Lysophosphatidylserine enhances the transfer of 22:6n3 to lysophosphatidic acid in rat brain microsomes.

Although the acyl groups of phosphatidylserine in brain are uniquely enriched in docosahexaenoic acid (22:6n3), the mechanism for this enrichment is not well understood. When rat brain homogenates and microsomes were incubated in the presence of lysophosphatidylserine (LPS) together with [14C]22:6n3 and cofactors for activation to its acylCoA, very little radioactivity was incorporated into phosphatidylserine (PS). On the other hand, [14C]20:4n6 was more actively incorporated into PS. Addition of LPS (1-10 uM), however, resulted in a 2-5 fold enhancement of the transfer of labeled 22:6n3 and 20:4n6 to phosphatidic acid (PA). Kinetic analysis indicated the ability of LPS to lower the Km and increase the Vmax of the lysophosphatidic acid (LPA) acyltransferase reaction. Among other lysophospholipids tested, lysophosphatidylserine was most effective in enhancing PA biosynthesis. Since PA is an important intermediate for de novo biosynthesis of phospholipids, these results reveal a novel mechanism for promoting synthesis of PA enriched in polyunsaturated fatty acids in brain.     

Glucagon and epinephrine-stimulated phospholipid methylation in hepatic microsomes

Phospholipid methylation by hepatic microsomes was measured following glucagon or epinephrine administration either to intact rats or to the isolated perfused liver. Both hormones stimulated the methylation measured as the incorporation of S-adenosyl-L-[methyl-³H]methionine into phospholipids. The labeled products were identified by thin layer chromatography and most of the counts were found to be incorporated into phosphatidylcholine. The stimulatory effects of the hormones were evident already 5 minutes following hormone administration both in $\text{in vivo}$ and in $\text{in vitro}$. The observed stimulation of the methylation process by glucagon and epinephrine might be related to the previously reported stimulatory effect of these hormones on the microsomal Ca²⁺-ATPase, and indicate that methylation process(es) might mediate some of the effects of these hormones.     

The biosynthesis of phosphatidylserines by acylation of 1-acyl-sn-glycero-3-phosphoserine in rat liver

The conversion of 1-[14C]acyl-sn-glycero-3-phosphoserine into molecular species of [14C]phosphatidylserine was studied using rat liver homogenate and microsomal preparations in the absence of added fatty acyl moieties. In liver homogenates, 81% of the newly-formed phosphatidylserines were tetraenoic (arachidonoyl) species while saturated, monoenoic, dienoic, trienoic, pentaenoic, and hexaenoic (docosahexaenoyl) species each represented 2-5% of the total. A similar pattern of molecular species was produced in liver microsomes. The selectivity of the microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase towards different acyl-CoA derivatives was also investigated. The relative suitability of the various acyl-CoA esters as substrates was found to be of the following order:20:4 = 18:2 greater than 18:1 greater than 16:0 = 18:0. These results with endogenous acyl donors suggest that the acylation of 1-acyl-sn-glycero-3-phosphoserine may partly account for the enrichment of liver phosphatidylserine in arachidonic acid but does not appear to be primarily responsible for the preponderance of docosahexaenoic acid in this phospholipid. The fatty acid specificity of the acyl-CoA: 1-acyl-sn-glycero-3-phosphoserine acyltransferase may contribute to the preferential formation of arachidonoyl phosphatidylserine.     

Effects of norepinephrine and acetylcholine on 32P incorporation into phospholipids of the rabbit iris muscle following unilateral superior cervical ganglionectomy.

The effect of norepinephrine and acetylcholine on the ³²P incorporation into phospholipids of normal and sympathetically denervated rabbit iris muscle was investigated. (1) In the absence of exogenously added neurotransmitters sympathetic denervation exerted little effect on the incorporation of ³²P into the phospholipids of the excised iris muscle. $\text{In vivo}$ thr iris muscle incorporated ³²P into phosphatidylinositol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin in that order of activity while $\text{in vitro}$ phosphatidylinositol was followed by phosphatidylcholine. (2) Tension responses of iris dilator muscle from denervated irises exhibited supersensitivity to norepinephrine. Furthermore, norepinephrine at concentrations of 3 μM and 30 μM produced 1.6 times and 3 times stimulation of the phosphatidic acid of the denervated muscle respectively. In contrast at 30 μM it stimulated this phospholipid by 1.6 times in the normal muscle. This stimulation was completely blocked by phentolamine. (3) While in the normal muscle acetylcholine stimulated the labelling of phosphatidic acid and phosphatidylinositol by more than 2 times, in the denervated muscle it only stimulated 1.4 to 1.7 times. (4) Similarly when ³²Pi was administered intracamerally, the labelling found in the various phospholipids of the denervated iris was significantly lower than that of the normal. (5) It was concluded that denervation decreases the ³²P labelling in the presence of acetylcholine. (6) The norepinephrine-stimulated ³²P incorporation into phosphatidic acid appears to be post-synaptic.     

Interaction of bovine brain phospholipid exchange protein with liposomes of different lipid composition

The major phospholipid exchange protein from bovine brain catalyzes the transfer of phosphatidylinositol and phosphatidylcholine between rat liver microsomes and sonicated liposomes. The effect of liposomal lipid composition on the transfer of these phospholipids has been investigated. Standard liposomes contained phosphatidylcholine-phosphatidic acid (98:2, mol%); in general, phosphatidylcholine was substituted by various positively charged, negatively charged, or zwitterionic lipids. The transfer of phosphatidylinositol was essentially unaffected by the incorporation into liposomes of phosphatidic acid, phosphatidylserine, or phosphatidylglycerol (5–20 mol%) but strongly depressed by the incorporation of stearylamine (10–40 mol%). Marked stimulation (2–4-fold) of transfer activity was observed into liposomes containing phosphatidylethanolamine (2–40 mol%). The inclusion of sphingomyelin in the acceptor liposomes gave mixed results: stimulation at low levels (2–10 mol%) and inhibition at higher levels (up to 40 mol%). Cholesterol slightly diminished transfer activity at a liposome cholesterol/phospholipid molar ratio of 0.81. Similar effects were noted for the transfer to phosphatidylcholine from microsomes to these various liposomes. Compared to standard liposomes, the magnitude of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ tended to increase for liposomes which depressed phospholipid transfer and to decrease for those which stimulated; little change was observed in the values of $\text{V}$. Single phospholipid liposomes of phosphatidylinositol were inhibitory when added to standard liposomes.     

Incorporation of 18 O into homovanillic acid of rat brain during exposure to oxygen 18 containing atmospheres

Rats were exposed to air containing ¹⁸O₂ at atmospheric pressure. $\text{In vivo}$ incorporation of ¹⁸O in brain homovanillic acid (HVA) was determined by gas chromatography-mass spectrometry. One ¹⁸O atom was incorporated into each molecule of HVA indicating that tyrosine is the predominant precursor of brain dopamine and that the oxygen in the 3-position is of atmospheric origin. Intraperitoneal administration of ¹⁸O-enriched water did not alter the ¹⁸O content of brain HVA Mass fragmentography (2) was used to measure the increase in ¹⁸O and the decrease in ¹⁶O in HVA from rat brain over several hours of exposure to an ¹⁸O enriched atmosphere. These experiments demonstrate the possibility to pulse label brain dopamine and its metabolites by $\text{in vivo}$ inhalation of stable oxygen isotopes. The procedure should be useful for quantitative determinations of the turnover of brain dopamine in animals and man.     

Acyltransferases and the biosynthesis of pulmonary surfactant lipid in adenoma alveolar type II cells.

Acyltransferases are present in microsomes from alveolar type II cell adenomas (produced by urethan injections) that transfer palmitic acid in the presence of CoA, ATP, and Mg⁺⁺ to $\text{sn}$-glycerol-3-P to form phosphatidic acid, to dihydroxyacetone-P to form acyldihydroxyacetone-P, and to 1-acyl-$\text{sn}$-glycero-3-phosphocholine to form 3-$\text{sn}$-phosphatidylcholine. The data clearly demonstrate that the microsomal preparations can catalyze significant incorporation of palmitic acid into the 2-position of the disaturated species of 3-sn-phosphatidylcholine independently of phosphatidic acid formation as evidenced by the fact that $\text{sn}$-glycerol-3-P and calcium ions (which inhibit choline phosphotransferase) did not influence the incorporation of palmitic acid into the main surfactant lipid. Thus, a deacylation-acylation reaction involving 2-lysophosphatidylcholine appears to be an important pathway for the synthesis of surfactant lipid in alveolar type II cells; the control of acyl specificity at the 2-position is determined by the relative concentrations of the coparticipating substrates, l-palmitoyl-$\text{sn}$-glycero-3-phosphocholine and palmitoyl-CoA.     

Incorporation of linoleic acid into membrane glycerophospholipids from rat brain submitted to ischemia and hypoxia

In the presence of ATP, Mg, and CoA-SH[1-¹⁴C]linoleic acid was incorporated into membrane phospholipids (P₂ fraction and synaptosomes) prepared from rat brain cortex. The relative order for linoleate incorporation was: phosphatidylcholine >phosphatidylethanolamine>phosphatidylinositol>ethanolamine plasmalogen >phosphatidylserine. The incorporation of labeled linoleate into P₂ fraction phospholipids was investigated in rats, aged 4, 16, and 90 days, after being subjected to ischemic and hypoxic conditions. With the exception of a small increase in the incorporation of the radioactivity into diacyl-GPC, little change in incorporation profile was observed with 4-day-old rats submitted to ischemic and hypoxic conditions. However, the incorporation of labeled linoleate into membrane phospholipids was decreased in 16-and 90-day-old rats after being subjected to ischemic and hypoxic conditions. Among the phospholipids, the decrease in incorporation of radioactivity was most prominent with ethanolamine plasmalogens and phosphatidylinositol although the radioactivity of phosphatidylcholine seemed to remain relatively constant. The decreased incorporation activity in these two age groups was noted along with concomitant increase in the FFA content, whereas an increase in FFA was not observed in the 4-day-old brain samples. Thus, the specific decrease in labeling of ethanolamine plasmalogens and phosphatidylinositol may be the result of increased enzymic degradation of these compounds after ischemic and hypoxic treatment. Furthermore, the decrease in incorporation of linoleate into membrane phospholipids may be due to an increase in the membrane, FFA pool which subsequently, gave a dilution of the labeled precursor.     

Transfer of phospholipids between the endoplasmic reticulum and mitochondria in rat hepatocytes in vivo

The transfer of phospholipids from the endoplasmic reticulum to the inner mitochondrial membrane was investigated by pulse labeling $\text{in}\text{vivo}$. With [³H]glycerol microsomal phosphatidylethanolamine and phosphatidylcholine were rapidly labeled during the first 30 min; while maximum incorporation into the inner mitochondrial membrane occurred only after about 5 hours. It appears that the $\text{in}\text{vivo}$ transfer of these phospholipids between the two membrane compartments is a relatively slow process.     

The synthesis of proteolipid protein by isolated rat liver mitochondria

About 15% of the total (³H)leucine incorporated into protein by isolated rat liver mitochondria $\text{in}\text{vitro}$ could be extracted by chloroform:methanol. This incorporation was inhibited by chloramphenicol and carbomycin, both specific inhibitors of mitochondrial protein synthesis. SDS-gel electrophoresis of the mitochondrial membrane revealed 6–7 labeled bands. Label in the proteolipid fraction was present mainly in a band of 40,000 molecular weight. Several labeled bands observed in gels of the mitochondrial membrane were not removed or changed by extraction with chloroform:methanol suggesting that some, but not all, of the proteins synthesized by rat liver mitochondria are proteolipids.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

Effects of γ-aminobutyric acid on 33Pi incorporation into rat brain phospholipids in vivo

The effects of γ-aminobutyric acid (GABA), bicuculline and strychnine on the incorporation $\text{in vivo}$ of ³³Pi into phospholipids of rat brain were studied at 10 and 30 minutes after intracisternal injection of the radionuclide. GABA inhibited labeling of phospholipids in the three brain regions studied at both times. Bicuculline by itself had no significant effect on ³³Pi incorporation, but totally blocked the inhibitory effect of GABA in all three brain regions. Strychnine by itself inhibited phospholipid labeling in the brain stem and forebrain, had no significant effect on GABA inhibition of ³³Pi incorporation in the cerebellum and forebrain, and partially blocked the GABA effect in the brain stem. GABA inhibited ³³Pi incorporation into phosphatidic acid, phosphatidylinositol, phosphatidyl choline and phosphatidyl ethanolamine but had no effect on phosphatidyl serine. The data suggest that the inhibitory effects of GABA on CNS phospholipid labeling are mediated specifically through GABA receptor sites.     

Nonenzymatic incorporation of prostaglandin A1 into phospholipids in rat liver microsomes

The incorporation of [5,6(n)-3H]prostaglandin A1 (PGA1) and [1-14C]oleic acid into membrane phospholipids of rat liver microsomes was studied. It was shown that PGA1 is incorporated into phospholipids in a much lesser degree than oleic acid. PGA1 is incorporated into phosphatidylethanolamine and, in a lesser degree, into phosphatidylcholine and phosphatidylinositol + phosphatidylserine. The exogenous cofactors of fatty acid acylation (ATP, CoA, Mg2+) exert no marked influence on the incorporation of PGA1 into the phospholipids. PGA1 interacts with isolated rat liver phospholipids; the PGA1-phospholipid conjugate formed is not destroyed in the course of one- or two-dimensional thin-layer chromatography. On the other hand, PGA1 binding to unsaturated phosphatidylcholines is strictly dependent on the phospholipid oxidation index. It is concluded that PGA1 incorporation into rat liver phospholipids is a result of interaction of PGA1 with peroxidized phospholipids.     

Alternative pathways of glucose utilization in brain. Changes in the pattern of glucose utilization in brain during development and the effect of phenazine methosulfate on the integration of metabolic routes.

The activities of alternative pathways of glucose metabolism in developing rat brain were evaluated by measurement of the yields of ¹⁴CO₂ from glucose labeled with ¹⁴C on carbons 1, 2, 3 + 4, 6 and uniformly labeled glucose, from the detritiation of [2-³H]glucose and from the incorporation of ¹⁴C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. The glycolytic route and tricarboxylic acid cycle (¹⁴CO₂ yield from carbons 3, 4, and 6 of glucose) increased during development. The flux through the glutamate-γ-aminobutyric route (¹⁴CO₂ yield from carbon 2-carbon 6 of glucose) also showed an increase with development. In contrast, the proportion of glucose metabolized via the pentose phosphate pathway was markedly decreased as development progressed. The artificial electron acceptor, phenazine methosulfate, was used as a probe to investigate the effect of alterations in the redox state of $\text{NADP}{}^{\mathrm{+}}\text{NADPH}$ couple on a number of NADP-linked systems in developing brain. Phenazine methosulfate produced a massive (20- to 50-fold) stimulation of the pentose phosphate pathway, in contrast, the incorporation of glucose carbon into fatty acids and flux through the glutamate-γ-aminobutyrate shunt were sharply decreased. The effects of phenazine methosulfate on the incorporation of glucose into glyceride glycerol, on the flux of glucose through the pyruvate dehydrogenase reaction and tricarboxylic acid cycle, all processes linked to the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ couple, appeared to be minimal in the brain at the stages of development studied, i.e., 1, 5, 10, 20 days, and in the adult rat. The significance of the massive reserve potential of the pentose phosphate pathway in the developing brain is discussed.     

Effect of experimental diabetes and insulin onphosphatidyl-inositol synthesis in rat sciatic nerve.

The rate of incorporation of [2-³H]myo-inositol into phosphatidylinositol was found to be significantly decreased in sciatic nerve from both alloxan and streptozotocin-diabetic rats. The rates of incorporation into phospholipid of tritiated serine and ethanolamine were unchanged while choline showed an upward trend in sciatic nerve from alloxan-diabetic rats. Insulin added $\text{in}\text{vitro}$ significantly increased [2-³H]myo-inositol incorporation into phospholipids by normal rat sciatic nerve; only small changes were recorded with high concentrations of glucose, and galactose. The results are discussed in relation to the physiological functions of phosphatidylinositol and the role of free myo-inositol in the regulation of cellular processes.     

Modification of glycerolipid metabolism in L-M fibroblasts by an unnatural amino-alcohol, N-isopropylethanolamine.

The unnatural amino-alcohol, N-isopropylethanolamine, is incorporated into a phospholipid by monolayers of L-M fibroblasts. This phospholipid was identified as 1,2-diacyl-$\text{sn}$-glycero-3-phosphoisopropylethanolamine by using chemical and enzymatic procedures combined with thin-layer and gas-liquid chromatography. Since the phospho-N-isopropylethanolamine moiety is removed by phospholipase C, the stereochemistry of the phospholipid analog is identical to naturally occurring phosphoglycerides. Incubation of cells in 10 mM N-isopropylethanolamine inhibited the incorporation of [¹⁴C]choline and [¹⁴C]ethanolamine into phospholipids and stimulated the incorporation of [1-¹⁴C]palmitic acid and [1-¹⁴C]hexadecanol into triacylglycerols and alkyldiacylglycerols. These results indicate that N-isopropylethanolamine affects glycerolipid synthesis at the diradylglycerol branch point.     

32P incorporation into different membranous structures separated from rat cerebral cortex

Abstract— The time course of incorporation, between 3 hr and 16 days, of ortho[³²P]phosphate into different membranous structures isolated from the rat cerebral cortex was studied. After subarachnoideal administration into the CSF it was found that myelin, mitochondria, microsomes and purified nerve-ending membranes and synaptic vesicles incorporate ³²P at the same rate. Most of the individual phospholipids of the synaptic vesicles and nerve-ending membranes also have similar rates of incorporation. Only phosphoinositides and/or phosphatidylserine may have a more rapid metabolism. The incorporation of ³²P into phosphoproteins follows a different pattern from that of the phospholipids. The intraperitoneal route is less effective in the ³²P incorporation and differences among the fractions may be found. These results are discussed in relation to the problem of the blood-brain barrier to phosphate and to the labelling of individual phospholipids in the different membranes.     

Comparative rates of protein synthesis of rat kidney medulla and cortex in vitro

The $\text{in}\text{vitro}$ rate of ¹⁴C-leucine incorporation into protein has been examined in rat kidney tissue. The presence of a marked gradient was observed. Thus, the white medulla was the most active in this respect followed by, in descending order, red medulla and cortex. ¹⁴C-Leucine incorporation into protein was completely abolished in the presence of cycloheximide. The distribution of labeled protein between the medium and slice suggests a high degree of cellular integrity and little secretion of labeled protein from slice to medium. The pattern of ¹⁴C-leucine incorporation amongst the different zones of kidney of hypophysectomized rats was similar to that noted in normal rats.     

Simultaneous inhibition of guinea pig brain 2′, 3′-cyclic nucleotide 3′-phosphohydrolase and myelin protein synthesis by 2′-adenosine monophosphate

Although the function of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) in myelin is unknown, the enzyme has been implicated in the metabolism of myelin proteins. Using 2′-AMP to inhibit CNPase, we examined the effect of reduced enzyme activity on the $\text{in vitro}$ incorporation of ¹⁴C-leucine into brain proteins. The results of this study revealed that (1) guinea pig brain homogenates incorporate leucine into protein from a sucrose medium in a linear fashion, (2) all brain fractions (cytosol, myelin, and microsomes) are labelled within 1 hr, (3) 2′-AMP inhibition of CNPase by 50% results in a similar inhibition of brain protein synthesis, and (4) the reduced protein synthesis is accompanied by a shift in label from myelin proteins to those found in the microsomes. These results are consistent with a role for CNPase in myelin protein synthesis.     

ACYLATION OF LYSOPHOSPHATIDYLSERINE BY RAT BRAIN MICROSOMES

Abstract— The acylation of lysophosphatidylserine, prepared by snake venom digestion of phosphatidylserine, by rat brain microsomes is described. Acylation was monitored by spectrophotometric assay and by measuring the incorporation of radioactively labelled acyl CoA thioesters. Acylation was time dependent, showed an approximately linear response to enzyme concentration and had a pH optimum of 9.0. Maximum acylation was attained at a concentration of about 100 μM for lysophosphatidylserine and about 40μM for acyl CoA thioesters. Positional distribution studies with [¹⁴C]oleoyl CoA and [¹⁴C]arachidonoyl CoA showed incorporation was predominantly at position -2, but with significant labelling at position–1, particularly with oleoyl CoA, possibly as a result of isomerization of the 1–acyl isomer of lysophosphatidylserine. Both saturated and unsaturated thioesters could serve as acyl group donors. Myristoyl CoA was considerably superior to palmitoyl CoA and stearoyl CoA, which were poor acyl group donors. Some selectivity was shown among the long chain unsaturated thioesters, linoleoyl, linolenoyl and arachidonoyl CoA being the most effective acylating agents. Although docosahexaenoic acid is a major unsaturated fatty acid in brain phosphatidylserine, its CoA ester was a relatively poor acyl group donor. Relative acylation rates remained essentially constant over a wide range of lysophosphatidylserine concentrations. It is concluded that acyl transfer mechanisms are active in brain for the regulation of the fatty acid profile of phosphatidylserine.