cell-size (10-70 microns) unilamellar vesicles composed of gel-or fluid-phase phospholipid. Using an inverted microscope with epi-illumination optics and an intensified silicon intensified target camera interfaced to a minicomputer, fluorescence images of single vesicles were obtained at emission polarizer orientations of 0 degrees, 45 degrees, 90 degrees, and 135 degrees relative to the excitation light polarization direction. Fluorescence intensity ratios F90 degrees/F0 degrees (= F perpendicular/F parallel) and F135 degrees/F45 degrees were calculated on a pixel-by-pixel basis from digitized image pairs. Theoretical expressions were derived for collected polarized fluorescence as a function of position on the membrane surface as well as the degree of lipid order, in terms of the fluorophore's maximum angular motional freedom in the bilayer (identical to theta max), using a modification of the method of D. Axelrod (1979. Biophys. J. 26:557-574) together with the "wobbling-in-a-cone" model of probe rotational diffusion. Comparison of experimental polarization ratios with theoretical ratios yielded the following results. In gel-phase dipalmitoyl-phosphatidylcholine, the data for all three probes correspond to a model in which the cone angle theta max = 17 +/- 2 degrees and there exists a collective tilt of the phospholipid acyl chains of 30 degrees relative to the bilayer normal. In addition, approximately 5% of DPH and TMA-DPH molecules are aligned parallel to the plane of the bilayer. In fluid-phase palmitoyloleoyl-phosphatidylcholine, the data are well fit by models in which theta max = 60 +/- 2 degrees for DPH and DPH-PC and 32 +/- 4 degrees for TMA-DPH, with approximately 20% of DPH molecules and 10% of TMA-DPH molecules aligned parallel to the bilayer plane, and a net phospholipid tilt at or near the headgroup region of approximately 30 degrees. The results demonstrate that lipid order can be measured with a spatial resolution of approximately 1 micron2 in cell-size vesicles even with high aperture observation through a microscope.

On the use of partition coefficients to characterize the distribution of fluorescent membrane probes between coexisting gel and fluid lipid phase: an analysis of the partition behavior of 1,6-diphenyl-1,3,5-hexatriene.

The distribution of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene between coexisting gel and fluid phospholipid phases in multilamellar vesicles has been examined using fluorescence quenching by spin-labeled phosphatidylcholine. For both thermally-induced and Ca2+-induced lipid phase separation, the ratio of probe concentration in the fluid liquid-crystal phase to that in the gel phase is found to be independent of either the probe concentration or the relative amounts of gel and fluid lipid phases, and hence is an equilibrium concentration ratio, or partition coefficient.

Effect of fluorophore linkage position of n-(9-anthroyloxy) fatty acids on probe distribution between coexisting gel and fluid phospholipid phases.

The quenching of probe fluorescence by spin-labeled phospholipid has been used to determine the distribution of a series of n-(9-anthroyloxy) fatty acids between coexisting gel and fluid liquid-crystal phases in multilamellar phospholipid vesicles. The phase distribution ratio in every case is found to favor the fluid lipid phase, but is much greater between fluid and Ca2+-induced gel than between fluid and thermal gel. For a given gel type, n-(9-anthroyloxy)stearic acids with n = 3, 6, 9 or 12 as well as 11-(9-anthroyloxy)undecanoic acid all exhibit similar behavior, favoring the fluid phase by about a factor of 4 over thermally-induced lipid gel phase and by 18 over Ca2+-induced gel phase. 16-(9-Anthroyloxy)palmitic acid, with the bulky probe at the terminus of the 16-carbon chain, favors the fluid phase less strongly, by a factor of 1.5 or 11 over thermally-induced or Ca2+-induced gel phase, respectively, indicating better packing of this probe in phospholipid gel phases.