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PubMedDate not supplied

Activation and inhibition of Na/K-ATPase by filipin-cholesterol complexation. A correlative biochemical and ultrastructural study on the microsomal and purified enzyme of the avian salt gland.

The Na/K-ATPase-rich microsomal fraction and purified Na/K-ATPase membranes of the salt-stressed avian salt gland were studied at defined filipin/cholesterol molar ratios (F/C) using enzyme assay and electron microscopy including negative staining, thin sectioning and freeze fracturing. Comparative examinations of detergent-treated microsomal fractions and the use of electron microscopic tracers revealed that F/C up to 2 activated latent Na/K-ATPase in sealed right-side-out vesicles by increasing membrane permeability without disrupting the vesicular membrane. Therefore, filipin offers an alternative to the detergents for the activation of latent vectorial membrane enzymes and a possible tool to examine their subcellular localization and sidedness in the membrane. The same F/C had no stimulatory effect on the microsomal anion-ATPase suggesting that the 2 ATPases are not located in the same membrane. Increasing F/C applied to the unfixed Na/K-ATPase membranes caused an increase in the number of structural F-C-complexes and a progressive lateral displacement of the enzyme particles which finally led to a separation of the areal distribution of these structures at F/C = 10. Such displacements did not occur in unfixed microsomes and were prevented by glutaraldehyde fixation of the purified membranes. F/C exceeding 2 progressively and temperature-dependently inhibited the Na/K-ATPase in its membrane-bound states, whereas the solubilized enzyme was rather insensitive. The structural and biochemical data suggest that inhibition results from the perturbation of the lipidic microenvironment of the enzyme caused by filipin-cholesterol complexation.

Animals↗
PubMed1983

Evolution of filipin-sterol complexes and intramembrane particle distribution during ciliogenesis.

Membrane events during ciliogenesis were studied in the quail oviduct cells. Two membrane domains differentiate during ciliogenesis: the ciliary necklace progressively composed of one to six rows of intramembranous particle (IMPs), and the shaft membrane poor in IMPs but characterized by a high content of sterol. Membrane sterol can be detected by the use of filipin which forms filipin-sterol complexes (f-c complexes) visible in electron microscopy on both freeze fracture replicas and thin sections. During ciliary growth, the necklace IMP rows are progressively organized in an area completely devoid of f-c complexes whereas the membrane of growing shaft shows a high density of f-c complexes except at the ciliary tip.

Animals↗
PubMed1993

Filipin test for diagnosis of Niemann-Pick disease type C.

The test is based on the reaction of unesterified cholesterol with fluorescent antibiotic filipin giving a strongly fluorescent, stable cholesterol-filipin complex suitable for in situ detection. The test is useful for the evaluation of impaired cholesterol esterification in cultured cells (fibroblasts or others) in Niemann-Pick disease type C.

Cells, Cultured↗
PubMed2001

Quantified increases of cholesterol, total lipid and globotriaosylceramide in filipin-positive Niemann-Pick type C fibroblasts.

BACKGROUND: Niemann-Pick disease type C (NPC) is a neurovisceral lysosomal lipidosis caused in most cases by mutations in the NPC1 gene that codes for the cholesterol regulating NPC1 protein. METHODS: Cultured skin fibroblasts from 11 NPC patients aged 0.25 to 34 years at diagnosis with different severity of neurologic and visceral involvement, diagnosed by the cytochemical filipin test for lysosomally stored cholesterol, were analyzed for lipid composition. Cholesterol and other lipids were separated on thin-layer chromatography from fibroblast total lipid extracts, quantified by densitometry and compared with the total cell lipid mass. RESULTS: Cholesterol concentration in the patient cells was 1.5 to 5-fold higher than normal and total lipids up to 2.4-fold normal. Cholesterol and total lipids were particularly high in cells from NPC patients aged less than about 6 years, and for the whole patient series the abundance of fibroblast cholesterol was correlated with the tentatively assessed clinical disease severity. The findings in NPC suggested that NPC1 protein has a role not only in the balance of cholesterol but also the distribution of the total cell lipid mass. Another increase found in the NPC cells was that of a minor lipid fraction, globotriaosylceramide (Gb3, known as a cell signalling glycolipid). Gb3, in the average of its very variable individual concentrations, was about 2.5-fold higher in the NPC cell group as compared to normal or pathologic control group, but there was no correlation of Gb3 with the other lipid concentrations studied. CONCLUSIONS: For NPC diagnosis, the fibroblast cholesterol and total lipid quantification can be used as an alternative to the usual filipin test for lysosomal cholesterol, but both test methods are prone to equivocal results in cells from a small fraction of atypical NPC patients, where chemical testing in organ biopsies or mutational analysis of the NPC1 gene should be tried.

Adult↗
PubMed1979

Effects of filipin and steroids on phytochrome pelletability.

Red light given to dark-grown etiolated leaves of Hordeum vulgare L. in vivo or to crude homogenates increases the phytochrome content of the 20,000 g pellet on centrifugation. The steroids cholesterol and stigmasterol inhibit this red light-induced phytochrome pelletability. Filipin (a polyene antibiotic, which is known to combine with steroids) inhibits red light-induced phytochrome pelletability. Filipin and steroids at the appropriate concentration applied together prevent the inhibition caused by either when applied alone. These results suggest that phytochrome may bind to a steroid component of membranes. The phospholipid phosphatidyl choline dipalmitoyl has no effect on red light-induced phytochrome pelletability. Preliminary evidence demonstrates a direct association of soluble phytochrome in its active form and steroids. The physiological significance of red light-induced pelletability and the primary mechanism of phytochrome action are discussed in terms of a hypothetical steroid-binding site.

Journal Article↗
PubMed1992

Changes in the distribution of filipin-sterol complexes in the boar sperm head plasma membrane during epididymal maturation and in the uterus.

The distribution of filipin-sterol complexes (FSCs) and intramembranous particles (IMPs) in the plasma membrane of the late spermatid of the boar and of the sperm obtained from the epididymides, ejaculates, and uterus 2 hours after mating was examined by a freeze-fracture replica technique. In the late spermatid, the FSC density was found to be very low. A majority of the FSCs in the acrosomal plasma membrane (APM) appeared as protuberances on the E face in the epididymal, ejaculate, and uterine sperm. The density of the FSCs in the principal segment (PS) of the APM was 291 +/- 44 FSC/microns2 (mean +/- standard deviation, S.D.), 322 +/- 41 FSC/microns2 and 355 +/- 31 FSC/microns2 in the caput, corpus, and cauda epididymidis, respectively. In comparison with the cauda epididymal sperm, the FSC density gradually decreased in the PS of the ejaculated (277 +/- 39 FSC/microns2) and uterine sperm (243 +/- 50 FSC/microns2). The reduction was especially remarkable in the equatorial segment (ES), where the density of FSCs in ejaculated and uterine sperm decreased to about half and less than half of that in the cauda epididymal sperm, respectively. Large (13 nm) and small (8 nm) IMPs were distributed evenly and densely in the P face of the APM in the late spermatid, epididymal, and ejaculated sperm. In the uterine sperm, IMP-free areas were observed in the P face of the plasma membrane, a feature thought to represent one of the capacitation changes of the boar sperm.

Animals↗
PubMed1987

Filipin-sterol complexes at Schmidt-Lanterman incisures.

Employing the freeze-fracture technique, the distribution of filipin-sterol complexes was determined for membranes of peripheral nerve myelin. A heterogeneous distribution of complexes was observed with the greatest abundance on membranes associated with the cytoplasmic channels of Schmidt-Lanterman and longitudinal incisures. In addition there was an irregular network of well-labelled membrane bands in compact myelin. The results are related to a possible role for these channels and bands in the biochemical turnover of cholesterol in myelin.

Animals↗
PubMed1985

Intercellular junctions in human tooth-pulp cells in culture in vitro revealed by freeze-fracture, lanthanum impregnation and filipin treatment.

Three kinds of intercellular junctions were detected between human dental pulp cells in explant culture with electron microscopy included filipin detection for cholesterol; desmosome-like junctions observed on ultrathin sections probably contribute to the cohesiveness between cells in culture. Gap junctions, responsible for intercellular communication, exhibited two morphologies on freeze-fracture replicas: a conventional arrangement of their intramembranous particles and a crystalline array corresponding to the formation stage of junctions. Primitive tight junctions were detected on freeze-fracture replicas but not on ultrathin sections. It is likely that they contribute to the cell-to-cell adhesion under culture conditions.

Cells, Cultured↗
PubMed1987

Accumulation of unesterified cholesterol in limbal cornea and conjunctiva of rabbits fed a high-cholesterol diet. Detection with filipin.

Progressive cholesterol deposition frequently occurs in the corneas, tendons, and arteries of those individuals who have hypercholesterolemia. Many investigators have noted histochemical and chemical similarities in the nature of the lipid that accumulates in these tissues. Lipid-soluble dyes have been used to stain hydrophobic lipids such as cholesteryl ester and triglyceride in previous studies of corneal lipid deposits. In this report, deposits of unesterified cholesterol have been detected in the limbal cornea and conjunctiva of hypercholesterolemic rabbits using the fluorescent dye filipin. Whereas, oil red O-stained lipid accumulated both intra- and extracellularly, unesterified cholesterol accumulated extracellularly, similar to its predominant location in tendon xanthoma and atherosclerotic lesions. The source and mechanism by which unesterified cholesterol accumulates in limbal cornea and conjunctiva, tendons, and arteries remains to be determined.

Animals↗
PubMed1987

Filipin and digitonin studies of membrane cholesterol in frog atrial fibers with unusual gap junction configurations.

The gap junctions of frog myocardium present, in freeze-fracture, an atypical organisation of their junctional particles. Freeze-fracture cytochemistry with the cholesterol probes filipin and digitonin has been used to investigate whether the particular arrangement of the particles involves a lipid segregation in the plane of the membrane. Both probes labeled uniformly the non-junctional membrane, but no deformations were ever found inside the smooth membrane area circumscribed by the circle of junctional particles. At the level of junction formation zones almost no sterol probe complexes were found in the intramembranous particle free area which surrounded small clusters of junctional particles. These results suggest a regional variation in cholesterol related to the necessity of membrane fluidity during junction morphogenesis.

Animals↗
PubMed1980

Influence of extracellular K+ or Mg2+ on the stages of the antifungal effects of amphotericin B and filipin.

The macrolide heptaene amphotericin B (AmB) induced concentration-dependent effects on Saccharomyces cerevisiae which were separable into two distinct stages. At low concentrations the drug inhibited the growth of the yeast and reversible changed cell permeability to Na+ and K+. At high levels it was lethal. The intracellular K+ concentration of cells with reversible damage (stage I) could be increased by addition of K+ to the medium, but cells irreversibly damaged (stage II) were not able to retain K+. The addition of K+ to the medium did not influence the growth-inhibitory or killing action of AmB. Addition of Mg2+ to cultures increased S. cerevisiae resistance to the killing effects of AmB. At low concentrations of AmB, growth inhibition was also decreased by extracellular Mg2+, but at higher concentration of AmB, growth inhibition was increased, probably because the prevention by Mg2+ of the lethal effect allowed expression of the inhibitory effect in a greater range. Simultaneous addition of K+ and Mg2+ markedly decreased both the inhibitory and lethal action of AmB at all concentrations. Filipin, a pentaene macrolide, had only lethal effects, which were unaffected when K+ was added to the medium but were diminished when medium was supplemented with Mg2+.

Amphotericin B↗
PubMed1989

Membrane segregation in atrioventricular nodal myocytes of the golden hamster (Mesocricetus auratus). A cytochemical study using filipin and tomatine.

The membrane systems of hamster general atrial and ventricular myocytes were studied and their reactivity with the sterol probes, filipin and tomatine, compared with that of atrioventricular nodal myocytes. The sarcolemma of atrial and atrioventricular nodal myocytes was considerably more reactive to sterol probes than the sarcolemma of general ventricular myocytes. Atrial myocyte sarcolemma was also more reactive than previously reported in rat atrial myocytes. Particle-free regions of annular nexuses were consistently unreactive with all combinations of sterol probes whereas much smaller particle-free regions of macular nexuses and desmosomes were reactive with the same probes. Intracellular membrane systems of all three types of cardiac myocyte reacted in a similar manner with sterol probes. These results indicate variation in comparable sarcolemma composition of cardiac myocytes between the rat and hamster and between cardiac regions within the same species.

Animals↗
PubMed1985

Rates of amphotericin B and filipin association with sterols. A study of changes in sterol structure and phospholipid composition of vesicles.

The influence of structural modifications in sterols and phospholipids on the rate of polyene antibiotic-sterol interaction was studied. For filipin and amphotericin B association with sterols in vesicles, a preferential interaction was found with sterols whose side chain length is close to that of cholesterol. Introduction of trans double bonds into the sterol side chain did not alter the rate of interaction in vesicles. The delta 7-bond of the sterol appears to be of critical importance in amphotericin B-sterol interaction, whereas the delta 5-bond is not essential. These observations are relevant to the well-known effects of amphotericin B on cell membranes containing ergosterol compared with those containing cholesterol. The dependence of the rates of sterol-polyene antibiotic interaction on the phospholipid composition of the vesicles indicates that phospholipid vesicles may be an inadequate model for reaching a comprehensive understanding of the effects exerted on biological membranes by these agents.

Amphotericin B↗
PubMed1984

The distribution of filipin-cholesterol complexes in secretory ameloblasts of rat incisor.

The number of filipin-sterol complexes, visualised as 25-35 nm protuberances on freeze-fracture replicas of secretory ameloblasts of rat incisor, was small on the plasma membrane and nil on internal membranes of the cell body. Distal infoldings and vesicles inside Tomes process presented a higher number of protuberances. Such membrane-induced deformations were especially abundant on membrane remnants left inside the forming enamel and in holes at the dentine-enamel junction. This reflects regional variations of cholesterol or accessibility of cholesterol. Increased rigidity of membranous remnants may play a role in the induction of local defects during rat enamel formation. These observations clarify the origin of phospholipids and cholesterol detected biochemically.

Ameloblasts↗
PubMed1982

Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. II. Taurolithocholate-induced alterations of canalicular morphology and of the distribution of filipin-cholesterol complexes.

Hepatocytes in primary monolayer culture treated with taurolithocholate showed distinct ultrastructural changes localized primarily to the bile canalicular membrane. These alterations comprised disturbance and proliferation of tight junctions, dilatation of the canaliculi, loss of microvilli, thickening of the pericanalicular ectoplasm, and bizarre lamellar transformations of canaliculi. In freeze-fracture replicas the lamellae projecting into the canalicular lumen were found to be devoid of intramembranous particles. Localization by the use of filipin of cholesterol in plasma membranes of taurolithocholate affected hepatocytes revealed an extensive accumulation of cholesterol in membranes of dilated canaliculi, and also in outpouchings of the contiguous membrane in the ultimate vicinity. Furthermore, a pronounced segregation of cholesterol-rich membrane material into the lumen of dilated canaliculi and into enlargements of the intercellular space could be observed in thin sections. In contrast, a total absence of cholesterol was noted in the lamellar projections of the bizarre transformed canaliculi. The reliability of these findings and their consequences for the mechanism of taurolithocholate-induced cholestasis are discussed and it is suggested that the incorporation of cholesterol into the canalicular membrane reflects only one aspect of the cholestatic effect of taurolithocholate. An additional aspect seems to comprise the dislocation of membrane bound proteins and perhaps other membrane components. This is probably caused by independent mechanisms.

Animals↗
PubMed1993

Cholesterol and 25-hydroxycholesterol retention in specimens of liver and aorta prepared for electron microscopy. II. Effect of filipin, osmium, digitonin and saponin.

Sprague-Dawley rats were injected intraperitoneally with [1 alpha, 2 alpha(n)-3H]cholesterol or 25-hydroxy-[26,27-3H]cholesterol, and one and five days later liver and aortic tissues were fixed. The extent to which these sterols were lost from the tissues during preparation for electron microscopy (EM) was examined utilizing different fixation procedures and various protective agents. Radioactive tracers, scintillation counting and standard EM techniques were used. Although most of the procedures examined caused major lipid losses, useful fixation procedures that allow retention of cholesterol or 25-hydroxy-cholesterol in liver and aortic tissues were found and are described here.

Animals↗