Phospholipid synthesis in rat liver endoplasmic reticulum after the administration of phenobarbitone and 20-methylcholanthrene

1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [(32)P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [(32)P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine-phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [(32)P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [(14)C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [(32)P]orthophosphate and [(14)C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [(32)P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [(32)P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.     

INCORPORATION OF 32P INTO THE PHOSPHOLIPIDS OF NEURONAL AND GLIAL CELL ENRICHED FRACTIONS ISOLATED FROM RABBIT CEREBRAL CORTEX: EFFECT OF NOREPINEPHRINE

Abstract— Glial cells isolated from rabbit cerebral cortex contained approximately one-third more phospholipids per unit protein than the neuronal cell bodies. The pattern of individual phospholipids was rather similar in both cell types. The incorporation of intracisternally administered ³²P into neuronal and glial phospholipid classes of rabbit brain was studied at intervals ranging from 5 to 60min. In general, for all investigated phospholipids the incorporation of the label was somewhat faster in neurons than in glial cells. Phosphatidylinositol showed the fastest and ethanolamine plasmalogen the slowest incorporation of ³²P in both neurons and glial cells. A lag phase of about 10 min could be observed before labelling of the glial phosphatidylcholine, phosphatidylethanolamine, ethanolamine plasmalogen, phosphatidylserine and sphingomyelin had occurred. Among the neuronal phospholipids a lag phase was found only for the labelling of the ethanolamine plasmalogen. Norepinephrine increased the incoropration of ³²P into phosphatidylinositol of both glia and neurons but had no effect on the specific radioactivity of ethanolamine plasmalogen and sphingomyelin. Labelling of phosphatidylcholine was slightly inhibited in both cell types by the administration of norepinephrine.     

Amino acid requirement for the growth-hormone stimulation of incorporation of precursors into protein and nucleic acids of liver slices

1. Incorporation of [(14)C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [(3)H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.     

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.     

Lipids of rabies virus and BHK-21 cell membranes.

The lipid composition of highly purified Flury strain of rabies virus (HEP) propagated in BHK-21 cells in a chemically defined medium was observed to be 6.7% neutral lipids, 15.8% phospholipids, and 1.5% glycolipids. In the virion, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the most abundant phospholipids, accounting for 90% of the total, and the molar ratio of cholesterol to phospholipid was 0.48. Uninfected BHK-21 cell membranes were obtained by nitrogen cavitation techniques and separated by density gradient centrifugation, and the membranes were assayed for purity using 5'-nucleotidase, cytochrome oxidase, and reduced nicotinamide adenine dinucleotide phosphate diaphorase activities. Lipids of the plasma membrane were enriched in cholesterol, phosphatidylcholine, and phosphatidylethanolamine. In contrast, membranes of the endoplasmic reticulum were enriched in phosphatidylcholine, but contained smaller amounts of phosphatidylethanolamine and sphingomyelin. Comparison of the fatty acyl chains of virus and membranes from uninfected cells revealed the virion to have the lowest ratio of C18:1 to C18:0 (1.771), compared with values of about 3.0 for the plasma membrane and endoplasmic reticulum. Total polyenoic fatty acids were enriched in the plasma membrane, whereas the virus contained higher amounts of total saturates than either of the two membrane preparations. Analysis of the polar and neutral lipid fractions as well as the acyl chain analysis suggests the virion has a lipid composition that is intermiediate to that of the plasma membrane and endoplasmic reticulum and is consistent with the view that numerous viral particles are synthesized de novo by not utilizing a preexisting membrane template. From the ratio of cholesterol to phospholipid of 0.48, we calculated that 1.92 X 10(5) molecules of lipid would cover 4.14 X 10(4) nm2 in the form of a bilayer. Considerations of the molecular dimensions of the rabies envelope (total surface area, 5 X 10(4) nm2) as a bilayer suggest that some penetration of lipids by envelope proteins (M and G) is necessary.     

Phospholipid exchange reactions within the liver cell

1. Isolated rat liver mitochondria do not synthesize labelled phosphatidylcholine from CDP-[(14)C]choline or any phospholipid other than phosphatidic acid from [(32)P]phosphate. The minimal labelling of phosphatidylcholine and other phosphoglycerides can be attributed to microsomal contamination. However, when mitochondria and microsomes are incubated together with [(32)P]phosphate, the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of the reisolated mitochondria become labelled, suggesting a transfer of phospholipids between the two fractions. 2. When liver microsomes or mitochondria containing labelled phosphatidylcholine are independently incubated with the opposite un-labelled fraction, there is a substantial and rapid exchange of the phospholipid between the two membranes. Exchange of phosphatidylinositol also occurs rapidly, whereas phosphatidylethanolamine and phosphatidic acid exchange only slowly. There is no corresponding transfer of marker enzymes. The transfer of phosphatidylcholine does not occur at 0 degrees , and there is no requirement for added substrate, ATP or Mg(2+), but the omission of a heat-labile supernatant fraction markedly decreases the exchange. 3. After intravenous injection of [(32)P]phosphate, short-period labelling experiments of the individual phospholipids of rat liver microsomes and mitochondria in vivo give no evidence for a similar exchange process. However, the incubation of isolated microsomes and mitochondria with [(32)P]phosphate also fails on reisolation of the fractions to demonstrate a precursor-product relationship between the individual phospholipids of the two membranes. 4. The intraperitoneal injection of [(32)P]phosphate results in a far greater proportion of the dose entering the liver than does intravenous administration. After intraperitoneal administration of [(32)P]phosphate the specific radioactivities of the individual phospholipids are in the order microsomes > outer mitochondrial membrane > inner mitochondrial membrane. 5. The incorporation of (32)P into cardiolipin is very slow both in vivo and in vitro. After labelling in vivo the radioactivity in the cardiolipin persists compared with that of the other phospholipids, whose specific radioactivities in the microsomes and mitochondrial fragments decay at a similar rate to that of the acid-soluble phosphate pool. 6. The possibility of phospholipid exchange processes occurring in the liver cell in vivo is discussed, and it is suggested that only a small but highly labelled part of the endoplasmic-reticulum lipoprotein pool is involved in the transfer.     

Phenobarbital-induced alterations in phosphatidylcholine and triglyceride synthesis in hepatic endoplasmic reticulum

Biosynthetic pathways of phosphatidylcholine and triglyceride were studied in proliferating hepatic endoplasmic reticulum of rats pretreated with phenobarbital. Phosphatidylcholine accounted for the major increment in membrane phospholipid. In vitro measurements of hepatic microsomal enzymes which catalyze phosphatidylcholine biosynthesis revealed a significant increase in specific activity of the enzyme governing phosphatidylcholine synthesis by sequential methylation of phosphatidylethanolamine. The specific activity of phosphorylcholine-glyceride transferase, which catalyzes phosphatidylcholine synthesis from d-1,2-diglyceride and CDP-choline, was not altered. Specific activity of diglyceride acyltransferase, which catalyzes triglyceride biosynthesis, was increased to a degree comparable to the increase in specific activity found in the phenobarbital-induced drug-metabolizing enzyme which oxidatively demethylates aminopyrine. In vivo incorporation of methyl-(3)H from l-methionine-methyl-(3)H into microsomal phosphatidylcholine was significantly increased, resulting in an increased methyl-(3)H to choline-1,2-(14)C incorporation ratio of more than three times that found in control animals. A comparable increase in this incorporation ratio was noted in serum phospholipids. The in vitro enzyme studies, in agreement with in vivo incorporation data, indicate that the increase in phosphatidylcholine content of phenobarbital-induced proliferating endoplasmic reticulum is related to increased activity of the pathway of phosphatidylcholine biosynthesis involving the sequential methylation of phosphatidylethanolamine.     

Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.     

Effects of fatty acid deficiency on the lipid composition and physical properties of guinea pig rough endoplasmic reticulum

Twenty-six days of fat deficiency brought about a decrease of linoleic and an increase of oleic acid in rough endoplasmic reticulum (RER) of guinea pig liver. Arachidonic acid was only slightly decreased in some phospholipids whereas eicose-5,8,11-trienoic acid was not enhanced except in phosphatidyl-inositol. All these changes were relevant specifically in phosphatidylinositol molecules and less important in phosphatidylcholine and phosphatidylethanolamine. Fat deficiency did not modify the relative proportion of phospholipids and cholesterol. Therefore, fat deficient guinea pig microsomes are a good model to study the effect of unsaturated fatty acids on membrane properties. Fluorescent anisotropy of RER membranes, lipids and phospholipids labeled with diphenylhexatriene, was increased by the fat deficiency. The most important increase was observed in liposomes of a mixture of RER phosphatidylinositol, phosphatidylserine and sphingomyelin. A small change was found in phosphatidylcholine and phosphatidylethanolamine dispersions at 37°C. The modification of the lipid unsaturation evoked fluorescent anisotropy changes. Temperature-dependent fluorescent polarization curves of RER membranes labeled with trans-parinaric acid did not show inflections in the temperature range from 5 to 45°C but, RER lipids and phospholipids presented a phase separation at about 20°C. This inflection point was not modified by the fat deficient diet. In those liposomes prepared with a mixture of RER phosphatidylinositol, phosphatidylserine and sphingomyelin, the inflection point was produced at about 37°C.The author is member of the Carrera del Investigador Cientifico, Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina.     

The formation, distribution and function of ribosomes and microsomal membranes during induced amphibian metamorphosis

1. A lag period of about 4 days preceded the onset of metamorphosis precociously induced by tri-iodothyronine in tadpoles of the giant American bullfrog (Rana catesbeiana). It was established by the accelerated synthesis or induction of carbamoyl phosphate synthetase and cytochrome oxidase in the liver, serum albumin and adult haemoglobin in the blood, acid phosphatase in the tail, and the increase in the hindleg/tail length ratio. 2. A 4- to 6-fold stimulation, 2 days after the induction of metamorphosis, of the rate of synthesis of rapidly labelled nuclear RNA in liver cells was followed by an increasing amount of RNA appearing in the cytoplasm. Most of the newly formed RNA on induction of metamorphosis was of the ribosomal type. An accelerated turnover at early stages of development preceded a net accumulation of RNA in the cytoplasm, with no change in the amount of DNA per liver. 3. Most hepatic ribosomes of the pre-metamorphic tadpoles were present as 78s monomers and 100s dimers; metamorphosis caused a shift towards larger polysomal aggregates with newly formed ribosomes that were relatively more tightly bound to membranes of the endoplasmic reticulum. 4. The appearance of new polyribosomes in the cytoplasm on induction of metamorphosis was co-ordinated in time with a stimulation of synthesis of phospholipids of the smooth and rough endoplasmic reticulum, followed by a gradual shift in preponderance from the smooth to the rough type of microsomal membranes. 5. Electron- and optical-microscopic examination of intact hepatocytes revealed a striking change in the distribution and nature of ribosomes and microsomal membranes during metamorphosis. 6. Ribosomes prepared from non-metamorphosing and metamorphosing animals were identical in their sedimentation coefficients and in the structural ribosomal proteins. The base composition and sedimentation coefficients of ribosomal RNA were also identical. Induction of metamorphosis also did not alter the incorporation of (32)P into the different phospholipid constituents of microsomal membranes. 7. Nascent (14)C-labelled protein with the highest specific activity was recovered in the ;heavy' rough membrane fraction of microsomes, whereas little (14)C was associated with ;free' polysomes. Protein synthesis in vivo was most markedly stimulated during metamorphosis in the tightly membrane-bound ribosomal fraction after the appearance of new ribosomes. 8. The rate of synthesis of macromolecules in vivo could not be followed beyond 7-8 days after induction because of variable shifts in precursor pools due to regression of larval tissues. 9. The stimulation of RNA and ribosome formation was specifically associated with the process of metamorphosis since no similar response to thyroid hormones occurred in those species (Axolotl and Necturus) in which the hormones failed to induce metamorphosis.     

Intracellular transfer of phospholipids in the yeast, Saccharomyces cerevisiae

In Saccharomyces cerevisiae, unlike in higher eukaryotic cells, most of the reactions involved in phospholipid biosynthesis occur both in mitochondria and in the endoplasmic reticulum. Some of the key enzymes involved, however, are restricted to one compartment. Thus, the formation of phosphatidylethanolamine by decarboxylation of phosphatidylserine occurs only in mitochondria, while phosphatidylcholine synthesis via methylation of phosphatidylethanolamine is restricted to microsomes. When yeast cells were pulse labelled with [3H]serine,[3H] phosphatidylethanolamine formed in mitochondria was found not only in the organelle but also, with even higher specific radioactivity, in the endoplasmic reticulum. Translocation of phosphatidylethanolamine between organelles was blocked immediately after poisoning cells with cyanide, azide and fluoride. Part of the [3H]phosphatidylcholine formed in the endoplasmic reticulum by methylation of [3H]phosphatidylethanolamine was transferred to mitochondria. This process continued in deenergized cells, although at a lower rate as compared to metabolizing cells. This result indicates rapid movement of both phosphatidylethanolamine and phosphatidylcholine requires metabolic energy, but that phosphatidylinositol-specific phospholipid transfer protein that has been found in saccharomyces cerevisiae (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 784, 385-391). The mechanism of movement of phospholipids from internal membranes to the cell surface was studied with temperature-sensitive secretory mutants (Schekman, R. (1982) Trends Biochem. Sci. 7, 243-246) of Saccharomyces cerevisiae. A shift from the permissive to the restrictive temperature, which blocks the flow of vesicles involved in the secretion of proteins, had no effect on the transfer of phosphatidylinositol to the plasma membrane.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

Carbon tetrachloride target lipids in the rat liver microsomes. Effect of cystamine administration on their pattern of labeling by 14CCl4

¹⁴C from ¹⁴CCl₄ irreversibly binds to lipids from the smooth (SER) and rough (RER) endoplasmic reticulum. Most of the label is associated with the phospholipid fraction (> 95%). Prior cystamine administration decreased the extent of that binding but does not change its pattern of distribution. About the half of the label in phospholipids is in the phosphatidylcholine fraction; the other half is distributed similarly among lysophosphatidylcholine, sphingomyelin and phosphatidylethanolamine, while only a very minor fraction is associated with diphosphatidyl glycerol. No differences were found in the pattern of labeling of phospholipids in SER and RER.     

Sidedness of Phospholipid Synthesis on Brain Membranes

Abstract: We have investigated the localization of the site of incorporation and the subsequent equilibration of newly synthesized phospholipids in brain membranes. Rats were injected intracranially with [³H]glycerol; the animals were killed at varying times afterwards, and microsomal fractions were isolated from the brains. In some cases, microsomes were subfractionated on sucrose gradients. Initially, most of the radioactive phosphatidylethanolamine appeared in a pool that reacted with the impermeable reagent trinitrobenzene sulfonic acid (TNBS). This probe presumably modified only the lipid on the outer face of microsomal vesicles (which may, in large part, consist of pinched-off endoplasmic reticulum). At 5 min after injection, the specific radioactivity of the TNBS-modified phosphatidylethanolamine (cytoplasmic face) was four times that of the unmodified (luminal or inner face) phosphatidylethanolamine. With time, the ratio of the specific activities in the modified and unmodified pools of phosphatidylethanolamine approached 1.0, with kinetics that suggested a half-time on the order of 30 min form vivo conversion of the TNBS-accessible to the -inaccessible pool. This equilibration in specific activities could be the result of either translocation of phospholipids across endoplasmic reticulum membranes or conversion with time of initially labeled endoplasmic reticulum to other membranous organelles which form randomly oriented vesicles upon homogenization. A similar experimental design, using phospholipase C to hydrolyze outer face phospholipids preferentially, verified this conclusion for phosphatidylethanolamine and yielded similar results for phosphatidylcholine. Control studies measuring radioactive sucrose permeability indicated that neither TNBS nor phospholipase C treatment significantly disrupted microsomal vesicles under the conditions used.     

Phospholipid asymmetry in rough- and smooth-endoplasmic-reticulum membranes of untreated and phenobarbital-treated rat liver.

Phospholipase C was used as a probe for the distribution of phospholipids about the membrane of rough and smooth microsomal fractions from normal and phenobarbital-treated rat liver. All membranes exhibited an asymmetric distribution, with phosphatidylethanolamine and phosphatidylserine concentrated in the inner leaflet of the bilayer and phosphatidylcholine and sphingomyelin concentrated in the outer leaflet. The only phospholipid showing a significant difference in distribution between fractions was phosphatidylcholine, which was shifted towards the outer leaflet in the smooth microsomal fraction compared with the rough microsomal fraction, and towards the outer leaflet in both rough and smooth microsomal fractions from phenobarbital-treated liver compared with the same preparations from untreated rat liver. Apart from this small change, the asymmetric distribution of phospholipids was conserved in microsomal fractions which had proliferated in response to phenobarbital and in which the protein composition had changed.     

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.     

Adrenocorticotropic hormone-mediated changes in rat adrenal mitochondrial phospholipids

We examined the subcellular localization of ACTH (adrenocorticotropic hormone)-induced changes in adrenal phospholipids using dexamethasone-treated rats. In adrenal mitochondrial fraction, ACTH significantly enhanced both concentrations and contents of phosphatidylinositol (37%), phosphatidylcholine (22%), and phosphatidylethanolamine (20%). Other mitochondrial phospholipids including cardiolipin did not change upon administration of ACTH. In adrenal plasma membrane, endoplasmic reticulum, and peroxisomes, no increase in phospholipids was observed. The ACTH-induced increases in mitochondrial phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine were specific to adrenal among tissues tested. These changes were observed specifically in cortical cells rather than medulla. Nonsteroidogenic ACTH fragments and related peptides were unable to induce the change in adrenal mitochondrial phospholipids. From the dose-response profile with ACTH, the changes in mitochondrial phospholipids were closely related to ACTH-dependent stimulation of steroidogenesis. Furthermore, in vitro treatment with cyclic AMP enhanced both concentrations and contents of mitochondrial phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine similar to those by the in vivo administration of ACTH. Both in vivo and in vitro experiments revealed that the hormone-induced changes in mitochondrial phospholipids were sensitive to a protein-synthesis inhibitor, cycloheximide. However, aminoglutethimide and cytochalasin B, which strongly inhibited the hormone-induced formation of corticosterone, did not affect the increases in mitochondrial phospholipids. These results suggest that the hormone-induced increases in these phospholipids occur between ACTH-mediated ribosomal protein synthesis and corticosterone formation.     

Effects of analogues of ethanolamine and choline on phospholipid metabolism in rat hepatocytes

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [(14)C]ethanolamine incorporation into phospholipids, whereas the incorporation of [(3)H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [(3)H]glycerol and hepatocytes, the appearance of (3)H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [(3)H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of (3)H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-(14)C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [(14)C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.     

The phospholipid composition of embryonic chick liver microsomes

1. The phospholipid composition of hepatic microsomal fractions from different developmental stages of embryonic chick was established. The major components were phosphatidylcholine (approx. 66%), phosphatidylethanolamine plus phosphatidylserine (approx. 21%) and sphingomyelin (approx. 9%). 2. There were no significant changes in the phospholipid composition during embryonic development from 9 to 20 days. 3. When microsomal subfractions were prepared it was found that the smooth-microsomal fractions (Ia and Ib) had a significantly greater sphingomyelin content than the rough-microsomal fraction (II). This was compensated by a lower phosphatidylcholine content in fractions Ia and Ib and an increase of phosphatidylcholine in fraction II. 4. The significance of the differences in the phospholipid composition of smooth and rough microsomes is discussed with particular reference to the origin and interrelation of smooth and rough endoplasmic reticulum.     

Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.