Fasting enhances taurine transport by rat liver plasma membrane vesicles.

Hepatic taurine stores are maintained by biosynthesis from the sulfur-containing amino acids, methionine and cysteine, and by uptake via a Na(+)- and Cl(-)-dependent transport system, which is specific for beta-amino acids. We hypothesized that liver stores of taurine are maintained by enhanced hepatic transport during fasting when dietary sources for taurine and its precursors are diminished. Liver plasma membrane vesicles, enriched for the basolateral domain, were prepared from adult male rats fasted for 72 h and from control rats. The maximum velocity for Na(+)-dependent taurine uptake was twofold greater for the fasted group compared with the control group (0.87 +/- 0.09 vs. 0.31 +/- 0.03 nmol.mg protein-1.min-1). The apparent Michaelis constant for taurine was also greater for fasted compared with control (154.0 +/- 0.5 vs. 80.0 +/- 2.0 microM). gamma-Aminobutyric acid, but not alanine or glutamine, abolished the effect of fasting on hepatic taurine transport. To determine the effect of fasting independent of changes in the lipid microenvironment, taurine uptake was measured in proteoliposomes reconstituted by inserting detergent-solubilized membrane proteins into asolectin vesicles. Taurine uptake by proteoliposomes reconstituted from membranes prepared from the fasted group was significantly greater than from the control group. We conclude that Na(+)-dependent taurine transport is enhanced in liver plasma membranes prepared from fasted rats. Our findings imply that enhanced taurine uptake with fasting is due to either an increased number of functional carriers or activation of existing transporters.

The use of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipids in determining transmembrane lipid distribution.

Transbilayer lipid distribution of small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) was measured using 31P-nuclear magnetic resonance (NMR) spectroscopy, chemical modification with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dithionite reduction of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipid (NBD-lipid). The dithionite assay was the most reproducible of the three assays, with 1.2% error for SUVs and 3.9% error for LUVs. The dithionite assay also agreed best with theoretical inner:outer leaflet ratios, based on vesicle diameters determined by electron microscopy (Thomas et al. (1989) Biochem. Biophys. Acta 978, 85-90). Dithionite assay measurements were within 2.7% of theoretical ratios for SUVs and 2.3% for LUVs, while the NMR assay for SUVs was 14% lower than theoretical ratios and 23% lower for LUVs. The accuracy of NBD-lipids as markers for total transbilayer lipid was investigated. NBD-labeled phosphatidylserine, phosphatidylcholine and phosphatidylglycerol were accurate markers for total transbilayer lipid distribution, as their distributions were in close agreement with theoretical ratios. However, NBD-labeled phosphatidylethanolamine displayed a slight preference for the inner leaflet at low mole fractions of phosphatidylethanolamine, while native phosphatidylethanolamine showed a preference for the outer leaflet at the same concentration. NBD-labeled phosphatidic acid also showed a slight preference for the inner leaflet. We conclude that although dithionite-based assessment of NBD-labeled lipids across membrane bilayers can be a powerful analytical tool, caution must be used in the interpretation of results.

Fusogenic virosomes prepared by partitioning of vesicular stomatitis virus G protein into preformed vesicles.

Virosomes were prepared by the insertion of vesicular stomatitis virus glycoprotein, a pH-sensitive fusion protein, into preformed liposomes. The fusogenic activity of these virosomes was characterized in cell-free fusion assays using liposomal targets. Fusion was monitored by concentration-dependent changes in the efficiency of resonance energy transfer between N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine and N-(4-nitrobenzo-2-oxa-1,3-diazol)-phosphatidylethanolamine and by electron microscopy. The fusogenic activity was dependent on the presence of vesicular stomatitis virus glycoprotein, was pH-sensitive, and had a pH threshold of activation similar to that of the native virus. The extent of fusion was dependent upon the lipid composition of the vesicles. This technique will allow vesicles prepared by any method to be made fusogenic.

Fractionation of Pneumocystis carinii developmental stages by counterflow centrifugal elutriation and sequential filtrations.

Immunomagnetic sorting, sequential filtrations, and counterflow centrifugal elutriation were compared for their ability to obtain enriched populations of Pneumocystis carinii developmental stages from infected rat-lung homogenates. Elutriation combined with sequential filtrations resulted in highly (> 95%) enriched populations of P. carinii cysts and trophozoites with excellent viability. This approach offers advantages over previously described methods of obtaining enriched P. carinii cell populations and should have important applications to research on this organism.

Synthesis and characterization of fluorescent neutral lipid analogs containing N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-aminohexanoic acid.

The synthesis, identification and characterization of neutral lipid analogs containing N-(7-nitro-2,1,3-benzoxadiazoi-4-yl)-aminocaproic acid are reported. The acyl-imidazole derivative of the fluorescent fatty acid was used to esterify L-alpha-glycerophosphorylcholine. Fluorescent phosphatidylcholines were converted to the corresponding diacylglycerols by phospholipase C digestion. Triacylglycerols were formed by esterification with either fluorescent fatty acid-imidazole or non-fluorescent fatty acid anhydride. The 11 compounds synthesized were identified by a combination of thin layer chromatography, liquid secondary ion mass spectrometry and enzymatic digestion. A solvent system for identifying all eleven analogs by thin layer chromatography is presented. The fluorescence characteristics of these analogs are consistent with previously observed parameters of NBD-lipid analogs, including the density-dependent quenching of analogs containing multiple NBD fluorophores. These analogs mimic native lipids, as evidenced by digestions with the enzymes, porcine pancreatic lipase, phospholipase C and phospholipase A2.

Association of cytoplasmic free Ca2+ gradients with subcellular organelles.

Previous investigations have identified gradients of intracellular free (Ca2+)i (Ca2+i) in the cytoplasm of human fibroblasts. In this study we have compared the spatial distribution of these gradients with the subcellular distribution of cytoplasmic organelles. Using the Ca(2+)-sensitive dye fura-2 and organelle-specific fluorescent dyes, we have found that the highest Ca2+ concentrations are found in the perinuclear cytoplasm and that these regions co-localize with the Golgi apparatus. The area occupied by the endoplasmic reticulum, which includes the Golgi region plus an adjacent area, is also significantly elevated above the average cellular (Ca2+)i. Most mitochondria are located in regions different from those with the highest (Ca2+)i. A variety of phenomena which could have given rise to artifactual (Ca2+)i gradients have been ruled out, including compartmentalization of fura-2 in subcellular organelles, incomplete hydrolysis of fura-2AM esters, and the presence of pH gradients which might change the Ca2+ binding characteristics of fura-2. The existence of gradients in (Ca2+)i between ER and Golgi containing regions of the cytoplasm supports the hypothesis (Sambrook: Cell 61:197-199, 1990) that the traffic of membrane bound vesicles from ER to Golgi is directed by local variations in (Ca2+)i.

Isolation of Pneumocystis carinii cysts by flow cytometry.

Pneumocystis carinii cysts were separated as enriched populations from suspensions of lung homogenate obtained from infected rats containing all developmental stages of this organism. Isolation of cyst populations was achieved by incubating filtered lung homogenate with the fluorescent phospholipid analog 1-palmitoyl-2-C6-NBD-phosphatidylcholine. Whereas cysts did not fluorescence as a result of outer-wall restraint of lipid integration, trophozoites and young intermediate stages readily incorporated the fluorescently labeled lipid analog into their outer membrane. The two distinct labeling patterns displayed by cysts and other developmental phases of P. carinii constitute a novel, easy, and reproducible means of isolating cysts from infected lung homogenate by flow cytometry.

Fluorescence assay for phospholipid membrane asymmetry.

Highly fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogues are widely used to examine lipid transport and membrane structure. We have developed a method for chemically modifying NBD-labeled lipids in both artificial and biological membranes. This was achieved by treating fluorescently labeled membranes with dithionite (S2O4(-2)). When small unilamellar vesicles containing NBD-labeled phospholipids were reacted with dithionite, only the fluorescent lipid located on the outer leaflet of the vesicles' bilayer was reduced. Seven different NBD-lipid analogues, including a fluorescent sterol, were reduced by treatment with dithionite to nonfluorescent 7-amino-2,1,3-benzoxadiazol-4-yl-lipid derivatives. To assess the feasibility of using this reagent in biological systems, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanol ami ne was inserted into the outer leaflet of the plasma membrane of CHO-K1 cells. Subsequent incubation of these cells with a nontoxic concentration of dithionite resulted in the complete loss of fluorescence from the plasma membrane. In contrast, when cells were permitted to endocytose some of their fluorescently labeled plasma membrane and then treated with dithionite, fluorescence at the plasma membrane was eliminated, while intracellular labeling was not affected. These data suggest that dithionite reacts with NBD-labeled lipids in the outer leaflet of membrane bilayers, producing nonfluorescent derivatives. We demonstrate how reduction of NBD-lipids with dithionite can be used to prepare asymmetrically labeled liposomes and to measure transverse-membrane asymmetry in vesicles. This method should be useful in many biochemical investigations, including the measurement of phospholipid translocase activity.

Evidence for the activity of a phospholipid exchange protein in vivo.

Liposomes containing a self quenching concentration of a fluorescent phosphatidylethanolamine analog, were microinjected into Chinese hamster ovary cells. Immediately after microinjection, little intracellular fluorescence was observed. 10 min post-injection, labeling of the nuclear envelope and mitochondria became evident. Combined with control studies, our results suggest that phospholipid exchange protein(s) facilitates phosphatidylethanolamine movement in vivo.

Liposomes for the transformation of eukaryotic cells.

Gene therapy of human disease is a method of treatment under active development. DNA-loaded liposomes exhibit great promise for use in this field. Liposome-based transfection vectors have many inherent advantages that will likely lead to their wide in vivo use. Vectors with low toxicity and a high degree of targetability can now be easily prepared. These vectors are also free of the length constraints governing retroviral vectors. In this review we discuss recent developments in the use of liposomes for transfection of eukaryotic cells.

Large-scale preparation of asymmetrically labeled fluorescent lipid vesicles.

A method for producing lipid vesicles containing fluorescent phospholipid analogues localized to the inner leaflet of their membrane was developed. Incubation of a 450-fold molar excess of serum albumin with lipid vesicles symmetrically labeled with 1 mol % 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazolyl)amino-caproyl phosphatidylcholine resulted in the removal of 99% of the fluorescent lipid from the outer leaflet. Asymmetrically labeled vesicles were separated from albumin/lipid complexes by gel filtration chromatography. Vesicles prepared in this manner were unable to transfer fluorescent lipid to cells during liposome-cell incubations. Liposomes asymmetrically labeled with other 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-phospholipid analogues were also prepared. Removal of amino-dodecanoyl-NBD-labeled lipids from the outer leaflet of liposomes required three times more bovine serum albumin, and 48 h of incubation. This method can be used to produce large amounts of asymmetrically labeled liposomes suitable for use in investigating a variety of membrane phenomena.

Analysis of Pneumocystis carinii cyst wall. I. Evidence for an outer surface membrane.

It has long been thought that the cyst form of Pneumocystis carinii, which can resist host defenses and antimicrobial drugs, is responsible for relapses of P. carinii pneumonia. The thick wall of the cyst is immunogenic and rich in glucosyl/mannosyl and N-acetyl-D-glucosamine residues. In this study we have demonstrated the presence of a hitherto unreported outer membrane in the cyst wall of P. carinii. This membrane was detected by a combination of techniques, including transmission electron microscopy, freeze-fracture electron microscopy, and membrane labeling with fluorescent lipid analogs following treatment of P. carinii cysts from infected rats for 30 min with Zymolyase, a beta-1-3 glucanase. As in gram-negative bacteria and blue-green algae, this 2nd membrane may have an important role in osmoregulation and nutrient utilization; it may also mediate the interaction of P. carinii with its host and serve as a target for drug therapy.

Synergistic activation of CTP:phosphocholine cytidylyltransferase by phosphatidylethanolamine and oleic acid.

CTP:phosphocholine cytidylyltransferase present in rat liver cytosol was activated almost 30-fold when assayed in the presence of liposomes containing 60 mole % dioleoyl phosphatidylethanolamine (DOPE). During the assay, some of the DOPE was degraded to lysoPE and oleic acid. Whereas cytidylyltransferase activity was not affected when assayed in the presence of liposomes containing lysoPE, liposomes containing oleic acid activated the enzyme. Activation by oleic acid could be eliminated by the addition of fatty acid-free bovine serum albumin (BSA) to the assay. When cytidylyltransferase activity was measured in the presence of both BSA and liposomes containing DOPE, enzyme activity was increased almost 20-fold, as compared with assays performed in the absence of added lipid. The 1.5-fold difference in cytidylyltransferase activity observed when cytosol was assayed with DOPE containing liposomes in the absence or presence of BSA (30-fold stimulation vs 20-fold stimulation) cannot be explained by the loss of activation attributable to oleic acid alone. Activation of the enzyme in the presence of liposomes containing DOPE and oleic acid is several-fold greater than the sum of the activations caused by the individual compounds. These data suggest that PE and oleic acid act synergistically in activating the cytidylyltransferase.

Differences in intracellular transport of a fluorescent phosphatidylcholine analog in established cell lines.

The transport and metabolism of a fluorescent phosphatidylcholine analog, 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)- aminocaproyl-phosphatidylcholine [palmitoyl, C6-NBD)-PC), in BHK, CHO-K1, CHO-15B, MDCK, VA-2, Vero, V79 and WI-38 cells has been investigated. When liposomes containing (palmitoyl, C6-NBD)-PC were incubated with cells at 2 degrees C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells' plasma membranes occurred. Most of the lipid transferred to the cells could be removed by incubating the cells in the presence of nonfluorescent liposomes or media containing 10% serum, suggesting that the fluorescent probe resided exclusively in the outer leaflet of the plasma membrane at 2 degrees C. After insertion into the plasma membrane, internalization of (palmitoyl, C6-NBD)-PC occurred when the cells were warmed to 37 degrees C. This resulted in four different labeling patterns: (1) little or no internalization of (palmitoyl, C6-NBD)-PC into punctate vesicles was observed in Vero cells. (2) Transport of (palmitoyl, C6-NBD)-PC to the region of the Golgi apparatus and to a small number of intracellular vesicles was observed in both V79 and CHO-K1 cell lines. (3) A large number of fluorescently labeled intracellular vesicles with little or no labeling in the region of the Golgi apparatus appeared after the internalization of (palmitoyl, C6-NBD)-PC in BHK, CHO-15B, MDCK and WI-38 cell lines. (4) Accumulation of (palmitoyl, C6-NBD)-PC in small vesicles, mitochondria and the nuclear envelope was observed in VA-2 cells. In addition, cells having a defect in glycoprotein processing and those transformed with simian virus 40 (SV40) internalized the fluorescent lipid probe differently compared with parental lines. Neither differences in rates of endocytosis nor rates of (palmitoyl, C6-NBD)-PC degradation between cell types appears to cause the observed dissimilarities in intracellular lipid transport. We suggest that these different cell types may have dissimilar pathways of intracellular lipid trafficking or differential regulation of a common transport pathway, and that the predominant pathway of lipid translocation can be altered in cells by changing the composition of their glycoproteins or by viral transformation.

Defining lipid transport pathways in animal cells.

A new technique for studying the metabolism and intracellular transport of lipid molecules in living cells based on the use of fluorescent lipid analogs is described. The cellular processing of various intermediates (phosphatidic acid and ceramide) and end products (phosphatidylcholine and phosphatidylethanolamine) in lipid biosynthesis is reviewed and a working model for compartmentalization during lipid biosynthesis is presented.

Transbilayer movement of a fluorescent phosphatidylethanolamine analogue across the plasma membranes of cultured mammalian cells.

The internalization of a fluorescent analogue of phosphatidylethanolamine following its insertion into the plasma membrane of cultured Chinese hamster fibroblasts was examined. When liposomes composed of 50 mol % 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylethanolamine (C6-NBD-PE) and dioleoylphosphatidylcholine were incubated with monolayer cell cultures at 2 degrees C, a spontaneous transfer of the fluorescent lipid from liposomes to cells occurred. As long as the cells were kept at 2 degrees C, the fluorescent lipid remained at the plasma membrane. However, if, after removing the fluorescent liposomes, the cultures were warmed to 37 degrees C, the C6-NBD-PE was internalized and resided in the nuclear envelope, mitochondria, and Golgi apparatus in addition to the plasma membrane. Delivery of the fluorescent lipid to the Golgi apparatus could be blocked by the addition of 2-deoxyglucose plus sodium azide to the incubation medium. Evidence is presented suggesting that while delivery of the fluorescent lipid to the Golgi apparatus was mainly dependent on endocytosis, delivery to the nuclear envelope and mitochondria occurred by rapid transbilayer movement of the lipid across the plasma membrane followed by translocation of lipid monomers. Rapid transbilayer movement of C6-NBD-PE across the plasma membrane was found to be a temperature-dependent process that was blocked below 7 degrees C.

Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus.

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD-PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.