Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes

Incorporation of dioleoyl N-(monomethoxy polyethyleneglycol succinyl)phosphatidylethanolamine (PEG-PE) into large unilamellar liposomes composed of egg phosphatidylcholine:cholesterol (1:1) does not significantly increase the content leakage when the liposomes are exposed to 90% human serum at 37 degrees C, yet the liposomes show a significant increase in the blood circulation half-life (t1/2 = 5 h) as compared to those without PEG-PE(t1/2 less than 30 min). The PEG-PE's activity to prolong the circulation time of liposomes is greater than that of the ganglioside GM1, a well-described glycolipid with this activity. Another amphipathic PEG derivative, PEG stearate, also prolongs the liposome circulation time, although its activity is less than that of GM1. Amphipathic PEGs may be useful for the sustained release and the targeted drug delivery by liposomes.     

Mechanism of hepatocellular uptake of albumin-bound bilirubin

We previously demonstrated that unconjugated bilirubin spontaneously diffuses through phospholipid bilayers at a rate which exceeds albumin dissociation, suggesting that solvation from albumin represents the rate-limiting step in hepatic bilirubin clearance. To further examine this hypothesis, we studied the uptake of bovine serum albumin (BSA)-bound bilirubin by cultured hepatoblastoma (HepG2) cells. Uptake of bilirubin was saturable, with a K(m) and V(max) of 4.2+/-0.5 microM (+/-S.E.M.) and 469+/-41 pmol min(-1) mg(-1) at 25 degrees C. Substantial bilirubin uptake also was observed at 4 degrees C (K(m)=7.0+/-0.8 microM, V(max)=282+/-26 pmol min(-1) mg(-1)), supporting a diffusional transport mechanism. Consistent with reported solvation rates, the cellular uptake of bilirubin bound to human serum albumin was more rapid than for BSA-bound bilirubin, indicative of dissociation-limited uptake. Counterintuitively, an inverse correlation between pH and the rate of bilirubin flip-flop was observed, due to pH effects on the rate of dissociation of bilirubin from albumin and from the membrane bilayer. The identification of an inflection point at pH 8.1 is indicative of a pK(a) value for bilirubin in this range. Taken together, our data suggest that hepatocellular uptake of bilirubin is dissociation-limited and occurs principally by a mechanism involving spontaneous transmembrane diffusion.     

Interaction of immunoglobulin-coupled liposomes with rat liver macrophages in vitro

The interaction between liposomes coated with covalently linked rabbit immunoglobulin (RbIg-liposomes), and rat liver macrophages (Kupffer cells) in monolayer culture was studied biochemically with radioactive tracers and morphologically by electron microscopy. The attachment of immunoglobulin (Ig) to liposomes caused a five-fold increase in liposome uptake by the Kupffer cells at 37 degrees C, in comparison with uncoated liposomes. The uptake was linear with time for at least 4 h and linear with liposome concentration up to a lipid concentration of 0.2 mM. At 4 degrees C uptake, probably representing cell surface-bound liposomes, was reduced to a level of approx. 20% of the 37 degrees C values. Involvement of the Fc receptor in the uptake process was indicated by the reduction of RbIg-liposome uptake by more than 75% as a result of preincubating the cells with heat-aggregated human or rabbit Ig at concentrations (less than 2 mg/ml) at which bovine serum albumin (BSA) had virtually no effect on uptake. At high concentrations (10-35 mg/ml), however, albumin also reduced liposome uptake significantly (20-30%), which suggests an interaction of the RbIg-liposomes with the Kupffer cells that is partially non-specific. RbIg-liposome uptake was dependent on the amount of RbIg coupled to the liposomes. Maximal uptake values were reached at about 200 micrograms RbIg/mumol liposomal lipid. Electron microscopic observations on cells incubated with horseradish peroxidase-containing RbIg-liposomes demonstrated massive accumulation of peroxidase reaction product in intracellular vacuoles, showing that the uptake observed by label association represents true internalization.     

Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa,with its application to cleaning lipid-stained contact lenses

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.     

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.     

Ligase-deficient yeast cells exhibit defective DNA rejoining and enhanced gamma ray sensitivity.

Yeast cells deficient in DNA ligase were also deficient in their capacity to rejoin single-strand scissions in prelabeled nuclear DNA. After high-dose-rate gamma irradiation (10 and 25 krads), cdc9-9 mutant cells failed to rejoin single-strand scissions at the restrictive temperature of 37 degrees C. In contrast, parental (CDC9) cells (incubated with mutant cells both during and after irradiation) exhibited rapid medium-independent DNA rejoining after 10 min of post-irradiation incubation and slower rates of rejoining after longer incubation. Parental cells were also more resistant than mutant cells to killing by gamma irradiation. Approximately 2.5 +/- 0.07 and 5.7 +/- 0.6 single-strand breaks per 10(8) daltons were detected in DNAs from either CDC9 or cdc9-9 cells converted to spheroplasts immediately after 10 and 25 krads of irradiation, respectively. At the permissive temperature of 23 degrees C, the cdc9-9 cells contained 2 to 3 times the number of DNA single-strand breaks as parental cells after 10 min to 4 h of incubation after 10 krads of irradiation, and two- to eightfold more breaks after 10 min to 2.5 h of incubation after 25 krads of irradiation. Rejoining of single-strand scissions was faster in medium. After only 10 min in buffered growth medium and after 10 krads of irradiation, the number of DNA single-strand breaks was reduced to 0.32 +/- 0.3 (at 23 degrees C) or 0.21 +/- 0.05 (at 37 degrees C) per 10(8) daltons in parental cells, but remained at 2.1 +/- 0.06 (at 23 degrees C) or 2.3 +/- 0.07 (at 37 degrees C) per 10(8) daltons in mutant cells. After 10 or 25 krads of irradiation plus 1 h of incubation in medium at 37 degrees C, only DNA from CDC9 cells was rejoined to the size of DNA from unirradiated cells, whereas at 23 degrees C, DNAs in both strains were completely rejoined.     

High-activity radio-iodine labeling of conventional and stealth liposomes

A new method to label preformed liposomes with high activities of radiohalogenated compounds has been developed. It uses activated esters of simple synthetic molecules that may be readily halogenated, such as Bolton-Hunter reagent (BH), and arginine-containing liposomes. BH, in the form of an activated ester, crosses the liposome membrane to react with arginine inside the liposomes, as demonstrated by thin-layer chromatography and by the fact that saline-containing liposomes, or hydrolyzed BH or the water soluble sulfo-BH afforded only marginal encapsulation yields. Under optimized conditions, between 37 and 55 degrees C, 62 +/- 4% (mean +/- SD) of radiolabeled BH were consistently encapsulated in the liposomes within 30 min. In molar amounts, this corresponds to a mean of 56 nmol of BH per micromol of lipids. Based on achievable specific activity, up to 2.8 GBq of iodine-131 could be entrapped per micromol of lipids. Leakage of radioactivity was very low, with less than 5% of the encapsulated activity released within 6 days at 4 degrees C in phosphate-buffered saline and less than 50% within 24 h in human serum at 37 degrees C. The labeling stability, and the fact that both conventional and PEGylated liposomes can be readily labeled with high doses of radioactivity, will make this technique useful for in vivo targeting applications, such as tumor detection (using iodine-123 or iodine-124) or therapy (with iodine-131 or astatine-211).     

A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells

We have previously shown that when cultured fibroblasts are briefly incubated at 2 degrees C with a fluorescent (NBD) analogue of ceramide, N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]-D-e rythro- sphingosine, fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occurs. During further incubation at 37 degrees C, the Golgi apparatus and later the plasma membrane become intensely fluorescent. Concomitantly, the fluorescent ceramide is metabolized to fluorescent analogues of sphingomyelin and glucosylceramide [Lipsky, N. G., & Pagano, R. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2608-2612]. In the present study we synthesized fluorescent N-acylsphingosine analogues using various long-chain bases (D-erythro-sphingosine, L-erythro-sphingosine, D-threo-sphingosine, L-threo-sphingosine, D-erythro-dihydrosphingosine, L-threo-dihydrosphingosine, phytosphingosine, and 3-ketosphingosine) and fluorescent fatty acids (epsilon-NBD-aminohexanoic acid; D- or L-alpha-OH-epsilon-NBD-aminohexanoic acid; D- or L-alpha-NBD-aminohexanoic acid). Using previously described resonance energy transfer assays, we examined the rates of spontaneous transfer of these compounds between liposomes and their ability to undergo transbilayer movement. The fluorescent N-acylsphingosine analogues had half-times for spontaneous transfer of 0.3-4.0 min at 25 degrees C, and all were capable of transbilayer movement in lipid vesicles. The metabolism and intracellular distribution of analogues in cultured fibroblasts were also studied. While most of the fluorescent N-acylsphingosines were significantly metabolized to the corresponding sphingomyelin analogues, metabolism to glucosylceramide was strongly dependent on the long-chain base and the stereochemistry of the fluorescent fatty acid moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of phospholipase activities in chromaffin granule ghosts isolated from the bovine adrenal medulla

Highly purified chromaffin granule membranes contain high levels (100 nmol/mg protein) of long-chain free fatty acids (Husebye, E.S. and Flatmark, T. (1984) J. Biol. Chem. 259, 15272-15276), as well as lysophosphatidylcholine (268 nmol/mg protein) and lysophosphatidylethanolamine (92 nmol/mg protein). The release of saturated and unsaturated long-chain fatty acids from endogenous phospholipids was 38 and 28 nmol/mg protein per h, respectively, at 37 degrees C and pH 7.5 (alkaline pH optimum). p-Bromophenacyl bromide inhibited the release of palmitate and oleate by 88 and 65%, respectively. The deacylation of membrane phospholipids was not significantly affected by micromolar free Ca2+. Based on experiments with pancreatic phospholipase A2, stearate and arachidonate were found to be suitable markers for deacylation at the sn-1 and sn-2 positions, respectively. Experiments with exogenously added labeled phosphatidylcholines confirmed that chromaffin granule ghosts contain a phospholipase A2 activity (alkaline pH optimum). The preparations also revealed a phospholipase A1 activity (acid pH optimum). Finally, the ghosts contain a lysophospholipase activity (alkaline pH optimum), that accounts for the major part of the deacylation of membrane phospholipids, notably the release of saturated fatty acids (stearate and palmitate). It is unlikely that the high content of lysophospholipids is an artifact of the procedure by which the granule ghosts are isolated.     

Simultaneous determination of nitroglycerin and dinitrate metabolites in metabolism studies using liquid chromatography-mass spectrometry with electrospray ionization

We have developed a liquid chromatographic-mass spectrometric method for the simultaneous determination of nitroglycerin (NTG) and its active metabolites, glyceryl 1,2-dinitrate (1,2-GDN) and glyceryl 1,3-dinitrate (1,3-GDN), for metabolism studies in cell cultures. 1,2,4-Butanetriol-1,4-dinitrate was chosen as an internal standard. Using a linear gradient of water/methanol containing 0.025 mM NH(4)Cl, the compounds were eluted within 12.5 min on an Allure Aqueous C(18) column (100 mm x 2.1 mm). Detection and quantification was achieved with multiple reaction monitoring in the negative ion mode. Intra- and inter-day variabilities for simultaneous determination of the three nitrates were below 10 and 18%, respectively, over a range of NTG and GDN concentrations of 0.5-15 ng/ml. The lower limit of quantification was found to be about 0.01 ng on column. Application of this method was illustrated through in vitro metabolism studies of NTG in culture media bathing LLC-PK1 cells and human vascular smooth muscle cells (HA-VSMC) at 37 degrees C. The degradation half-life of NTG was found to be 4.5 +/- 0.4 h and 39.2 +/- 0.02 h, respectively, for LLC-PK1 cells versus HA-VSMC. At 5 h, the 1,2-GDN versus 1,3-GDN metabolite distribution ratio in the bathing medium was found to be 1.5 +/- 0.1 and 0.2 +/- 0.02 for LLC-PK1 and HA-VSMC cells, respectively. With this method, the degradation half-life of NTG in rat plasma at 37 degrees C was shown to be 26.8 +/- 1.8 min, consistent with previous values obtained using gas chromatography.     

Interaction of liposomes with Kupffer cells in vitro

We investigated the interaction of liposomes with rat Kupffer cells in monolayer maintenance culture. The liposomes (large unilamellar vesicles, LUV) were composed of 14C-labelled phosphatidylcholine, cholesterol and phosphatidylserine (molar ratio 4:5:1) and contained either 3H-labelled inulin or 125I-labelled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. After 2-3 days in culture the cells exhibited optimal uptake capacity. The uptake process showed saturation kinetics, maximal uptake values amounting to 2 nmol of total liposomal lipid/h/10(6) cells. This is equivalent to 1500 vesicles per cell. The presence of fetal calf serum (FCS) during incubation increased uptake nearly two-fold, whereas freshly isolated rat serum had no effect. The binding of the liposomes to the cells caused partial release of liposomal contents (about 15-20%) both at 4 degrees C and at 37 degrees C. In the presence of metabolic inhibitors the uptake at 37 degrees C was reduced to about 20% of the control values. Inulin and lipid label became cell-associated at similar rates and extents, whereas the association of albumin label gradually decreased after attaining a maximum at relatively low values. When, after 1 h incubation, the liposomes were removed continued incubation for another 2 h in absence of liposomes led to an approx. 30% release of cell-associated lipid label into the medium in water-soluble form. Under identical conditions as much as 90% of the cell-associated albumin label was released in acid-soluble form. Contrarily, the inulin label remained firmly cell-associated under these conditions. From these results we conclude that Kupffer cells in monolayer culture take up liposomes primarily by way of an adsorptive endocytic mechanism. This conclusion was confirmed by morphological observations on cells incubated with liposomes containing fluorescein isothiocyanate (FITC) dextran or horseradish peroxidase as markers for fluorescence microscopy and electron microscopy, respectively.     

A simple and convenient procedure for the hydrogenation of lipids on the micro- and nanomole scale

A glass tube of a special design has been used as a vessel for the hydrogenation of lipids under slight excess pressure at 50 degrees C. Methyl [1-(14)C]linolenate was quantitatively hydrogenated to methyl stearate in less than 30 min. High yields were obtained on both the micromole scale (mean and standard deviation observed for quadruplicate analyses was 98.2 +/- 4.8%) and the nanomole scale (94.3 +/- 7.0%). The applicability of the method is demonstrated by radio-gas-liquid chromatographic analyses of nanomole amounts of (14)C-labeled fatty acid methyl esters from photosynthetic tissue analyzed before and after hydrogenation.     

Effects of cooling rate and storage temperature on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.     

Long-term follicular dynamics and biochemical characteristics of dominant follicles in dairy cows subjected to acute heat stress

The objective of this study was to examine the quality of successive dominant follicles (DFs) after induced heat stress. Non-lactating dairy cows expressing estrus at normal intervals were allocated randomly to heat stress (HS; n=8) and control (C; n=8) groups. Cows received GnRH (100 microg, i.m.) on Day 0, a progesterone CIDR-B device on Day 4 and prostaglandin (PGF(2alpha); 25mg, i.m.) on Day 7 upon removal of the CIDR device. The DF and follicles >5mm were aspirated on Day 8, and GnRH (100 microg) injected following aspiration, to initiate a new follicular wave. In this manner, a DF was aspirated every 8 days (one "follicular cycle") for 10 cycles. After the first follicular cycle, HS cows were placed in environmental chambers for 7 days during the second follicular cycle (8h per day at 43.3 degrees C set point and 16h per day at 24 degrees C for 4 days, and 8h per day at 43.3 degrees C set point and 16h per day at 32.2 degrees C set point for 3 days; relative humidity, 40%) and thereafter maintained outdoors with control cows at a mean ambient temperature (18.5 degrees C; range 12.7-26 degrees C). Rectal temperature increased (P<0.001) in HS as compared with C cows (39.28+/-0.01 degrees C versus 38.78+/-0.01 degrees C). Concentrations of estradiol (E(2); 1662+/-189 versus 1493+/-188ng/ml) and progesterone (P(4); 44.7+/-5 versus 54.1+/-5.1ng/ml) in follicular fluid (FF) of DF did not differ between C and HS treatments, respectively. Total FF protein concentration was greater (P<0.05) in HS (99.7+/-2.3mg/ml) than in C (92.7+/-2.3mg/ml). Heat shock protein 90 (Hsp 90) in FF was not altered by heat stress. IGF-II ligand blots were conducted with FF samples (n=79) from four HS and four C cows. There was a predominance of IGFBP-3 in 76 of 79 FF samples, indicating healthy follicular status, and only three FF samples had the lower molecular weight IGFBP-2 indicative of a poor quality follicle. Plasma P(4) and E(2) concentrations did not differ between C and HS groups. The number of class 1 and 3 follicles increased during and just after heat stress, but the number of class 2 follicles did not differ between C and HS cows. Heat stress appeared to induce a decrease in follicular dominance, but GnRH-induced follicular cycles resulted in development of healthy preovulatory follicles in both groups.     

Effect of reduced concentrations of glycerol and various macromolecules on the cryopreservation of mouse and cattle embryos

The effect of different macromolecules [bovine serum albumin (BSA), Pluronic F-68, (ET surfactant), or sodium hyaluronate (SH)] on postthaw survival of mouse morulae and in vivo- and in vitro-derived bovine blastocysts frozen in 10, 5, or 1% glycerol solutions was investigated. Embryos were equilibrated with cryoprotectant solution at 25 degrees C for 10 min, seeded at -5 degrees C, cooled at 0.5 degrees C/min to -35 degrees C, and plunged into liquid nitrogen. Embryos were thawed in a 35 degrees C water bath, glycerol was removed with 0.6 M sucrose at 25 degrees C for 5 min, and postthaw viability was evaluated after 1, 24, and 48 h in culture. The addition of BSA supplementation improved postthaw survival of mouse morulae frozen in 5% glycerol, but not in 10% glycerol. All three macromolecular supplements were effective in increasing survival of mouse morulae in 5% glycerol but only BSA and SH were effective in increasing postthaw survival of in vivo- and in vitro-derived bovine blastocysts. None of the macromolecular supplements improved postthaw survival of embryos frozen in 1% glycerol.     

Conformational changes in human serum albumin studied by fluorescence and absorption spectroscopy. Distance measurements as a function of pH and fatty acids.

pH- and fatty acid-induced conformational changes in human serum albumin were investigated by fluorescence-energy transfer, determining the distance between Trp-214 and bound bilirubin at 25 degrees C. This distance changes significantly with the pH, being 2.52 +/- 0.01 nm at pH 6, 2.31 +/- 0.04 nm at pH 9, 2.13 +/- 0.07 nm at pH 11.0 and 2.77 nm at pH 11.9. The influence of different fatty acids on the distance was also determined. At pH 7.4 medium-chain fatty acids seem to increase this distance, whereas long-chain fatty acids, at low concentrations, decrease the distance between the two chromophores. The contraction of the protein carrying long-chain saturated fatty acids is even more pronounced at pH 9.     

Insulin Binding and Effects on Pyrimidine Nucleoside Uptake and Incorporation in Cultured Mouse Astrocytes

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.     

Binding of anandamide to bovine serum albumin

The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium with anandamide bound to BSA inside resealed human red cell membranes at low molar ratios below one. Data were obtained at 0 degrees C, 10 degrees C, 23 degrees C, and 37 degrees C. The equilibrium dissociation constant (Kd) increases with temperature from 6.87 +/- 0.53 nM at 0 degrees C to 54.92 +/- 1.91 nM at 37 degrees C. Regression analyses of the data suggest that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (DeltaG0) is calculated to -43.05 kJ mol-1 with a large enthalpy (DeltaH0) contribution of -42.09 kJ mol-1. Anandamide has vasodilator activity, and the binding to albumin may mediate its transport in aqueous compartments.     

Comparisons of the effects of temperature on the liver fatty acid binding proteins from hibernator and nonhibernator mammals.

Hibernating mammals rely heavily on lipid metabolism to supply energy during hibernation. We wondered if the fatty acid binding protein from a hibernator responded to temperature differently than that from a nonhibernator. We found that the Kd for oleate of the liver fatty acid binding protein (1.5 microM) isolated from ground squirrel (Spermophilus richardsonii) was temperature insensitive over 5-37 degrees C, while the rat liver fatty acid binding protein was affected with the Kd at 37 degrees C being about half (0.8 microM) that found at lower temperatures. This same trend was observed when comparing the specificity of various fatty acids of differing chain length and degree of unsaturation for the two proteins at 5 and 37 degrees C. At the lower temperature, ground squirrel protein bound long-chain unsaturated fatty acids, particularly linoleate and linolenate, at least as well as at the higher temperature and matched requirements for these fatty acids in the diet. The most common long-chain fatty acid, palmitate, was a more effective ligand for ground squirrel liver fatty acid binding protein at 5 degrees C than at 37 degrees C, with the opposite occurring in the eutherm. Rat protein was clearly not adapted to function optimally at temperatures lower than the animal's body temperature.     

Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.