The rat retina is a useful in vivo model to study membrane lipid synthesis: Rates of biosynthesis of neutral glycerides and phospholipids

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segmentssynaptic bodies (P₁) and synaptosomal-mitochondrial (P₂) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-³H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-³H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-³H]glycerol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.     

Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-(3)H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-(14)C]methionine and CDP-[Me-(14)C]choline appeared to be localized in the microsomal fraction.     

Fatty acid and glycerol labeling of glycerolipids of leukocytes in response to ionophore A23187.

The effect of divalent cation ionophore, A23187, on the incorporation of [1-14C]palmitic acid, [1-14C]linoleic acid and [U-14C]glycerol into glycerolipids of polymorphonulcear leukocytes was examined. Ionophore A23187 stimulated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. [1-14C]Palmitic acid and [1-14C]linoleic acid incorporation into phosphatidylcholine and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [U-14C]glycerol labeling of these lipids was not significantly changed under identical conditions. These data reflect that the acylation of sn-glycerol 3-phosphate is activated, and the acylations of lysophosphatidyl-choline and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23187. External calcium was not required for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the metabolism of the fatty acid and glycerol moieties of glycerolipids by changing the distribution of intracellular calcium of leukocytes.     

A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro

During long-term dietary n-3 fatty acid supplementation, eicosapentaenoic acid (EPA) is not incorporated into phosphatidylinositol or -serine of human platelets in vivo and is not detectable in phosphatidic acid upon stimulation with thrombin. However, EPA is released from platelet phospholipids and metabolized to thromboxane B3 (TXB3). In contrast, in vitro, platelets incorporate [14C]EPA into phosphatidylinositol, whether they contain endogenous EPA in their cellular lipids or not. Following platelet stimulation, [14C]EPA appears in phosphatidic acid, as free fatty acid, and is transformed to TXB3. We conclude that the fatty acid compositions of platelet phospholipid subclasses are regulated with a high degree of specificity in vivo. Qualitative differences exist between in vivo and in vitro uptake of EPA into platelet phospholipid subclasses. After in vivo incorporation, EPA is released by action of a phospholipase A2.     

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.     

Metabolic regulation during early frog development: flow of glycolytic carbon into phospholipids in Xenopus oocytes and fertilized eggs

32P-labeled glucose 6-phosphate, [32P]phosphoenolpyruvate, and [gamma-32P]ATP were injected into oocytes and fertilized eggs of Xenopus laevis, and the incorporation of the 32P label was followed into phospholipids. Several classes of phospholipids incorporated 32P label from the injected glycolytic intermediates, including lysophosphatidic acid, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol phosphates, inferring de novo synthesis of these lipids from dihydroxyacetone phosphate or glycerol 3-phosphate. Injection of [gamma-32P]ATP into oocytes and fertilized eggs led to labeling of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, indicating an active phosphatidylinositol cycle in resting oocytes and fertilized eggs. Maturation and fertilization of the oocyte led to a qualitative change in phosphatidylinositol metabolism, increased labeling of phosphatidylinositol phosphate compared to phosphatidylinositol bisphosphate (either from glycerol 3-phosphate or from ATP). This change occurs late in the maturation process, and the new pattern of phosphatidylinositol metabolism is maintained during the rapid cleavage stages of early embryogenesis.     

Accumulation of phosphatidic acid in microsomes from propranolol-treated retinas during short-term incubations

Abstract: The pool size and synthesis of phosphatidic acid derived from [2-³H]glycerol were studied in bovine whole retinas and subcellular fractions. Microsomal preparations from retinas incubated with [2-³H]glycerol displayed the highest percentage labeling of phosphatidic acid at 5 min of incubation; labeling decreased rapidly thereafter. In drug-treated retinas,0.5 m M propranolol increased the endogenous content of phosphatidic acid and stimulated [2-³H]glycerol labeling in whole retina and microsomal and postmicrosomal supernatant fractions. This effect was observed during short-term incubations and was reversible. In pulse-chase experiments, 60 min of reincubation greatly reduced the labeling effect, although propranolol still enhanced phosphatidic acid labeling. At the same time, endogenous phosphatidic acid accumulated and reincubation without propranolol reversed the effect. During accumulation, the amount of palmitate increased and that of oleate decreased, whereas the relatively high level of docosahexaenoate in phosphatidic acid remained unchanged. It was concluded that this propranolol-induced effect is due to cationic amphiphilic drug activity in the endoplasmic reticulum that results in a partial inhibition of phosphatidic acid degradation and a stimulation of its de novo synthesis. Hence, net synthesis of phosphatidic acid can be assessed in the retina during short-term incubation with propranolol.     

Enhanced turnover of arachidonic acid-containing species of phosphatidylinositol and phosphatidic acid of concanavalin A-stimulated lymphocytes

The incorporation of [32P]orthophosphate into phosphatidylinositol (PI) of pig lymphocytes was markedly increased by stimulation with concanavalin A. The labeling of PI with [3H]glycerol was also enhanced significantly, indicating that both de novo synthesis and recircular system (PI response) of PI were accelerated. This rapid labeling of PI might be related to the rapid breakdown of phosphatidylinositol 4,5-bisphosphate which was observed in various stimulated tissues. Concanavalin A also accelerated the labeling of phosphatidic acid with 32P and [3H]glycerol. To determine the dependence of this phenomenon on the fatty acid composition of both phospholipids, we separated PI and phosphatidic acid into individual molecular species. The predominant molecular species in PI was tetraene (81.6%) and those in phosphatidic acid were monoene (53.0%), diene (15.8%) and tetraene (19.2%), respectively. Interestingly, the incorporation of 32P into arachidonic acid-containing species (tetraene) was most rapidly elevated. On the other hand, the increment of 32P into saturated + monoene, diene and triene was relatively smaller and resembled that of [3H]glycerol. Similarly, the incorporation of 32P into tetraene of phosphatidic acid was preferentially accelerated. This is the first report concerning the metabolism of molecular species of phosphatidic acid in stimulated cells. These results indicate that the PI recirculating system is virtually dependent on tetraenoic species and that the participation of other molecular species is small. The increased de novo synthesis mainly depends upon molecular species other than tetraene. Arachidonic acid-containing species which turn over rapidly via the PI cycle may have an important role in the mitogenic triggering.     

The influence of myo-inositol on phosphatidylglycerol synthesis by rat type II pneumonocytes.

Type II pneumonocytes isolated from adult rat lung were incubated in a serum-free medium containing [14C]glycerol and the incorporation of 14C into glycerophospholipids was measured. After 24 h, more than 80% of the 14C incorporated into total lipids or into phosphatidylcholine and approx. 90% of the 14C incorporated into phosphatidylglycerol after 24 h was recovered in the glycerophosphoester moieties of these molecules. Supplementation of the incubation medium with foetal-bovine serum (10%, v/v) did not alter the incorporation of [14C]glycerol by type II pneumonocytes after 24 h into either a total lipid extract or phosphatidylcholine. In the presence of foetal-bovine serum, however, the incorporation of 14C into phosphatidylglycerol was decreased and the incorporation of 14C into phosphatidylinositol was increased. In the absence of foetal-bovine serum, the incorporation of 14C into phosphatidylglycerol was decreased progressively as the concentration of myo-inositol in the incubation medium was increased. The range of concentration (0.04-0.50 mM) over which myo-inositol had the greatest influence on [14C]glycerol incorporation into phosphatidylglycerol by type II pneumonocytes in vitro encompassed the concentration range measured in foetal-rat serum late in gestation. At 4 days before birth, the concentration of myo-inositol in foetal-rat serum was 0.36 mM and decreased to 0.23 mM 1 day before birth. The concentration of myo-inositol in adult rat serum increased from 0.03 mM to 0.06 mM during pregnancy. Isolated rat type II pneumonocytes were found to take up myo-inositol by a saturable process. A half-maximal rate of myo-inositol uptake occurred at a concentration of myo-inositol of 0.29 mM. The results of this investigation are consistent with the hypothesis that late in gestation there is a decreasing availability of myo-inositol to the foetal lungs and that this favours the biosynthesis of phosphatidylglycerol for surfactant at the expense of phosphatidylinositol biosynthesis.     

Insulin and oxytocin effects on phosphoinositide metabolism in adipocytes

The effects of hormones on phosphoinositide metabolism were examined in rat adipocytes prelabeled with 32Pi or [3H]inositol. Oxytocin and vasopressin produced large decreases in labeled polyphosphoinositides and increases in phosphatidic acid and inositol phosphates, whereas insulin was without effect, although it stimulated lipogenesis from glucose. Likewise, insulin did not elevate 1,2-diacylglycerol measured chemically by high pressure liquid or thin-layer chromatography in fat cells or pads. It also did not increase the radioactivity in 1,2-diacylglycerol in ghosts prepared from fat cells previously labeled with [3H]arachidonic acid, although oxytocin and vasopressin increased this. It is therefore concluded that insulin does not stimulate the breakdown of polyphosphoinositides to yield 1,2-diacylglycerol and inositol phosphates in adipocytes and that the insulin-like actions of oxytocin must be due to other changes. Insulin induced small, but significant and equal increases (40% at 30 min) in the incorporation of [3H] inositol into phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in adipocytes. The effects were not dependent upon glucose and were not evident before 15 min. Oxytocin also produced large increases in the labeling of the three phosphoinositides. Insulin stimulated the incorporation of [3H]glycerol into the three phosphoinositides and also phosphatidic acid, phosphatidylserine, and phosphatidylethanolamine by 50-100% in cells incubated without glucose. No changes in the labeling of glycerol 3-phosphate, lysophosphatidic acid, phosphatidylcholine, and triacylglycerol were detected, and there was a small increase (30%) in 1,2-diacylglycerol labeling. It is concluded that insulin increases the synthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylethanolamine, and phosphatidylserine in fat cells partly by stimulating a reaction(s) located between glycerol 3-phosphate and phosphatidic acid in the biosynthetic pathway.     

Molecular species of phosphatidylinositol, phosphatidic acid and diacylglycerol in a phytohemagglutinin-stimulated T-cell leukemia line

Addition of phytohemagglutinin to JURKAT cells, a human T-cell leukemia line, induced a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (and may also be phosphatidylinositol 4-phosphate) and an accumulation of phosphatidic acid. The accumulation and disappearance of the various molecular species of phosphatidic acid, diacylglycerol and phosphatidylinositol (PtdIns) in response to phytohemagglutinin was studied in JURKAT cells. The cells were prelabeled with [2-3H]glycerol for 2 days and 3H-labeled lipids were isolated from the cells after incubation for 2 min at 37 degrees C in the absence or in the presence of phytohemagglutinin. The isolated 3H-labeled lipids were separated into individual molecular species by reverse-phase HPLC after conversion to their 1,2-[3H]diacylglycerol acetate derivatives either by acetolysis or by acetylation. Stimulation with phytohemagglutinin induced a 2-fold increase in [3H]phosphatidic acid. The molecular species of the accumulated [3H]phosphatidic acid consisted of polyenoic species, which were almost absent in the [3H]phosphatidic acid of the unstimulated cells. Stearoylarachidonoyl species of [3H]phosphatidic acid accumulated most prominently. Although an accumulation of [3H]diacylglycerol was hardly measurable in the phytohemagglutinin-stimulated cells, the HPLC analysis of the molecular species of [3H]diacylglycerol showed a 2-fold increase in the stearoylarachidonoyl species in the stimulated cells. Stimulation with phytohemagglutinin had almost no effect on the composition of molecular species of [3H]PtdIns. The stearoylarachidonyl species is the most abundant molecular species of PtdIns in JURKAT cells. These results suggest that the [3H]diacylglycerol moiety of [3']phosphatidic acid originates from inositol lipid(s). The results also suggest a rapid and preferential phosphorylation of the diacylglycerol formed by receptor-stimulated hydrolysis of inositol lipid(s).     

Composition and labeling by [3H]glycerol and [3H]serine of phospholipids of the chicken retina and optic tectum

The content and fatty acid composition of phospholipids and the in vivo labeling of lipids by [3H]glycerol and [3H]serine was studied in the retina and the optic tectum of young chickens. The tectum had a higher content of phospholipids and a significantly lower ratio of choline (CGP) to ethanolamine (EGP) glycerophospholipids than the retina. Lipids of the chicken optic system were characterized by a high proportion of polyenoic fatty acids of the n-6 series compared to other species. Intravitreally injected [3H]glycerol was incorporated into all glycerol-containing lipids of the retina, especially in CGP and EGP. Most of the label from [3H]serine was found in serine glycerophospholipids (SGP). The time-dependent distribution of both precursors among retinal lipids was consistent with de novo synthesis as well as metabolic interconversions of lipids. Thus, [3H] from serine also appeared in EGP and CGP, indicating the presence and activity of SGP decarboxylase and EGP-n-methyl transferase. Lipids labeled with both precursors in retina were subsequently found in the tectum, via axoplasmic transport. Even though different lipid classes were labelled by each precursor the proportion of lipids transported to the tectum was similar in both cases (about 1% of the label present in retina).     

Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization

Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [¹⁴C]acetate or [¹⁴C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.

The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP-diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [¹⁴C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid → CDP-diacylglycerol →→ phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane.

     

Phytohemagglutinin induces rapid degradation of phosphatidylinositol 4,5-bisphosphate and transient accumulation of phosphatidic acid and diacylglycerol in a human T lymphoblastoid cell line, CCRF-CEM

The human T lymphoblastoid cell line designated CCRF-CEM responds to phytohemagglutinin with a 3.7-fold enhancement of the 32PO4 incorporation into phosphatidylinositol. In myo-[2-3H]inositol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a 3.3-fold accumulation of myo-[2-3H]inositol phosphate during 15 min incubation at 37 degrees C in the presence of 5 mM LiCl. Since Li+ is a potent inhibitor of myo-inositol-1-phosphatase, the results indicate that phytohemagglutinin induces the hydrolysis of inositol lipids in CCRF-CEM cells. In 32PO4-prelabeled CCRF-CEM cells, phytohemagglutinin induced a breakdown of 28% of [32P]phosphatidylinositol 4,5-bisphosphate 40-60 s after the stimulation. The decrease of [32P]phosphatidylinositol 4,5-bisphosphate was found as early as 10 s after the stimulation. This decrease was followed by an increased 32P-labeling of phosphatidic acid. In [2-3H]glycerol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [3H]phosphatidic acid and [3H]diacylglycerol. The amount of [3H]phosphatidic acid in the stimulated cells was 3.7-times the control value at 2 min after the stimulation, whereas the amount of [3H]diacylglycerol in the stimulated cells was 1.5-times the control value at 5 min after the stimulation. In [3H8]arachidonate-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [3H]phosphatidic acid; the amount was 2.5-times the control value at 2 min after the stimulation. Quinacrine (1 mM) caused 41% reduction in the amount of [3H]phosphatidic acid accumulated by the stimulation in [2-3H]glycerol-prelabeled cells. Stimulation in a Ca2+-free saline containing 1 mM EGTA caused 53% reduction in the amount of [3H]phosphatidic acid accumulated by the stimulation. The results presented in this paper indicate that a human T lymphoblastoid cell line, CCRF-CEM, responds to phytohemagglutinin with a rapid turnover of inositol lipids.     

Rhythms of glycerophospholipid synthesis in retinal inner nuclear layer cells

The present study demonstrates that the biosynthesis of phospholipids in the inner nuclear layer cells of the chicken retina displays daily rhythms under constant illumination conditions. The vertebrate retina contains circadian oscillators and photoreceptors (PRCs) that temporally regulate its own physiology and synchronize the whole organism to the daily environmental changes. We have previously reported that chicken photoreceptors and retinal ganglion cells (RGCs) present significant daily variations in their phospholipid biosynthesis under constant illumination conditions. Herein, we demonstrate that cell preparations highly enriched in inner nuclear layer cells also exhibit a circadian-regulated phospholipid labeling after the in vivo administration of [(32)P]phosphate or [(3)H]glycerol both in animals maintained under constant darkness or light for at least 48h. In constant darkness, there was a significant incorporation of both precursors into phospholipids with the highest levels of labeling around midday and dusk. In constant light, the labeling of (32)P-phospholipids was also significantly higher during the day and early night whereas the incorporation of [(3)H]glycerol into phospholipids, that indicates de novo biosynthesis, was greater during the day but probably reflecting a higher precursor availability at those phases. We also measured the in vitro activity of phosphatidate phosphohydrolase and diacylglycerol lipase in preparations obtained from the dark condition. The two enzymes exhibited the highest activity levels late in the day. When we assessed the in vitro incorporation of [(14)C]oleate into different lysophospholipids from samples collected at different phases in constant darkness, reaction catalyzed by lysophospholipid acyltransferases II, labeling showed a complex pattern of daily activity. Taken together, these results demonstrate that the biosynthesis of phospholipids in cells of the chicken retinal inner nuclear layer exhibits a daily rhythmicity under constant illumination conditions, which is controlled by a circadian clock.     

Lipid Metabolism in Electroplax

The in vivo labeling of electrocyte lipids is followed after injection of radioactive glycerol and two fatty acids, oleate and arachidonate, into the electric organ of an elasmobranch (Discopyge tschudii). De novo synthesis of lipids and acyl-exchange reactions are operative in the electrocyte. The three precursors are preferentially incorporated into phosphatidylcholine, phosphatidylinositol, and triacylglycerols. The highest specific activities are attained by triacylglycerols and polyphosphoinositides. Electrocyte stacks from electric organ show an efficient and continuous esterification of oleate and arachidonate into lipids after several hours of incubation. Except for an apparently more active labeling of triacylglycerols, which is attributed to the larger availability of free fatty acid precursors under the in vitro experimental conditions, the pattern of lipid labeling is similar to that attained in vivo. 32P-labeled lipids are also steadily produced in electrocyte stacks (24 h of incubation with [32P]phosphate) using glucose as the sole exogenous source of energy. Polyphosphoinositides are the lipids preferentially labeled. The ability to sustain the labeling of lipids under in vitro conditions renders isolated electrocyte stacks an interesting model for future research on lipid involvement in cholinergic function.     

SELECTIVE SYNTHESIS OF MOLECULAR CLASSES OF PHOSPHATIDIC ACID, DIACYLGLYCEROL AND PHOSPHATIDYLINOSITOL IN RAT BRAIN

—Phosphatidic acids of rat brain were shown to be predominantly of the monoenoic class while diacylglycerols and phosphatidylinositols were constituted mainly by tetraenes. Metabolic inter-relationships were examined after intraventricular injection of [¹⁴C]glycerol, [³H]arachidonate and [9,10-³H₂]stearate. In each case, diacylglycerols were most highly labelled, although a small pool of arachidonate was located in brain triacylglycerols, mainly esterified to a primary carbinol, with extremely high turnover rate. Fractionation of the lipids showed a preferential synthesis of disaturated, monoenoic and polyenoic classes (>4 double bonds) of phosphatidic acid, diacylglycerol and phosphatidylinositol. The high flux of [³H]stearate through disaturated species of phosphatidic acid and diacylglycerol could be partially suppressed by simultaneous injections of unsaturated fatty acids, both probably consequences of perturbing the very small brain pool of free fatty acids. Kinetics of labelling of phosphatidylinositols were consistent with formation of arachidonoyl-containing species by acyl transfer mechanisms with disaturated and oligoenoic classes serving as precursors. Although the profile of molecular classes of diacylglycerol and phosphatidylinositol strongly suggests a metabolic relation, there was no obvious evidence for this in the kinetic studies of the whole brain lipids. Such relation, however, may have been masked by the rapid flow of radioactivity from phosphatidic acids to diacylglycerols.     

Phosphatidic Acid and Phosphoinositide Turnover in Myelin and Its Stimulation by Acetylcholine

Brain slices obtained from the forebrains of adult female rats were incubated with [32P]phosphate and [3H]glycerol for 60 min, and lipids extracted and analyzed by TLC. The 32P in brain slice lipids was primarily in polyphosphoinositides, phosphatidylinositol (PI), and phosphatidate (PA). Distribution of the 32P-labeled lipids in isolated myelin was biased toward PA, 38%, relative to 16% in whole tissue slice lipids. About 33% of the total labeled PA in brain slices was accounted for by that in myelin. On a per milligram protein basis, PA labeling in myelin is about 2.5-fold greater than that of whole brain slice. Since incorporation of [3H]glycerol (indicative of synthesis by the de novo synthetic pathway) was at very low levels, we conclude that [32P]phosphate entered into myelin PA primarily through a pathway involving phospholipase C activity. Much of the production of PA relates to hydrolysis of phosphoinositides, yielding diacylglycerol which is then phosphorylated within myelin. The distribution of label among the inositol-containing lipids suggests that only a fraction of the myelin polyphosphoinositides serve as substrate for rapid diglyceride production. In the presence of 10 mM acetylcholine (ACh) there was a 20-60% stimulation of [32P]phosphate incorporation into PA and PI of brain slice lipids and purified myelin. Stimulation by ACh was blocked by atropine. The observed increase in the 32P/3H ratio, relative to controls, indicated that for both total lipids and myelin lipids there was selective stimulation of a phospholipase C-dependent cycle relative to de novo biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like effects of epidermal growth factor and insulin-like growth factor-I on [3H]2-deoxyglucose uptake, diacylglycerol generation and protein kinase C activation in BC3H-1 myocytes.

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) were found to provoke increases in [3H]2-deoxyglucose uptake, diacylglycerol (DAG) generation and membrane-bound protein kinase C activity in BC3H-1 myocytes. These effects were similar to those provoked by insulin. The increases in DAG did not appear to be derived from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol, but may have been derived from synthesis of phosphatidic acid de novo, and hydrolysis of phosphatidylcholine, as revealed by studies with [3H]glycerol and [3H]choline respectively. Accordingly, both EGF and IGF-I increased acute [3H]glycerol labelling of DAG (and other lipids) and [3H]choline labelling of phosphocholine. These labelling responses were similar in time course, suggesting that they are closely coupled. Our findings suggest that EGF and IGF-I, like insulin, increase DAG-protein kinase C signalling, apparently by activating co-ordinated lipid-synthesis and -hydrolysis responses, which are distinctly different from the PIP2-hydrolysis response.     

Reversibility of Propranolol-Induced Changes in the Biosynthesis of Monoacylglycerol, Diacylglycerol, Triacylglycerol, and Phospholipids in the Retina

Abstract: The biosynthesis and metabolism of phospholipids and neutral glycerides were studied in the bovine retina. Radioactive glycerol was used as a precursor. Phentolamine and d - and dl -propranolol were found to produce similar effects on lipid metabolism in the retina. Marked stimulation of phosphatidylinositol (PhI) synthesis and maximal inhibition of phosphatidylcholine (PhC), diacylglycerol (DG), and triacylglycerol (TG) formation were observed within 5 min after exposure to 0.5 m M dl -propranolol. Pulse-chase experiments showed a high turnover rate in DG and a reversibility of the propranolol-induced changes produced during the synthesis of PhC, TG, DG, monoacylglycerol (MG), and phosphatidylserine. All reversals of the drug-induced biosynthetic profiles approached control values 60 min after incubation in drug-free medium. However, complete reversal was not achieved in any of the cases under these conditions. Propranolol appeared to inhibit both the formation of DG from phosphatidic acid and the further metabolism of DG, probably to MG. Phosphatidylethanolamine biosynthesis showed some recovery from this inhibition. Synthesis of Phi was greatly stimulated by preincubation with propranolol and was further enhanced by reincubation in the presence of propranolol. However, this effect was not reversed by reincubation without the drug. The active de novo biosynthesis of retinal phospholipids and glycerides is a very dynamic pathway that may be redirected by amphiphilic drugs. In addition, the partial reversal of modifications induced in the flux of [2-³H]glycerol through the lipids can occur during short-term reincubations of retinas in drug-free medium.