Effect of liposomes containing cholesterol on adenylate cyclase activity of cultured mammalian fibroblasts.

Liposomes prepared with cholesterol and dipalmitoyl phosphatidylcholine were incubated with a clone of normal rat kidney fibroblast of cells in culture. The cells took up [14C]cholesterol in proportion to the concentration of liposomes in the incubation medium, and the uptake increased with time over the four hours of study. Two cell membrane enzymes, adenylate cyclase and (Na+ + K+)-ATPase, exhibited decreased activity after treatment with cholesterol-containing liposomes. The decrease in adenylate cyclase activity was directly proportional to the uptake of [14C]cholesterol. When a variety of subclones of NRK 5W were examined some were found to respond to cholesterol treatment and some did not. These data are consistent with the view that membrane cholesterol content plays a role in controlling the activity of some plasma membrane enzymes.     

Postnatal changes in dolichol-pathway enzyme activities in rat liver.

The activity of hepatic protein N-glycosylation was compared in rats of different ages by incubating UDP-[14C]glucose with liver microsomes. Dolichyl-phosphate [14C]glucose, [14C]glucosyl-oligosaccharide-lipid and [14C]glycoproteins formed were increased after birth to maximal levels at 2 weeks; thereafter dolichylphosphate [14C]glucose remained constant, while [14C]glucosyl-oligosaccharide-lipid and [14C]glycoproteins were decreased to constant levels at 4 weeks. The postnatal change in the formation of [14C]glycoproteins was similar to the change in the hexosamine content of N-glycans in liver microsomes and plasma, suggesting that the N-glycosylation of proteins in rat liver increases after birth to a maximum at 2 weeks, and thereafter decreases to a constant level at 4 weeks. The possibility of a regulatory role for dolichyl phosphate in glycoprotein synthesis in rat liver during postnatal development was eliminated by demonstrating the inefficiency of exogenous dolichyl phosphate on the postnatal changes in [14C]glycoprotein formation. The transfer of [14C]glucose from UDP-[14C]glucose to denatured alpha-lactalbumin in liver microsomes increased to a maximum at 2 weeks and then decreased to a constant level, as with transfer to endogenous proteins (i.e. the formation of [14C]glycoproteins). On the other hand, the transfer of oligosaccharide from exogenous [14C]glucosyl-oligosaccharide-lipid to denatured alpha-lactalbumin reached a maximum at 2 weeks and then remained constant. These results strongly suggest that oligosaccharide-lipid available for N-glycosylation is limiting in rat liver after 2 weeks post partum. The activities of dolichyl-phosphate glucose, dolichyl-phosphate mannose and dolichyl-pyrophosphate N-acetylglucosamine synthases increased until 2 weeks post partum. Thereafter, the activity of dolichyl-pyrophosphate N-acetylglucosamine synthase decreased to a constant level at 4 weeks, while the activities of dolichyl-phosphate glucose and dolichyl-phosphate mannose synthases remained constant. These results suggest that N-glycosylation of proteins in rat liver increases until 2 weeks post partum, and that this depends on the activities of dolichol-pathway enzymes as a whole rather than on the activity of specific enzymes. N-Glycosylation then decreases to a constant level at 4 weeks due to decreases in the activities of enzymes responsible for oligosaccharide assembly on lipids, including dolichyl-pyrophosphate N-acetylglucosamine synthase.     

Leishmania plasma membrane Mg2+-ATPase is a H+/K+-antiporter involved in glucose symport. Studies with sealed ghosts and vesicles of opposite polarity

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.     

Substantial glucose leakage from liposomes on filters and upon molecular-sieve chromatography in determinations of reconstituted glucose-transport activity and liposome volumes

Transport-protein activities are often determined by procedures that involve isolation of liposomes containing the transported radioactive solute. We determined the activity of the human red cell glucose transporter in liposomes and, by similar procedures, internal volumes of liposomes. For these purposes, we isolated freeze-thawed liposomes loaded with [14C]glucose, either by filtration on cellulose-nitrate and cellulose-acetate filters, or by chromatography on Sephadex. The interaction of liposomes with filters caused substantial leakage of [14C]glucose. About half of the internal [14C]glucose was released on the filters from glucose-transporter liposomes with inhibited transport. Chromatography at high flow rate provided higher and more accurate values than did the filtration procedure. Leakage corrections could be made by use of flow-cell scintillation elution profiles. The ratios between the corrected chromatographic volume values and the filtration values were 1.4-3.0 for liposomes without protein, 2.4-4.0 for glucose-transporter liposomes and 3.6-7.9 for liposomes with several human red cell integral membrane proteins. The D-glucose equilibrium exchange with glucose-transporter liposomes at 50 mM D-glucose was 2.0 nmol D-glucose per microgram transporter per second as determined by use of chromatography at high flow rate. The filtration procedure gave only 0.6 nmol.microgram-1.s-1 due to the [14C]glucose leakage. In our experiments, the chromatographic procedure thus proved superior.     

Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+.

The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.     

Alteration of Synaptosomal Plasma Membrane Cholesterol Content: Membrane Physical Properties and Cation Transport Proteins

The level of nerve membrane cholesterol was altered by in vitro incubation of rat brain synaptosomal plasma membrane with liposomes having varying cholesterol contents. The normal plasma membrane cholesterol/phospholipid ratio of 0.3-0.4 (mol/mol) could be decreased by about one-half or increased more than 100%. Fluorescence polarization measurements were made using the probe 1,6-diphenyl-1,3,5-hexatriene. At temperatures below 35 percent C, lowering membrane cholesterol led to increased apparent microviscosity, while raising cholesterol content produced little change. However, at 45 percent C a continuous direct relationship existed between experimental membrane cholesterol/phospholipid ratio (ranging from 0.18 to 0.73) and apparent microviscosity. Under standard liposome-synaptosomal plasma membrane exchange conditions, 80% of the initial specific [(3)H]saxitoxin binding activity to the voltage-dependent sodium channel and at least 95% of the (Mg2+,K+)-p-nitrophenylphosphatase activity were preserved. Our results indicate that neither the characteristics of toxin binding nor the kinetics of this enzyme activity is dependent upon membrane cholesterol content.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.     

Effect of liposomes containing cholesterol on adenylate cyclase activity of cultured mammalian fibroblasts

Liposomes prepared with cholesterol and dipalmitoyl phosphatidylcholine were incubated with a clone of normal rat kidney fibroblast of cells in culture. The cells took up [¹⁴C]cholesterol in proportion to the concentration of liposomes in the incubation medium, and the uptake increased with time over the four hours of study. Two cell membrane enzymes, adenylate cyclase and ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$, exhibited decreased activity after treatment with cholesterol-containing liposomes. The decrease in adenylate cyclase activity was directly proportional to the uptake of [¹⁴C]cholesterol. When a variety of subclones of NRK 5W were examined some were found to respond to cholesterol treatment and some did not. These data are consistent with the view that membrane cholesterol content plays a role in controlling the activity of some plasma membrane enzymes.     

Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg2+ (10 mM) or Ca2+ (1 mM), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 mM Mg2+ and 1 mM Ca2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent Km values for S-adenosyl-L-methionine were 20 and 400-500 microM for the conversion of PE to PME and for the conversion of PME to PC they were 5 microM and 40 microM. S-Adenosyl-L-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [14C]methyl incorporation into total lipid. The apparent Ki value for AdoHcy was 44.7 microM. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approximately 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A2 in the thymocyte plasma membrane.     

Function of alpha-D-glucosyl monophosphorylpolyprenol in biosynthesis of cell wall teichoic acids in Bacillus coagulans.

D-[alpha-14C]]glucosyl phosphorylpolyprenol ([ 14C]Glc-P-prenol) was formed from UDP-D-[14C]glucose in each of the membrane systems obtained from Bacillus coagulans AHU 1631 and AHU 1634 and two Bacillus megaterium strains. Membranes of these B. coagulans strains, which possess beta-D-glucosyl branches on the repeating units in their major cell wall teichoic acids, were shown to catalyze the transfer of the glucose residue from [14C]Glc-P-prenol to endogenous polymer. On the other hand, membranes of B. coagulans AHU 1366, which has no glucose substituents in the cell wall teichoic acid, exhibited neither [14C]Glc-P-prenol synthetase activity nor the activity of transferring glucose from [14C]Glc-P-prenol to endogenous acceptor. The enzyme which catalyzes the polymer glycosylation in the former two B. coagulans strains was most active at pH 5.5 and in the presence of the Mg2+ ion. The apparent Km for [14C]Glc-P-prenol was 0.6 microM. Hydrogen fluoride hydrolysis of the [14C]glucose-linked polymer product yielded a major fragment identical to D-galactosyl-alpha(1----2)(D-glucosyl-beta(1----1/3)) glycerol, the dephosphorylated repeating unit in the major cell wall teichoic acids of these B. coagulans strains. This result, together with the behavior of the radioactive polymer in chromatography on Sepharose CL-6B, DEAE-Sephacel, and Octyl-Sepharose CL-4B, led to the conclusion that [14C]Glc-P-prenol serves as an intermediate in the formation of beta-D-glucosyl branches on the polymer chains of cell wall teichoic acids in B. coagulans.     

Induction of increased calcium uptake in liposomes having membrane proteins of chicken erythrocytes by S-adenosylmethionine

Liposomes having membrane proteins of chicken erythrocytes were prepared and the effect of S-adenosylmethionine on ⁴⁵Ca²⁺ uptake into the liposomes was investigated. S-Adenosylmethionine, a donor of methyl groups in enzymatic methylation, induced an increase of ⁴⁵Ca²⁺ uptake into the proteoliposomes with membrane proteins but not into the liposomes without membrane proteins. Increased release of ⁴⁵Ca²⁺ from the inside of the proteoliposomes was also induced by S-adenosylmethionine. These increases of uptake and release of ⁴⁵Ca²⁺ were inhibited by S-adenosylhomocystein, an inhibitor of enzymatic methylation. Furthermore, membrane proteins from chicken erythrocytes showed protein and phospholipid methyltransferase activities. The uptake of other materials, 3-0-[methyl-³H]glucose, α-[1-¹⁴C]aminoisobutyric acid, ⁴²K⁺ and ⁵⁴Mn²⁺, into the proteoliposomes was not increased by S-adenosylmethionine. These results suggest that enzymatic methylation of membrane components may have an important role in the regulation of calcium transport in the chicken erythrocyte membrane and this regulation is rather specific for calcium.     

Differential distribution of liposome-entrapped [3H]methotrexate and labelled lipids after intravenous injection in a primate

Positive liposomes consisting of phosphatidylcholine, cholesterol and stearylamine and negatively charged liposomes consisting of phosphatidylcholine, cholesterol and phosphatidylserine, were double labelled with either ³H-labelled dipalmitoyl phosphatidylcholine and [¹⁴C]cholesterol or with [¹⁴C]cholesterol and [³H]methotrexate entrapped in the aqueous phase. The plasma levels and urinary excretion of radioactivity from sonicated and non-sonicated liposomes were then compared with the levels of radioactivity from free [³H]methotrexate during a 4 h experimental period after an initial intravenous injection in cynomolgous monkeys. Tissue uptake at the completion of the 4 h experimental period was also measured.It was found that plasma radioactivity from [³H]methotrexate and [¹⁴C]cholesterol in sonicated positive liposomes was cleared more slowly than from comparable non-sonicated liposomes, and considerably slower than from free [³H]methotrexate. Radioactivity from sonicated negative liposomes was cleared more rapidly than from positive sonicated liposomes. Positive liposomes captured considerably more [³H]methotrexate than negative liposomes and showed very low permeability to [³H]methotrexate in in vitro studies, even in the presence of high concentrations of serum.[¹⁴C]Cholesterol radioactivity was cleared more rapidly from plasma than ³H-radioactivity from liposome-entrapped [³H]methotrexate for double-labelled sonicated liposomes and generally showed greater uptake into tissues and red blood cells. ³H-labelled dipalmitoyl phosphatidylcholine in sonicated positive liposomes was cleared faster than [¹⁴C]cholesterol during the first 3 h. The more rapid disappearance of [¹⁴C]cholesterol from the plasma was complemented by greater uptake into a number of tissues, and positive non-sonicated liposomes were taken up to a greater extent by the spleen than equivalent sonicated liposomes.Renal excretion of ³H from liposome-entrapped [³H]methotrexate was considerably less than that of ³H from free [³H]methotrexate. There was insignificant excretion, however, of ¹⁴C from cholesterol in the urine.Entrapment in liposomes completely prevented the otherwise considerable breakdown of free methotrexate to ³H-containing products in plasma and partially prevented its breakdown in tissues.These studies indicate marked differences in the distribution of liposomes in vivo due to surface charge and size, and some degree of exchange of the lipid components of the liposome bilayer independent of the distribution of the entrapped species. They also show that entrapment in liposomes can reduce metabolic degradation of a drug, maintain high plasma levels and reduce its renal excretion.     

Differential distribution of liposome-entrapped [H]methotrexate and labelled lipids after intravenous injection in a primate

Positive liposomes consisting of phosphatidylcholine, cholesterol and stearylamine and negatively charged liposomes consisting of phosphatidylcholine, cholesterol and phosphatidylserine, were double labelled with either ³H-labelled dipalmitoyl phosphatidylcholine and [¹⁴C]cholesterol or with [¹⁴C]cholesterol and [³H]methotrexate entrapped in the aqueous phase. The plasma levels and urinary excretion of radioactivity from sonicated and non-sonicated liposomes were then compared with the levels of radioactivity from free [³H]methotrexate during a 4 h experimental period after an initial intravenous injection in cynomolgous monkeys. Tissue uptake at the completion of the 4 h experimental period was also measured.It was found that plasma radioactivity from [³H]methotrexate and [¹⁴C]cholesterol in sonicated positive liposomes was cleared more slowly than from comparable non-sonicated liposomes, and considerably slower than from free [³H]methotrexate. Radioactivity from sonicated negative liposomes was cleared more rapidly than from positive sonicated liposomes. Positive liposomes captured considerably more [³H]methotrexate than negative liposomes and showed very low permeability to [³H]methotrexate in in vitro studies, even in the presence of high concentrations of serum.[¹⁴C]Cholesterol radioactivity was cleared more rapidly from plasma than ³H-radioactivity from liposome-entrapped [³H]methotrexate for double-labelled sonicated liposomes and generally showed greater uptake into tissues and red blood cells. ³H-labelled dipalmitoyl phosphatidylcholine in sonicated positive liposomes was cleared faster than [¹⁴C]cholesterol during the first 3 h. The more rapid disappearance of [¹⁴C]cholesterol from the plasma was complemented by greater uptake into a number of tissues, and positive non-sonicated liposomes were taken up to a greater extent by the spleen than equivalent sonicated liposomes.Renal excretion of ³H from liposome-entrapped [³H]methotrexate was considerably less than that of ³H from free [³H]methotrexate. There was insignificant excretion, however, of ¹⁴C from cholesterol in the urine.Entrapment in liposomes completely prevented the otherwise considerable breakdown of free methotrexate to ³H-containing products in plasma and partially prevented its breakdown in tissues.These studies indicate marked differences in the distribution of liposomes in vivo due to surface charge and size, and some degree of exchange of the lipid components of the liposome bilayer independent of the distribution of the entrapped species. They also show that entrapment in liposomes can reduce metabolic degradation of a drug, maintain high plasma levels and reduce its renal excretion.     

Surface-active phospholipase A2 in mouse spermatozoa

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.     

The formation of sphingomyelin from phosphatidylcholine in plasma membrane preparations from mouse fibroblasts

The enzymatic formation of radioactive sphingomyelin from [14C]choline-labeled phosphatidylcholine was demonstrated to reside exclusively in the plasma membrane fraction of mouse fibroblasts. This activity has several properties in common with the phosphatidylcholine ceramide phosphocholine transferase of mouse liver microsomes. The enzyme has little if any phospholipase C activity and isotope dilution experiments suggest that phosphatidylcholine is the substrate rather than it is converted to CDP choline, phosphocholine, free choline or glycerophosphocholine prior to the transfer reaction. The activity is stimulated by the addition of bovine serum albumin and MnCl2 to the incubation mixtures. The plasma membrane localization of the enzyme suggests that it may have a central role in the biosynthetic pathways for sphingomyelin in mouse fibroblasts.     

The effect of 25-hydroxycholesterol on accumulation of intracellular calcium.

Liposomes prepared with 25-hydroxycholesterol and egg phosphatidylcholine (PC) were incubated with bovine arterial smooth muscle cells for 8 h at 37 degrees C. Cells incubated in the absence of liposomes or with liposomes containing cholesterol and PC were used as controls. The results indicated that calcium accumulated in the smooth muscle cells incubated in the presence of 25-hydroxycholesterol containing liposomes in an amount proportional to the time of incubation. The calcium accumulation, as indicated by kinetic analysis, resulted from an increased compartment size. (Ca(2+)+Mg2+)-ATPase exhibited decreased activity after pretreatment with 25-hydroxycholesterol containing liposomes and the increased intracellular calcium content was directly proportional to the decreased (Ca(2+) + Mg2+)-ATPase activity. When lipids in the cell membrane were examined, a failure to change the cholesterol/phospholipids ratio in the membrane was noted. The 25-hydroxycholesterol content in the membrane determined by HPLC did not increase. An increase in sphingomyelin and a decrease in phosphatidylethanolamine and acidic phospholipids in the membrane was noted. We suggest that the accumulation of intracellular calcium comes from both an increase of calcium influx and a decrease of (Ca(2+) + Mg2+)-ATPase activity, which may be the consequence of changes in membrane phospholipid composition.     

Sphingomyelin liposomes with defined fatty acids: metabolism and effects on reverse cholesterol transport

Small unilamellar liposomes prepared from sphingomyelins with defined 14C-labeled fatty acids were studied after injection into rats. The liposomes contained trace amounts of [3H]cholesteryl linoleyl ether (CLE), which served as a nonexchangeable and nonhydrolyzable marker. The liposomes were cleared from the circulation with an initial t1/2 of about 90 min. [14C]18:0- and [14C]18:1-containing sphingomyelins were cleared at a similar rate, but [14C]18:2-sphingomyelin disappeared much faster. The liver accounted for up to 70% of [3H]cholesteryl ether injected with 18:0-sphingomyelin liposomes, and for up to 50% with liposomes prepared from 18:1 or 18:2-sphingomyelin. The initial uptake of the liver appeared to be of the entire particle, and the loss of 14C label with time indicated metabolism of the sphingomyelins. With [14C]18:0-sphingomyelin liposomes, up to 8% of liver radioactivity was recovered in neutral lipids 6 h after injection, and this value was 17 and 22% with [14C]18:2- and [14C]18:1-sphingomyelins, respectively. The recovery in 'carcass' of [3H]cholesteryl ether 3 h after injection of [14C]18:2-sphingomyelin liposomes was 33% and of 14C label, 21%. Injection of 18:1- or 18:2-sphingomyelin liposomes (5.4 mumol/100 g body weight) resulted in a 2-fold increase of plasma unesterified cholesterol; a 30% increase was seen with 18:0 liposomes (2.63 mumol/100 g body weight). In experiments with cultured cells, the unsaturated sphingomyelin liposomes alone enhanced cholesterol efflux more extensively than the saturated ones, but their efficacies became similar when mixed with apoprotein (apo) A-I. At equimolar concentration, apo C-III1 or C-III2 had a smaller effect than apo A-I. It is concluded that 18:1- or 18:2-sphingomyelin tends to form small unilamellar liposomes which may reach also extrahepatic tissues. The liposomes able to enhance cholesterol release in vitro and in vivo. Since they are not a substrate for lecithin-cholesterol acyltransferase, they should be able to deliver the free cholesterol to the liver, where they are also rapidly metabolized.     

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.     

A neutral phospholipase D activity from rat brain synaptic plasma membranes. Identification and partial characterization

Rapid activation of phospholipase D (PLD) in response to cell stimulation was recently demonstrated in many systems, raising the hypothesis that PLD participates in transduction of extracellular signals across the plasma membrane. In the present study, we describe the identification of a neutral PLD activity in purified rat brain synaptic plasma membranes, and the in vitro conditions required to assay its catalytic activity with exogenous [3H]phosphatidylcholine as substrate. Production of [3H]phosphatidic acid, the natural lipid product of PLD and of [3H]phosphatidylethanol, catalyzed by PLD in the presence of ethanol via transphosphatidylation, were measured. The synaptic membrane PLD exhibited its highest activity at pH 7.2 and was thus defined as a neutral PLD. Enzyme activity was absolutely dependent on the presence of sodium oleate and was strongly activated by Mg2+ ions (at 1 mM). Ca2+ at concentrations up to 0.25 mM was as stimulatory as Mg2+, but at 2 mM it completely inhibited enzyme activity. Mg2+ extended the linear phase of PLD activity from 2 to 15 min, suggesting that it may stabilize the enzyme under our assay conditions. The production of [3H]phosphatidylethanol was a saturable function of ethanol concentration. Production of [3H] phosphatidic acid was inversely related to the concentration of ethanol and to the accumulation of phosphatidylethanol, indicating that the two phospholipids are indeed produced by the competing hydrolase and transferase activities of the same enzyme. beta,beta-Dimethylglutaric acid, utilized previously as a buffer in studies of rat brain PLD, inhibited enzyme activity at neutral pH but not at acidic pH. The properties of the neutral synaptic membrane PLD and its relationships with other in vitro, acid, and neutral PLD activities, as well as with the signal-dependent PLD detected in intact cells, are discussed.