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PubMed1988

Unesterified cholesterol-rich lipid particles in atherosclerotic lesions of human and rabbit aortas.

Unesterified cholesterol-rich lipid particles were isolated from human and cholesterol-fed rabbit aortas. These particles have been previously reported to constitute the initial lipid deposition in atherosclerotic lesion development. Purification of the particles was accomplished with microfiltration, gel filtration chromatography, and density gradient centrifugation. Particles from both human and rabbit aortas had a density of between 1.02 g/ml and 1.08 g/ml with a peak at d = 1.036 g/ml. These particles had a high molar ratio of unesterified cholesterol to phospholipid (2.4:1 in rabbit, 2.6:1 in human) and a high percentage of their cholesterol in an unesterified form (82% in rabbit, 76% in human). The particles had diameters between 700 and 3000 A and showed unilamellar and multilamellar structures. Freeze-fractured particles had smooth fracture faces and sometimes contained a smooth-surfaced core. Upon incubation with filipin, particles showed typical filipin-sterol complexes, demonstrating the presence of unesterified cholesterol. The particles we have isolated may constitute an early pathologic form of accumulated cholesterol in developing lesions and may represent a degradation product of infiltrated plasma low-density lipoprotein.

Animals↗
PubMed1987

Lack of cytochemically detectable cholesterol in rabbit vena cava endothelial plasma membrane.

The response of rabbit vascular endothelial plasma membranes to the cholesterol-binding agents filipin and tomatin was investigated by freeze-fracture electron microscopy. In both single and double labelling experiments, the endothelial plasma membranes of the posterior vena cava were remarkably resistant to the agents compared with the endothelial plasma membranes of other vessels (aorta, pulmonary artery, and pulmonary and cardiac capillaries). This suggests that vena cava endothelium differs markedly from other endothelia, either by having exceptionally low cholesterol levels in its plasma membranes, or with respect to other membrane properties that influence the reaction to filipin and tomatin.

Animals↗
PubMed1986

Prelesional events in atherogenesis. Accumulation of extracellular cholesterol-rich liposomes in the arterial intima and cardiac valves of the hyperlipidemic rabbit.

Biochemical, physiologic, and ultrastructural modifications which appear in the aortic intima and atrioventricular valves before monocyte diapedesis and foam cell formation were investigated in rabbits fed a cholesterol-rich diet. In the first 2 weeks of the diet, while plasma beta-VLDL cholesterol was increased up to 15-fold, the intima showed an enhanced uptake and deposition of dietary 3H-cholesterol, 125I-beta-VLDL, and the fluorescent beta-VLDL-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine conjugate. beta-VLDL-gold complex perfused in situ was transcytosed across endothelium by plasmalemmal vesicles. Concomitantly, within the intima, a progressive accumulation of extracellular densely packed uni- or multilamellar vesicles took place. These commonly occurred in cell-free subendothelial spaces and were not associated with any sign of cytolysis. In freeze-fracture preparations, these vesicles appeared as smooth surfaces, suggesting the absence of translamellar proteins. Upon incubation with filipin, these extracellular liposomes (EL) displayed characteristic approximately 20 nm filipin-sterol complexes, revealing the presence of preparations unesterified cholesterol in the phospholipid lamellas. EL deposition was paralleled by proliferation of basal lamina-like material, microfibrils, and proteoglycans, and continued to increase during foam cell formation. For the entire period of our experiments, the endothelium was morphologically intact, and no platelet involvement was detected. The results show that an early prelesional ultrastructural change in lesion-prone aortic and valvular areas is the accumulation of extracellular phospholipid liposomes rich in unesterified cholesterol.

Animals↗
PubMed1983

Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. IV. Disintegration of bile canaliculi and disturbance of tight junction formation caused by vinblastine.

Treatment of cultured hepatocytes with vinblastine or colchicine caused striking perturbations of the structural organization of the biliary pole and of the junctional complexes. During the early hours of cultivation the reassociation of the bile canaliculi was impaired by the drug, whereas at later times in culture preformed canaliculi were disintegrated to small vesicular remnants lacking microvilli. Vinblastine did not impair tight junction formation per se. However, under the influence of the drug, tight junctional strands associated in an atypic manner perpendicular to the upper surface of the hepatocytes, whereas those strands lining the canaliculi were decomposed to smaller entities and dislocated within the lateral membrane. Concomitantly to the structural disintegration of the biliary pole an accumulation of vesicles in the pericanalicular cytoplasm was noted. As indicated by numerous filipin-induced lesions, they were characterized by a high content of membrane cholesterol. The apical pole and the contiguous membrane on the other hand contained only very few filipin-cholesterol lesions. These findings suggest that antimicrotubular drugs impair the fusion of pericanalicular vesicles with the luminal membranes of the canaliculi, thus interrupting the delivery of membraneous material to the apical pole. In addition, microtubules seem to play an important role in the coordinated development and the structural fixation of the biliary pole of cultured hepatocytes.

Animals↗
PubMed1980

Freeze-fracture alterations in guinea pig sperm membranes preceding gamete fusion.

Normal fertilization in mammals depends on several events involving membrane fusion. One, a secretory phenomenon, is the fusion of the spermatozoan acrosomal granule membrane with the plasmalemma, and another concerns the union of this nascent hybrid membrane with that of the egg. Using freeze-fracture and ancillary cytochemical procedures, we can identify three general membrane constituents: proteins, apparent as intramembranous particles; phospholipids, visible as smooth, particle-free areas; and sterols, observed as 25-nm ellipsoid protuberances when complexed in situ with the polyene antibiotic filipin. Freshly removed from the epididymis, guinea pig sperm displays distinctive patterns of intramembranous particles and a nonhomogeneous distribution of sterols. When incubated in a Ca2+-free medium that permits processing spermatozoan membranes for fusion, these membrane components become altered. In the circular foci where union occurs, the particle pattern is modified from an orderly, geometric quilted design to a random distribution; filipin-reactive sterols are deleted, followed by the loss of particles from the nonesterified sterol-deficient patches. With the addition of Ca2+, the residual smooth areas meld as the number of particles equilibrates in the plane of the hybrid acrosomal-plasma membrane. Sterols appearing in the postacrosomal segment remain reduced in quantity. After acrosomal-plasma-membrane fusion, a second line of particle-cleared circles emerges behind the margin of the new membrane suture. These clearings appear to be identical to the circles observed in the acrosomal and plasma membranes preceding the acrosome reaction. We interpret this second series of clearings as the preparation of the spermatozoan membrane for fusion with the egg. In sperm, the focal alterations antecedent to organelle (acrosome) plasma membrane union are evidently comparable to those preceding plasma membrane (sperm) plasma membrane (egg) fusion. It will be of great interest to us to discover whether this type of protein and sterol/phospholipid juncture proves to be a unifying feature of fusions in all the various biological systems discussed in this volume.

Animals↗
PubMed2001

Murine high-fat diet and laser photochemical model of basal deposits in Bruch membrane.

OBJECTIVE: To examine the histologic, histochemical, and ultrastructural changes in Bruch membrane in mice on a high-fat diet with and without laser photochemical injury. METHODS: Five groups of C57BL/6 mice were studied. Group 1 included 2-month-old mice on a normal diet; group 2 included 8-month-old mice on a normal diet; group 3 included 8-month-old mice on a high-fat diet; groups 4 and 5 included 8-month-old mice on a normal diet or high-fat diet, respectively, that underwent laser application of one eye with argon blue laser (488 nm). The mice were killed and plasma lipid levels were measured. The eyes were examined by standard electron microscopy, filipin histochemistry for unesterified cholesterol (UC) and esterified cholesterol (EC), and the osmium-tannic acid-phenylenediamine method for preserving extracellular lipid particles. RESULTS: The plasma cholesterol level was significantly higher in the mice on the high-fat diet than the controls (P<.001). Bruch membrane was thicker in group 2 than group 1 (P =.04) and group 3 had a thicker Bruch membrane than group 2 (P =.003). All eyes in group 3 exhibited accumulation of electron-lucent debris. There was no histochemical and ultrastructural evidence that this material represented accumulated UC or EC. Seven of 9 laser-injured eyes in group 5 accumulated basal laminar deposit (BlamD)-like material in Bruch membrane (P =.02). CONCLUSIONS: Electron-lucent debris accumulates in murine Bruch membrane, and the amount correlates with age and high-fat diet. This debris has some similarities with basal linear deposits, although the debris does not form a discrete layer external to the basement membrane of the retinal pigment epithelium as occurs in basal linear deposits. These deposits do not appear to be UC or EC. Laser photochemical injury of the retinal pigment epithelium may result in the appearance of BlamD-like deposits in eyes with electron-lucent debris. The BlamD-like deposits in this model are similar to the basal laminar deposits that occur in age-related macular degeneration. CLINICAL RELEVANCE: This is an animal model of ultrastructural BlamD-like material in Bruch membrane that is very similar to the deposits that occur in age-related macular degeneration.

Animals↗
PubMed2000

Redistribution of cholesterol in oligodendrocyte membrane sheets after activation of distinct signal transduction pathways.

Cultured oligodendrocytes produce extensive membrane sheets that contain an internal lacy network of vein-like structures composed of microtubules, actin filaments, and 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). These cytoplasmic vein-like structures surround domains of myelin basic protein (MBP). Using the antibiotic filipin, that binds to cholesterol, the relationship between plasma membrane cholesterol and cytoskeleton in membrane sheets was examined. Our results show that cholesterol was relatively uniformly distributed within the plasma membranes of prefixed control oligodendrocyte membrane sheets. When live cultures were extracted with Triton X-100, however, a subpopulation of cholesterol molecules remained colocalized with cytoskeleton in the membrane sheets. Activation of two well-characterized signaling pathways that differentially affect microtubule and actin filament stability in membrane sheets resulted in an apparent massive lateral movement of cholesterol molecules away from membrane regions overlying internal MBP domains to membrane tracts directly overlying cytoplasmic cytoskeletal veins. Depolymerization of microtubules by colchicine resulted in redistribution of cholesterol directly over actin filaments, whereas depolymerization of actin filaments by cytochalasin B resulted in redistribution of cholesterol directly over CNPase/microtubular veins. These data suggest that cholesterol forms an association with cytoskeletal components or proteins associated with cytoskeleton. These data also suggest that cholesterol, via interactions with cytoskeleton, plays a role in signaling pathways in oligodendrocyte membrane sheets.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1985

Freeze-fracture observations on mammalian oocytes.

Freeze-fracture studies on mammalian oocytes have been hampered by the relatively small numbers of cells available at a given time as well as by difficulties encountered in effectively freezing these large, watery cells. We have nevertheless pursued this area because of the benefits of visualizing membrane faces involved in various fusion reactions by the freeze-fracture method. Our observations indicate no overall change in intramembranous particle (IMP) distribution before and after sperm penetration, although the question of possible alterations of these structures at the precise locus of sperm attachment remains open. Preliminary statistical analysis indicates that there is a much higher IMP density on the P face than on the E face of the plasma membrane and that the microvillar membranes bear more IMPs than those of the intermicrovillus regions. Probes of lipid subclasses were used to determine the distribution of cholesterol and anionic lipid in the egg plasma membrane. Filipin and tomatin showed extensive complex formation in microvillus as well as nonmicrovillus regions, whereas anionic lipids (using polymyxin B) have been difficult to detect on the oocyte surface. These results are discussed relative to current views of membrane fusion mechanisms.

Animals↗
PubMed1992

Modification of the amount of cholesterol in hepatic steatosis induced in susceptible and resistant mice infected with MHV3: a biochemical and ultrastructural study.

A mouse hepatitis virus-3 strain subcultured in our laboratory is a unique experimental model in which to study virus-induced liver steatosis. This strain produces massive lipid deposition not only in sensitive adult BALB/c mice but also (though less extensive) in virus-resistant adult A/J mice. Biochemical determinations have shown that this steatosis is characterized by an increased amount of neutral lipids (sterols and triglycerides) in infected livers of BALB/c mice and by a smaller increase in those of A/J mice. However, the relative percentage of cholesterol and triglycerides is similar in both strains. Liver phospholipid content was significantly decreased in both strains of mice. To discriminate between cytoplasmic and membrane cholesterol content in different types of liver cells, an ultrastructural study was performed with filipin, a specific cholesterol marker. This study shows on one hand an important increase in the cholesterol in the hepatocytes of BALB/c mice and a smaller increase in those of A/J mice, in agreement with biochemical data. However, marked cholesterol decrease and abnormal cholesterol distribution were observed in the endothelial liver cells of infected BALB/c mice. This decreased cholesterol content probably led to higher fluidity of these membranes, which could be related to the important drop in the number of endothelial cell fenestrae observed after mouse hepatitis virus-3 infection. Because in A/J infected mice neither a decrease in the amount and distribution of cholesterol nor decreased fenestration were observed in endothelial liver cells, these findings could be correlated with the resistance of these mice to the infection.

Animals↗
PubMed1978

Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations. I. Comparative effects of four classes of polyene macrolides on mammalian cells.

The relationship between polyene macrolide-induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by 51Cr release, followed by mediocidin (MED), amphotericin B-deoxycholate (Fungizone) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24-hour survival, 72-hour viability, and growth rate of cells. In evaluating polyene macrolide-induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.

Antibiotics, Antineoplastic↗
PubMed1979

Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations. II. Phenotypic expression in intraspecific and interspecific somatic cell hybrids.

Cytotoxicity and membrane permeability alterations induced by the polyene macrolide antibiotics filipin (FIL) and pimaricin (PIM) have been compared in parental intraspecific and interspecific somatic cell hybrids. B82 (mouse) and B1 (hamster) cells were found to be more resistant than RAG (mouse) parental cells to both polyene macrolides as indicated by 24-hour survival, 72-hour viability, and growth rate. Analysis of both intraspecific and interspecific somatic cell hybrids indicated that polyene macrolide resistance was being expressed even in the presence of the polyene macrolide-sensitive (RAG) genome. Where one of the two parental cell types is relatively polyene macrolide resistant, the use of specific polyene macrolides may prove efficacious as half-selective agents in cell hybridization.

Animals↗
PubMed1989

Advantages of the use of intact plant tissues in freeze-fracture electron microscopy.

The use of whole, intact plant tissue for freeze-fracture electron microscopy provides important information that cannot be obtained from the use of isolated biological membranes or of artificial (phospholipid) membrane preparations. This is not to imply that these examinations of such preparations are not useful, since it would be difficult to interpret our observations of intact cells and tissues without the analysis of these model systems. Analysis of intact tissue and cells reveals the relative densities of membrane proteins of the different membranes within a cell; the three-dimensional organization of various organelles, especially the endoplasmic reticulum; changes in intramembranous particle (IMP) distribution due to stress or injury; and, in conjunction with the use of filipin, membrane sterol content and relative distribution. It is our intention that this survey of freeze-fracture images of intact plant tissues will illustrate the uniqueness of the information gained from an analysis of whole plant tissues compared to isolated membrane fractions.

Filipin↗
PubMed1981

Oxidative metabolism of spermatozoa from inbred and random bred mice.

Epididymal spermatozoa from the random-bred CFW and from the inbred C57 BL/6 strains of mice were treated either hypotonically or with the antibiotic filipin in order to study the mitochondrial oxidative activities of the two strains in the absence of permeability barriers imposed by the plasma membrane. The percentage of motile spermatozoa from C57BL/6 mice was consistently higher than that of CFW mice, but sperm from the latter fertilized a higher percentage of eggs in vitro. In vivo, there was no apparent difference in fertilizing capacity in vivo: no significant difference between strains was observed. There is a strategy of oxidative metabolism in mouse spermatozoa which is common to the two genetic strains of the species tested, but which differs from that of rabbit and bull spermatozoa. The mitochondria of mouse spermatozoa oxidize L--3-glycerolphosphate but not glutamate in the presence of malate; both activities are present in bull spermatozoa but neither are present in rabbit spermatozoa. In common wit those of the mammalian species thus far studied, the mitochondria of mouse spermatozoa readily oxidize lactate and pyruvate in the presence of malate. They also oxidize acetyl CoA, acetyl carnitine, and long-chain acyl CoA esters directly, without the intermediacy of the carnitine esters. Mouse spermatozoa, therefore, have access to endogenous acyl CoA esters as a source of metabolic energy, which is consistent with their ability to maintain motility for 4-6 hours in the absence of added energy sources. Mouse spermatozoa are self-sufficient with regard to oxidative metabolism, which suggests that energy sources are not readily available to them in the mouse female reproductive tract.

Animals↗
PubMed1999

Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells.

The uptake of respiratory syncytial virus (RSV) antigen by cattle dendritic cells was investigated. Pathways of antigen uptake were monitored by flow cytometry using specific tracers and by proliferation assays, which were used to measure the presentation of RSV antigen and ovalbumin. Inhibitors that differentially affected pathways were used to distinguish them. Presentation of RSV antigen, but not ovalbumin, was inhibited by phorbol myristate acetate and filipin, which have been reported to inhibit caveolae, but not by cytochalasin D, amiloride, or mannose. These inhibitors have been reported to block macropinocytosis and other actin-dependent uptake mechanisms, endocytic pathways involving clathrin-coated pits, and the mannose receptor. Furthermore, co-localization of RSV antigen and caveolae was observed by confocal microscopy. Thus, the major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendritic cells to those established.

Amiloride↗
PubMed2003

Cholesterol storage and tau pathology in Niemann-Pick type C disease in the brain.

Niemann-Pick type C disease is an inherited neurovisceral storage disorder with intracellular accumulation of cholesterol. In affected brains, many ballooned neurons are seen. Considerable nerve cell loss of unknown pathogenesis leads to neurological deterioration and dementia. Chemical examination of brains has failed to demonstrate increased levels of cholesterol. Using filipin fluorometry of neuronal cells in tissue slices, we found massive accumulation of cholesterol in neurons in four out of five human Niemann-Pick type C cases including adult patients. Neurofibrillary tangles composed of aggregates of the otherwise highly soluble protein tau were present in three Niemann-Pick type C cases and were also immunologically identical to those associated with Alzheimer's disease. However, only a thin slab of spinal cord or a tiny piece of isocortex was available for examination in the two cases without tangles. In a further semi-quantitative analysis of 576 neurons, we determined higher cholesterol content in tangle-bearing neurons than in adjacent tangle-free neurons. The association of cholesterol accumulation with neurofibrillary degeneration in Niemann-Pick type C disease and Alzheimer's disease awakens interest in the role of impaired cholesterol metabolism in the development of neurofibrillary tangles in both diseases.

Adolescent↗
PubMed2001

High sensitivity of mouse neuronal cells to tetanus toxin requires a GPI-anchored protein.

Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis. Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP. Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin.

Animals↗
PubMed1999

Caveolar internalization of growth hormone.

Caveolae are plasma membrane specializations formed by caveolin and characterized by their dependence on membrane cholesterol for structural integrity. We have investigated the role of caveolae in the internalization of GH in CHO cells stably transfected with GH receptor cDNA (CHO-GHR1-638). We show by immunogold electron microscopy that a portion of the GH receptor at the cell surface is localized to or near caveolin-containing structures and upon GH stimulation the receptor aggregates in caveolae. Similarly the hormone is observed to be aggregated in caveolae and a portion of the hormone is internalized into the cell in caveolin-containing vesicles. Disruption of caveolar integrity by sterol-binding agents (filipin, nystatin) partially inhibits internalization of 125I-hGH whereas internalization of hormone is not affected by non-sterol-binding agents which also insert into the cell membrane (polymyxin B, xylazine). Transient transfection of caveolin cDNA into CHO cells concomitantly transfected with GH receptor cDNA increases both the internalization of hormone and the GH stimulation of STAT-mediated transcription. In conclusion, we demonstrate that caveolae constitute one pathway for the internalization of GH. Such an internalization pathway may also be utilized by other members of the cytokine receptor superfamily.

Animals↗
PubMed2001

Colocalization prostacyclin (PGI2) synthase--caveolin-1 in endothelial cells and new roles for PGI2 in angiogenesis.

In vascular cells, prostacyclin (PGI2) synthase (PGI2s) has been localized in the endoplasmic reticulum of endothelial cells and in the nuclear and plasma membrane of smooth muscle cells. In human umbilical vein endothelial (HUVE) cells, we detected the enzyme in abundant cytoplasmic vesicles apparently originating from the plasma membrane and similar to those stained by gold-albumin, which interacts with a caveolar receptor. This prompted us to try a direct confocal microscopy approach aimed at colocalizing gold-albumin, caveolin-1, and PGI2 synthase. Moreover, the staining of HUVE cells with an anti-BiP7Grp78 antibody (a marker of endoplasmic reticulum) shows a perinuclear localization, sharply separated from PGI2 synthase localization. The results indicate that more than 80% of the enzyme resides in cellular sites costaining with caveolin-1 antibody and gold-albumin. This evidence was confirmed by the demonstration that PGI2 synthase and caveolin-1 coimmunoprecipitate in HUVE cell lysates and that they are associated to detergent-insoluble membrane domains in the same low-density fractions of a sucrose gradient. In addition, depletion of cellular cholesterol by mevalonate and methyl-beta-cyclodextrin leads to the shift of PGI2 synthase and caveolin-1 to higher density fractions of the gradient. Biochemical evidence about colocalization was supported by the use of a fusion protein glutathione S-transferase (GST)/caveolin-1, which retained either PGI2s purified from ram seminal vesicles or PGI2s present in HUVE cell lysates. Binding of PGI2s to caveolin "scaffolding domain" and to C-terminal region was deduced by using full-length GST--Cav-1, GST--Cav 61--101, and GST C- and N-terminal fusion proteins. A double approach based on the usage of filipin as a specific caveolae-disrupting agent and antisense oligonucleotides targeting PGI2 synthase mRNA suggests that the production of PGI2 in caveolae is likely to be connected to the regulation of angiogenesis, at least in vitro.

6-Ketoprostaglandin F1 alpha↗