Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Increased concentrations of phosphatidylinositol (PI) and decreased esterification of arachidonic acid into phospholipids in platelets from patients with schizoaffective disorders or atypic phasic psychoses.

The concentration of various phospholipids (PLs) and sphingomyelin in platelets and the amount of [14C] arachidonic acid ([14C]-AA) esterified in phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were measured. The platelet-rich plasmas from unmedicated patients with psychiatric disorders and healthy controls were incubated for 30 min with 1 microM [14C]-AA. Platelets from patients with a schizoaffective disorder according to RDC criteria, a schizophreniform disorder (DSM III criteria) or an atypical phasic psychosis according to FC criteria contained twice as much PI and had significantly increased concentrations of PC as compared to controls (p less than 0.05, t-test). A highly significant (40-70%) reduced rate of esterification of [14C]-AA into PI/PS, PC and PE was found in platelets from patients with schizophreniform, schizoaffective and major depressive disorders but not in platelets from patients with chronic schizophrenia. The largest reduced esterification of [14C]-AA (about 70%) was found in PI/PS of platelets from patients with schizoaffective disorders (1.9 +/- 0.7 vs 6.3 +/- 1.7 mumol [14C]-AA/mol PI/PS; p less than 10(-4), t-test). The results indicate that changes in the metabolism of arachidonic acid and phosphatidylinositol and, to a lesser degree, of phosphatidylcholine in platelets are characteristic of patients with a likely favorable outcome of a psychotic episode.     

Bacterial lipopolysaccharide stimulates phospholipid synthesis and phosphatidylcholine breakdown in cultured human leukemia monocytic THP-1 cells.

1. De novo synthesis of phospholipid and its catabolism in human leukemia monocytic THP-1 cells were investigated. 2. Radiolabelled precursors: [methyl-3H]chloride, [1,2-14C]ethanolamine and myo-[2-3H]inositol were readily incorporated into CHCl3-MEOH extractable lipid fraction as a function of time. 3. The radiolabels derived from choline, ethanolamine and inositol were preferentially incorporated into PC, PE and PI fraction, respectively. The data indicate that de novo PL synthesis takes place, and the CDP-choline pathway is operative as a major pathway for PC synthesized in THP-1 cells. 4. Bacterial endotoxin dose-dependently stimulated the incorporation of radiolabelled precursors. Approximately 50% stimulation in PC and PE synthesis was obtained in 20 hr, while the incorporation of [3H]inositol was rapidly stimulated by 170% within 4 hr, and the stimulation declined drastically thereafter. 5. LPS did not alter the radiolabel distribution into PL in any of the three cases. 6. In pulse-chase studies, the cells prelabelled with radioactive PL were exposed to LPS (1 micrograms/ml). The breakdown of PC was enhanced about 30% within the first 2 hr followed by a stimulated PC synthesis observed in the next 4 hr. In contrast, LPS did not induce the hydrolysis of PE and PI. 7. The data indicate that LPS produces a broad spectrum of stimulatory effects on PL synthesis and selectively stimulates the hydrolysis of PC via phospholipase C/D reaction in THP-1 cells.     

Palmitate and oleate metabolism of rainbow trout in vivo

The turnover rates of palmitate and oleate were measured in vivo by continuous infusion of 1-[14C]palmitate and 9,10-[3H]oleate in rainbow trout. Our goals were: (1) to quantify the incorporation of a saturated and of a monounsaturated fatty acid into other classes of plasma lipids (neutral lipids, NL, and phospholipids, PL); and (2) to determine whether they could both be used as tracers to quantify fluxes of total non-esterified fatty acids (NEFA). We found that both acids play very different physiological roles because palmitate is preferentially channeled towards plasma PL, whereas oleate is mainly incorporated in circulating NL. Consequently, palmitate is predominantly involved in membrane PL turnover and oleate in the metabolism of circulating NL that may be used to shuttle oxidative fuel in teleosts. Despite this striking difference in their metabolism, palmitate and oleate have flux rates that are proportional to their relative abundance in plasma NEFA (i.e. they have the same fractional turnover rate). They can therefore both be used as reliable tracers to quantify the kinetics of total NEFA.     

Arachidonic acid turnover in peritoneal macrophages is altered in endotoxin-tolerant rats

Peritoneal macrophages from endotoxin-tolerant rats have been found to exhibit depressed metabolism of arachidonic acid (AA) to prostaglandins and thromboxane in response to endotoxin. The effect of endotoxin tolerance on AA turnover in peritoneal macrophages was investigated by measuring [14C]AA incorporation and release from membrane phospholipids. Endotoxin tolerance did not affect the amount of [14C]AA incorporated into macrophages (30 min-24 h). However, the temporal incorporation of [14C]AA into individual phospholipid pools (15 min-24 h) was altered. In endotoxin-tolerant macrophages, [14C]AA incorporation into phosphatidylcholine (PC) (2, 4, 24 h) and phosphatidylethanolamine (PE) (8 h) was increased, while the incorporation into phosphatidylserine (PS) (2-24 h) was reduced (P less than 0.005) compared to control macrophages. There was no change in [14C]AA incorporation into phosphatidylinositol (PI). Following 2 or 24 h of incorporation of [14C]AA, macrophages were incubated (3 h) with endotoxin (50 micrograms/ml) or A23187 (1 microM), and [14C]AA release was measured. Endotoxin-tolerant macrophages released decreased (P less than 0.05) amounts of [14C]AA in response to both endotoxin and the calcium ionophore A23187 compared to controls. Control macrophages in response to endotoxin released [14C]AA from PC, PI and PE. In contrast, tolerant cells released [14C]AA only from PC (P less than 0.05). A23187 released [14C]AA from all four pools in the control cells, but only from PC and PE in the tolerant cells. These data demonstrate that endotoxin tolerance alters the uptake and release of AA from specific macrophage phospholipid pools. These results suggest that changes in AA turnover and/or storage are associated with endotoxin tolerance.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Impact of endothelial lipase on cellular lipid composition

Using mass spectrometry (MS), we examined the impact of endothelial lipase (EL) overexpression on the cellular phospholipid (PL) and triglyceride (TG) content of human aortic endothelial cells (HAEC) and of mouse plasma and liver tissue. In HAEC incubated with the major EL substrate, HDL, adenovirus (Ad)-mediated EL overexpression resulted in the generation of various lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) species in cell culture supernatants. While the cellular phosphatidylethanolamine (PE) content remained unaltered, cellular phosphatidylcholine (PC)-, LPC- and TG-contents were significantly increased upon EL overexpression. Importantly, cellular lipid composition was not altered when EL was overexpressed in the absence of HDL. [¹⁴C]-LPC accumulated in EL overexpressing, but not LacZ-control cells, incubated with [¹⁴C]-PC labeled HDL, indicating EL-mediated LPC supply. Exogenously added [¹⁴C]-LPC accumulated in HAEC as well. Its conversion to [¹⁴C]-PC was sensitive to a lysophospholipid acyltransferase (LPLAT) inhibitor, thimerosal. Incorporation of [³H]-Choline into cellular PC was 56% lower in EL compared with LacZ cells, indicating decreased endogenous PC synthesis. In mice, adenovirus mediated EL overexpression decreased plasma PC, PE and LPC and increased liver LPC, LPE and TG content. Based on our results, we conclude that EL not only supplies cells with FFA as found previously, but also with HDL-derived LPC and LPE species resulting in increased cellular TG and PC content as well as decreased endogenous PC synthesis.     

Selective utilization of omega 6 and omega 3 polyunsaturated fatty acids by human skin fibroblasts

Incorporation of exogenous [14C] arachidonate by human skin fibroblasts was found to be significantly greater than that of either [14C]linoleate or alpha-[14C] linolenate. Arachidonate was preferentially esterified in the PI + PS and PE classes of phospholipids. Over 40% of the incorporated [14C] arachidonate was chain elongated in 24 hours. Cells were also grown in lipid-free medium to enhance PUFA desaturation and elongation and the utilization of various omega 6 and omega 3 metabolites examined. Whereas [14C] linoleate partitioned approximately 50:50 between PL and TAG, eicosatrienoate (20:3 omega 6) was selectively sequestered in TAG. Arachidonate and docosatetraenoate (22:4 omega 6) were preferentially incorporated into phospholipids; the PI + PS fraction was most highly enriched with arachidonate. Modification of alpha-[14C] linolenate was more extensive than that of [14C] linoleate. Docosapentaenoate (22:5 omega 3) was the major omega 3 [14C] PUFA of PI + PS and PE. Eicosapentaeonate was not selectively incorporated into phospholipids; within phospholipids the 20:5 omega 3 was primarily in PC. These results indicate that human skin fibroblasts exhibit acyl specificity in the esterification of polyunsaturated fatty acids, including preferential utilization of arachidonate rather than other prostaglandin precursors in the PI + PS fraction.     

Metabolic fate of plasma membrane diacylglycerols in NIH 3T3 fibroblasts.

We have examined the metabolism of three radiolabeled 1,2-diacylglycerols (DGs) in NIH 3T3 fibroblasts. Since the lipids used are not appreciably taken up by the cells, we used a phosphatidylserine (PS)-based liposome fusion system to rapidly associate the lipid species with the plasma membrane. When 1,2-[1-14C]dioleoyl-sn-3-glycerol ([14C]DOG) is delivered in this way, it is rapidly converted predominantly to phosphatidylcholine (PC) and triacylglycerol (TG) and to a lesser extent, to monoacylglycerol (MG) and fatty acids (FA), as well as phosphatidic acid (PA) and phosphatidylinositol (PI). We present evidence that [14C] DOG is largely utilized as an intact molecule rather than being broken down to FA and then incorporated to cell lipids. Examination of the metabolism of 1-stearoyl-2-[1-14C]myristoyl-sn-3-glycerol ([14C]SMG) and 1-stearoyl-2-arachidonoyl-sn-3-glycerol ([14C]SAG) reveal important differences. Both produce substantial labeling of PC but [14C]SMG gives rise to the highest proportion of TG and the lowest of PA and PI, whereas [14C]SAG yields the opposite pattern. When phosphatidic acid labeled on its glycerol backbone (1,2-dioleoyl-sn-[U-14C] glycero-3-phosphate) was supplied to the cells via the liposomes, rapid appearance of labeled DG was found which then decreased with concomitant labeling of cellular PC and TG. Only small amounts of the glycerol backbone were recovered in PI. Our experiments identify three types of processes involved in the metabolism of plasma membrane DGs: (i) transferase-catalyzed conversions to PC and TG, (ii) lipolytic breakdown to MG and FA, and (iii) phosphorylation to PA and then conversion to PI. The relative proportions of each DG species converted to these different products are strongly dependent on the fatty acyl composition of the particular DG molecular species, even though formation of PC is the major event in all cases. Since DGs are important second messengers, our study supports the view that conversion to PC and TG can play a key role in DG signal attenuation.     

Phospholipid and arachidonic acid metabolism in zymosan-stimulated human monocytes: modulation by cAMP

Receptor-ligand interaction in mononuclear phagocytes is intimately linked to alterations in membrane phospholipids and release of arachidonic acid (AA). In addition, synthesis of bioactive lipids from released AA can result in further modification of cell responses. Upon challenge with opsonized zymosan, [3H]-arachidonic acid ([3H]-AA)-labeled human monocytes released 25 +/- 2% of their incorporated radiolabel within 30 min. Pretreatment of the monocytes with 5 X 10(-4) M isobutylmethylxanthine (IBMX) or 1 X 10(-3) M dibutyryl cyclic AMP (d-cAMP) inhibited total [3H]-AA release in the presence of zymosan by 47% and 42%, respectively. Analysis of incorporated [3H]-AA in cellular phospholipid pools indicated that significant amounts of label were lost from both phosphatidylcholine (PC) and phosphatidylinositol (PI) during zymosan stimulation. Treatment with d-cAMP substantially inhibited the loss of label from PC, but had no affect on PI. HPLC analysis of cell supernatants from zymosan-treated cells indicated that 5-HETE was the predominant metabolite generated from [3H]-AA, and its production was depressed during treatment with d-cAMP. Phospholipase activity in human monocyte homogenates was not effected by d-cAMP or IBMX at the highest concentrations used, whether these were added directly to the homogenate or by pretreatment of whole cells, demonstrating that inhibition required an intact cell. These results suggest that human monocytes exposed to opsonized zymosan release AA via two mechanisms and that modulation by cAMP is indirectly effecting a phospholipase directed towards PC.     

Rapid remodeling of arachidonate from phosphatidylcholine to phosphatidylethanolamine pools during mast cell activation.

The objective of the present study was to better understand the remodeling of arachidonic acid (AA) in phospholipids of the mouse bone marrow-derived mast cell (BMMC) during Ag and ionophore A23187 activation. Initial studies were designed to understand the movement of AA in phospholipid classes under resting conditions. BMMC pulse labeled with AA incorporated greater than 95% of the label into the major phospholipid classes. Phosphatidylcholine (PC) subclasses, 1-acyl-2-arachidonoyl-(sn-glycero-3-phosphocholine (GPC)) in particular, initially accounted for most of the label incorporated into the cells with phosphatidylinositol/phosphatidylserine (PI/PS) and phosphatidylethanolamine (PE) subclasses containing much smaller quantities. Prolonged incubation of labeled BMMC resulted in a decrease in the radioactivity in PC with a concomitant increase in PE such that 1-alk-1-enyl-2-arachidonoyl-(sn-glycero-3-phosphoethanolamine (GPE)) became the single largest labeled AA pool by 24 h. Further experiments indicated that 24 h was the time required to reach isotopic equilibrium among AA-containing phospholipids of the BMMC. In the next series of experiments, BMMC phospholipids were labeled to different specific activities by either labeling the cells for 0.5 h or for 24 h followed by stimulation. Under isotopic equilibrium conditions (24 h), stimulation resulted in AA release from PE greater than PC much greater than PI/PS with 1-alk-1-enyl-2-arachidonoyl-GPE providing the bulk of AA released from the BMMC. By contrast, cells labeled for 0.5 h released AA from PC much greater than PI/PS, with 1-acyl-2-arachidonoyl-GPC accounting for most of the AA released from BMMC phospholipids. Label associated with PE subclasses under nonequilibrium conditions remained unchanged or slightly increased throughout a 10-min stimulation period. Finally, BMMC were double labeled with [14C]-AA for 24 h and then with [3H]-AA for 0.5 h. Cell stimulation resulted in a decrease in the [3H]/[14C] ratio in PC and PI and an increase in the ratio in PE. The decrease in [3H]/[14C] ratio in PC was mainly in 1-acyl-2-arachidonoyl-GPC, whereas the increase in PE subclasses was primarily in 1-alk-1-enyl-2-arachidonoyl-GPE. The [3H]/[14C] ratio in cellular neutral lipids and in supernatant fluid products were at values between PC and PE subclasses. Taken together, these data suggest that during Ag activation, the release of free arachidonic acid is from predominantly PE subclasses. Concomitant with the release of AA, there is a rapid remodeling of AA from PC subclasses into PE subclasses (1-alk-1-enyl-2-acyl-GPE).     

Incorporation of 2-fluorohistidine in murine protein in vivo.

The tissue distribution and time course of incorporation into acid insoluble (bound) and acid soluble (free) fractions of [3H]2-fluorohistidine is compared to that of U[14C]Histidine in mouse tissues in vivo. The cycloheximide-sensitive incorporation of 2-FHis is between 9 and 17 percent of that of His. Unlike [14C]His a major fraction, approximately 90% at 72 hrs, of isotope derived from [3H]2-FHis remains in tissues for a prolonged period in an acid soluble form. The excretion of isotope derived from [14C]His (T1/2 = 5 hr) is more rapid than from [3H]2-FHis (T1/2 = 11.4 hrs). 2-FHis, at doses from 100 to 250 mg/kg produce a reversible inhibition of growth in mice.     

Phospholipid molecular species, beta-oxidation, desaturation and elongation of fatty acids in Atlantic salmon hepatocytes: effects of temperature and 3-thia fatty acids

We have investigated the effects of a 3-thia fatty acid (TTA) and of temperature on the fatty acid (FA) metabolism of Atlantic salmon (Salmo salar). One experiment investigated the activity of the peroxisomal beta-oxidation enzyme, acyl-CoA oxidase (ACO), and the incorporation of TTA into phospholipid (PL) molecular species. Salmon hepatocytes in culture were incubated either without TTA (control(spades)) or with 0.8 mM TTA (TTA(spades)) in a short term (48 h) temperature study at 5 degrees C and at 12 degrees C. TTA was incorporated into the four PL classes studied: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). TTA was preferentially esterified with 18:1, 16:1, 20:4 and 22:6 in the PLs. Hepatocytes incubated with TTA had higher ACO activity at 5 degrees C than at 12 degrees C. In a second experiment salmon were fed a diet based on fish meal-fish oil without any TTA added (control) or a fish meal-fish oil diet supplemented with 0.6% TTA for 8 weeks at 12 degrees C and 20 weeks at 5 degrees C. At the end of the feeding trial, hepatocytes from fish acclimated to high or low temperatures were isolated from both dietary groups and incubated with either [1-(14)C]18:1 n-9 or [1-(14)C]20:4 n-3 at 5 degrees C or 12 degrees C. Radiolabelled 18:1 n-9 was mainly esterified into neutral lipids (NL), whereas [1-(14)C]20:4 n-3 was mainly esterified into PL at both temperatures. The rate of elongation of [1-(14)C]18:1 n-9 to 20:1 n-9 was twice as high in hepatocytes from fish fed the control diet than it was in hepatocytes from fish fed the TTA diet, at both temperatures. The amount of [1-(14)C]20:4 n-3 converted to 22:6 n-3 was approximately the same in hepatocytes from the two dietary groups, but there was a tendency to higher production of 22:6 n-3 at the lower temperature. Oxidation of [1-(14)C]18:1 n-9 to acid soluble products (ASP) and CO(2) was approximately 10-fold greater in hepatocytes kept at 5 degrees C than in those kept at 12 degrees C and the main oxidation products formed were acetate, oxaloacetate and malate.     

Lipid and membrane fluidity abnormalities in platelets and megakaryocytes of the hereditary macrothrombocytopenic Wistar Furth rat.

Biochemical and functional abnormalities of megakaryocytes and platelets were studied in Wistar Furth (WF) rats which have genetically determined macrothrombocytopenia and megakaryocytopenia, and were compared with their counterparts in Sprague-Dawley (SD) rats. Both megakaryocytes and platelets synthesized phospholipids from [14C]acetate. WF and SD megakaryocytes incorporated 0.27 and 0.29 nmol acetate per 10(6) cells, respectively. Phosphatidylcholine (PC) accounted for 64% and 58% of the PL radioactive label in megakaryocytes of SD and WF rats, respectively, (P less than 0.05), while 69% of labeled activity was associated with PC of SD platelets compared to 60% found in PC of WF platelets (P less than 0.01). In WF platelets a significant increase in the levels of lysophosphatidylcholine (6.1% vs. 3.0%) was observed. WF platelets had substantially higher levels of esterified cholesterol, triglycerides, ceramides and a 3-fold increase in the total protein per platelet compared to SD platelets. The fatty acid composition of WF platelet PC showed quantitative abnormalities. Plasma lecithin-cholesterol acyl transferase activity and platelet function monitored by the uptake and release of [14C] serotonin showed nonsignificant variations between SD and WF rats. Compared with the control, platelet membrane fluidity, measured by fluorescence polarization using platelets labeled with 1,6-diphenyl-1,3,5-hexatriene, was significantly decreased in the WF rats.     

Isolation and characterization of outer and inner membranes of Selenomonas ruminantium: lipid compositions.

The isolation procedure and characterization of the outer and inner membranes from Selenomonas ruminatium cells, a strictly anaerobic bacterium, are described. The metabolic fate of [14C]decanoate incorporated into the outer and inner membranes was examined. The percent distribution of radioactivities in the outer and inner membranes was about 40 and 50% of the total incorporated activity, respectively. Approximately 47% of the radioactivity incorporated into the outer membrane was recovered in the phospholipid fraction, and the remaining radioactivity was found in both aqueous and phenol layers when the outer membrane was treated with phenol-water. In contrast to [14C]decanoate, the percent distribution of [3H]glycerol in the outer and inner membranes was about 25 and 70% of the total incorporated activity, respectively. Most of the assimilated 3H was located in the phospholipid fraction of both membranes. However, no significant label was detected in either the protein or cell wall fraction. The following observations were made concerning lipid compositions in the outer and inner membranes by chemical and isotopic analyses. (i) The outer and inner membranes contained no detectable phosphatidyl glycerol or cardiolipin. (ii) A prominent radioactive compound, designated band III lipid, was found mainly in the outer membrane as a major radioactive spot when cells were grown with [14C]decanoate. This lipid contained phosphorus, 2-keto-3-deoxyoctulosonic acid and 3-OH fatty acid but no detectable glycerol. This lipid was identified tentatively to be 2-keto-3-deoxyoctulosonic acid-lipid A. (iii) Although the ubiquity of phosphatidyl ethanolamine plasmalogen in both outer and inner membranes was confirmed, the occurrence of the molecular species of phosphatidyl ethanolamine plasmalogen was quite different in the outer and inner membranes.     

Distribution and metabolism of arachidonic and docosahexaenoic acids in rat pineal cells. Effect of norepinephrine

The time-course incorporation of 10 μM [¹⁴C]arachidonic (AA) and docosahexaenoic (DHA) acids into glycerolipids was studied in rat pineal cells. The incorporation of both labeled fatty acids into total lipids was approximately equal, but their distribution profiles among the various cell lipids showed marked differences. The esterification of [¹⁴C]DHA in the neutral lipids, triacylglycerols (TAG) and cholesterol esters (CE), was 2-fold higher than that of [¹⁴C]AA whereas the opposite could be observed in total phospholipids (PL). The order of incorporation into PL was phosphatidylcholine (PC) > phosphatidylinositol (PI) = phosphatidylethanolamine (PE) for [¹⁴C]AA and PC = PE for [¹⁴C]DHA, the incorporation of both fatty acids being not detected in phosphatidylserine (PS) and that of DHA not in PI. When using 0.5 μM [³H] fatty acids, the respective distribution patterns resembled that of fatty acids at 10 μM, except for a lower proportion in TAG. The stimulation of ³H-labeled cells by 100 μM norepinephrine induced a 170% increase of basal release of [³H]AA into the medium, while [³H]DHA was virtually not released. However, the analysis of cell labeling revealed that both [³H] fatty acid levels were decreased in PL and increased in TAG. These findings suggest different involvement for AA and DHA in the pineal function. The preferential incorporation of DHA in TAG suggests that TAG might play an important role in the pineal enrichment with DHA. The absence of DHA release after NE stimulation, which however cannot be ascertained, may raise the question of the role of DHA in NE transduction.     

Lipid biosynthesis in warm- and cold-acclimatized sea anemones, Metridium senile (L.)

Sea anemones, Metridium senile (L.), naturally acclimatized to warm (18°C) and cold (0°C) conditions, were exposed to either [2-¹⁴C]acetate, [16-¹⁴C]palmitate, or [4-¹⁴C]cholesterol for periods up to 24 h. Isotope incorporation into triglyceride (TG), wax esters (WE), and polar lipid (PL) was recorded. Compared to warm-acclimatized groups, incorporation of [2-¹⁴C]acetate into WE of cold anemones was dramatically reduced, while TG incorporation remained at about the same levels. Highest values were recorded for PL in both groups. Using radiolabeled palmitate, incorporation values for WE were very low in both acclimatization groups though TG uptake remained comparatively high. Also noteworthy was a significant decrease in PL activity in cold anemones. Fatty acid analysis of total lipid, wax ester, triglyceride and phospholipid fractions showed a general shift towards increased chain length and unsaturation in cold-acclimatized anemones.     

Selective activation of cyclic AMP dependent protein kinase by calcitonin in a calcitonin secreting lung cancer cell line

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.3 and then gel-filtered, 1.25 mols of [14C]Nbf-O-Tyr and less than 0.1 mol of Nbf-N-Lys were formed per mol of enzyme. After adjusting the pH of the gel-filtered, modified enzyme to 9.0 and incubating it for 14 hrs. at 23 degrees C to promote O----N migration, 0.68 mol of Nbf-N-Lys were formed per mol of enzyme while about 16% of the original activity reappeared. Isolation of the subunits after the O----N migration showed that 90% of the incorporated 14C was present in the beta subunit, which contained 0.21 mols of [14C]Nbf-N-Lys per mol. A tryptic peptide which contained the majority of the 14C incorporated into the beta subunit was isolated and subjected to automatic amino acid sequence analysis contained 38 residues. The amino acid sequence immediately around the lysine residue labeled with [14C]Nbf-, K*, was found to be: ...I-G-L-F-G-G-A-G-V-G-K*-T-V-L-I-G... .     

Evidence that macrophages transfer arachidonic acid and cholesterol to tissues in vivo

Our previous studies have shown that [(14)C]-labelled cholesterol (CHOL) and arachidonic acid (AA) are transferred from macrophages (Mphi) to lymphocytes (LY) when these cells are co-cultured. In this study, we investigated whether these lipids can be transferred from control and thioglycollate-elicited Mphi (THIO-elicited Mphi) to various tissues and organs in vivo. For this purpose, control and THIO-elicited Mphi were pre-treated with [(14)C]-AA and [(3)H]-CHOL and then injected into the jugular vein of adult rats. More than 75% of the radioactivity injected was found in the liver of rats treated with [(14)C]-AA labelled-Mphi either control and THIO-stimulated. The radioactivity of [(3)H]-CHOL labelled Mphi was transferred mainly to the liver (51% in the control Mphi and 23% in the thioglycollate Mphi7) but it was also found in the kidney, lung and spleen. These results support the proposition that the transfer of lipids between cells also occurs in vivo. The full significance of this phenomenon however remains to be elucidated.     

Adaptational Changes in Lipids of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> SEMIA 6144 Nodulating Peanut as a Response to Growth Temperature and Salinity

Phospholipids provide the membrane with its barrier function and play a role in a variety of processes in the bacterial cell, as responding to environmental changes. The aim of the present study was to characterize the physiological and metabolic response of Bradyrhizobium SEMIA 6144 to saline and temperature stress. This study provides metabolic and compositional evidence that nodulating peanut Bradyrhizobium SEMIA 6144 is able to synthesize fatty acids, to incorporate them into its phospholipids (PL), and then modify them in response to stress conditions such as temperature and salinity. The fatty acids were formed from [1-¹⁴C]acetate and mostly incorporated in PL (95%). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) were found to be the major phospholipids in the bacteria analyzed. The amount and the labeling of each individual PL was increased by NaCl, while they were decreased by temperature stress. The amount of PC, PE, and PG under the combined stresses decreased, as in the temperature effect. The results indicate that synthesized PL of Bradyrhizobium SEMIA 6144 are modified under the tested conditions. Because in all conditions tested the PC amount was always modified and PC was the major PL, we suggest that this PL may be involved in the bacteria response to environmental conditions.