Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.     

Inositol Lipid Metabolism in Rat Hippocampal Formation Slices: Basal Metabolism and Effects of Cholinergic Agonists

Incubation of rat hippocampal formation slices under steady-state conditions with [3H]inositol leads to only three phospholipids becoming labelled: phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. All three lipids incorporate [32P]Pi into their phosphodiester phosphate group with the polyphosphoinositides also incorporating this tracer into their monoester phosphate groups. As the concentrations of these lipids remain constant during these labelling processes we conclude that the phosphodiester phosphate, the inositol moiety, and the monoester phosphate groups undergo metabolic turnover in hippocampal formation slices incubated in vitro. The rate of incorporation of [3H]inositol into all three inositol phospholipids was stimulated by the addition of methacholine to the medium. Moreover, following steady-state labelling of the inositol lipids with [3H]inositol, methacholine in the presence of 10 mM LiCl caused a transient fall of 13% in the radiochemical concentration of phosphatidylinositol 4,5-bisphosphate after only 30 s stimulation and a fall of 15% in the radiochemical concentration of phosphatidylinositol after 30 min. Concomitantly, there was an approximately stoichiometric rise in the radiochemical concentration of inositol phosphates. Thus, we suggest that methacholine stimulates an inositol phospholipid phosphoinositidase C in rat hippocampal formation slices.     

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.     

The turnover of myelin phospholipids in the adult and developing rat brain

1. Inorganic [(32)P]phosphate, [U-(14)C]glycerol and [2-(14)C]ethanolamine were injected into the lateral ventricles in the brains of adult rats, and the labelling of individual phospholipids was followed over 2-4 months in both a microsomal and a highly purified myelin fraction. 2. All the phospholipids in myelin became appreciably labelled, although initially the specific radioactivities of the microsomal phospholipids were somewhat higher. Eventually the specific radioactivities in microsomal and myelin phospholipids fell rapidly at a rate corresponding to the decline of radioactivity in the acid-soluble pools. 3. Equivalent experiments carried out in developing rats with [(32)P]phosphate administered at the start of myelination showed some persistence of phospholipid labelling in the myelin, but this could partly be attributed to the greater retention of (32)P in the acid-soluble phosphorus pool and recycling. 4. It is concluded that a substantial part of the phospholipid molecules in adult myelin membranes is readily exchangeable, although a small pool of slowly exchangeable material also exists. 5. A slow incorporation into or loss of labelled precursor from myelin phospholipids does not necessarily give a good indication of the rate of renewal of the molecules in the membrane. As presumably such labelled molecules originate by exchange with those in another membrane site (not necessarily where synthesis occurs) it is only possible to calculate the turnover rate in the myelin membrane if the behaviour of the specific radioactivity with time of the phospholipid molecules in the immediate precursor pool is known.     

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.     

NOREPINEPHRINE INCREASES THE [32P]LABELLING OF A SPECIFIC PHOSPHOLIPID FRACTION OF POST-SYNAPTIC PINEAL MEMBRANES

Abstract— Cultured pineal glands incorporated ³²P into membrane phospholipids. Treatment of cultured glands with norepinephrine, which is known to stimulate membrane- bound pineal adenyl cyclase and to increase the production and secretion of melatonin, stimulated the incorporation of ³²P into a phospholipid fraction of membranes and particulates containing phosphatidyl serine and phosphatidyl inositol. The labelling of other phospholipid fractions and the total ³²P in the gland were not changed by norepinephrine treatment. Experiments with chronically-denervated pineal glands indicated that the effect of norepinephrine on the [³²P]labelling of phospholipids occurred at a postsynaptic site. When norepinephrine-stimulated secretion of melatonin was partially inhibited by p -chlorophenylalanine (a compound which blocks the synthesis of melatonin precursors), the norepinephrine-stimulated labelling of phospholipids was still observed. Conversely, when melatonin secretion was stimulated in the absence of norepinephrine by treatment with the immediate precursor of melatonin, N -acetylserotonin, a stimulation of ³²P- labelling of phospholipids did not occur. These observations suggest that the increased [³²P]- labelling of a phospholipid fraction caused by the norepinephrine treatment is not related to the secretion of melatonin. This effect on phospholipids may be associated with the interaction of norepinephrine with a membrane-bound postsynaptic receptor. Stimulation by norepinephrine of [³²P]-incorporation into phospholipids has not been previously reported to occur in a tissue in which cholinergic fibres are absent.     

Phosphoinositide metabolism in rat superior cervical ganglion, vagus and phrenic nerve: effects of electrical stimulation and various blocking agents

Abstract— Paired vagus nerves, phrenic nerves or superior cervical ganglia from rats were incubated at 37 C for various times in a simple salt solution containing glucose and ³²P_i. One of the pair was usually stimulated electrically for 30 or 60 min. Stimulation of vagus nerve for 30 min increased phosphate incorporation into all the phospholipids studied but the increase was significant only in the case of triphos-phoinositide and diphosphoinositide. This increase was not accompanied by increased labelling of the nucleotide labile phosphate pool. Tetrodotoxin at concentrations sufficient to block transmission had no effect upon phospholipid labelling in vagus or phrenic nerve. Ouabain at blocking concentration did not affect polyphosphoinositide metabolism in vagus nerve but increased [³²P]labelling of the other phospholipids. Hemicholinium-3 increased the labelling of all three phosphoinositides in the sympathetic ganglia but the increase in phosphatidylinositol labelling due to electrical stimulation was not seen in the presence of this inhibitor.     

Comparison of muscarinic and alpha-adrenergic receptors in rat atria based on phosphoinositide turnover

The effects of muscarinic and alpha-adrenergic receptor stimulation on phosphoinositide turnover in rat atria have been compared. Despite the similar densities of muscarinic receptors in rat left and right atria, 0.1 mM carbachol increased [32P]phosphate incorporation into phosphatidylinositol (PI) by 35% (p less than 0.05) in left atria but had no effect in right atria. By contrast to the small muscarinic receptor effect, stimulation of alpha 1-adrenergic receptors by 0.1 mM methoxamine produced a more than two fold increase in [32P]phosphate incorporation into PI in both left and right atria, despite the reported smaller density of alpha-adrenergic receptors in rat atria compared to muscarinic receptors. Enhanced phosphate labelling by methoxamine did not occur in phospholipids other than PI, and was blocked by the alpha-adrenergic antagonist, phentolamine (20 microM). The results indicate that the majority of the muscarinic receptors in rat atria are not coupled to phosphoinositide turnover. If indeed the observed enhancement in [32P]-phosphate labelling by carbachol reflects phosphoinositide turnover, and assuming equal coupling efficiencies of muscarinic and adrenergic receptors, it is calculated that not more than 2% of the muscarinic receptors in rat left atria are coupled to this response.     

Sympathetic denervation impairs agonist-stimulated phosphatidylinositol metabolism in rat parotid glands.

Substance P, muscarinic and alpha-adrenoceptor agonists stimulated the incorporation of [3H]inositol into phosphatidylinositol in rat parotid gland slices. Surgical denervation of the sympathetic input to the rat parotid gland by superior cervical ganglionectomy produced marked reductions in these responses. The stimulated incorporation of radiolabelled precursors into phosphatidylinositol is a measure of its resynthesis after receptor-mediated breakdown of inositol phospholipids. We therefore examined the enzymic site of the lesion induced by sympathetic denervation using parotid gland slices labelled with either [3H]inositol or [32P]phosphate and stimulated with substance P. Receptor-activated phospholipase C attack upon [3H]inositol phospholipids was assayed by measuring the formation of [3H]inositol 1-phosphate in the presence of 10 mM-Li+ to inhibit further breakdown. It was not affected by denervation. Substance P elicited a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and this response was reduced in the denervated gland. The second step in stimulated phosphatidylinositol turnover, phosphorylation of diacylglycerol to phosphatidate was not affected by denervation. Sympathetic denervation appears to induce a specific enzymic lesion in the parotid gland that impairs receptor-stimulated resynthesis of phosphatidylinositol from phosphatidate. This change in membrane lipid metabolism may be related to a number of the effects of sympathetic denervation, such as agonist supersensitivity, reduced gland cell proliferation and induction of new surface receptors.     

Evidence for the incorporation of [32P]orthophosphate into leaf inositol phospholipids

Leaf discs of brinjal, tomato, sugar cane and maize rapidly incorporated [32P]orthophosphate into total phospholipids. Analyses of the labelled lipid extracts by thin-layer chromatography, autoradiography and comparison with inositol phospholipid standards demonstrated the labelling of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate in addition to other phospholipids. The presence of polyphosphoinositides was further confirmed by deacylation of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate and separation of the water-soluble products, glycerophosphoinositol phosphate and glycerophosphoinositol bisphosphate by formate exchange chromatography. Incorporation of [32P]orthophosphate into inositol phospholipids was time-dependent, with monoester phosphate groups attaining isotopic equilibrium within 90 min of incubation. After 2 h, incorporation of label into phosphatidylinositol, phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate was about 15, 10 and 3%, respectively, of the total phospholipids. The ratio of radioactivity in phosphatidylinositol/phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate was about 5:5:1 in brinjal leaves. However, this ratio may be an overestimate of the amounts of inositol phospholipids present, as other lysophospholipids may comigrate with standards.     

Lithium increases turnover of phospholipids in flight muscles of Periplaneta americana L

The effect of prolonged lithium administration on the phospholipid metabolism of flight muscles of the cockroach Periplaneta americana has been studied. Following daily injections of LiCl in a dose of 19.25 mumol LiCl per gram of wet weight [32P]- orthophosphate were injected and its incorporation into the phospholipids was measured 2, 12 and 24 h later. Lithium administration did not change the content of phospholipids but increased the 32P incorporation into phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine and sphingomyeline 1.87, 2.13, 2.02 and 1.87 times, respectively, as compared with the control values. These increases were neither due to an increased permeability of the tissue for inorganic phosphate nor to an increased turnover of gamma-P-ATP. It is concluded that prolonged lithium treatment increases the turnover of all phospholipids in insect flight muscle tissue.     

TURNOVER OF PROTEIN PHOSPHORUS IN RESPIRING SLICES OF GUINEA PIG CEREBRAL CORTEX: CELLULAR LOCALIZATION OF PHOSPHOPROTEIN SENSITIVE TO ELECTRICAL STIMULATION

Abstract— In experiments designed to localize the increased turnover of phosphoprotein-P which occurs in respiring brain slices as a result of electrical stimulation, a cell separation procedure was used to prepare a fraction enriched in neuronal cell bodies from incubated slices labelled with [³²P]phosphate. Labelled phosphoprotein was found to be twice as concentrated in the neuron-enriched fraction as in other fractions. Electrical stimulation for 10 s increased the rate of incorporation of [³²P]phosphate into phosphoproteins in the neuron-enriched fraction by 25 per cent ( P < 0.05), but had no effect on incorporation into a partially purified glial fraction contaminated with neuropil and cell debris.     

Phospholipid turnover in Torpedo marmorata electric organ during discharge in vivo.

One electric organ of anaesthetized Torpedo marmorata was stimulated through electrodes placed on the electric lobe of the brain. Nerves to the other electric organ were cut to provide an unstimulated control. Glucose 6-[32P]phosphate was injected into each organ 16h before electrical stimulation. After stimulation for 10 min at 5 Hz, the organs were removed homogenized and centrifuged on a density gradient for the preparation of subcellular fractions. Stimulation increased the incorporation of 32P into phosphatidate, phosphatidylinositol and phosphatidylcholine. The increased phosphatidate labelling, but not that of the other two lipids, was seen in fractions rich in synaptic vesicles. Stimulation had no effect on ATP labelling. The phosphatidate content of most fractions fell slightly after stimulation, but amounts of other phospholipids were not affected.     

In vitro study of adrenergic stimulation of 32P incorporation into phospholipids of brown adipose tissue of control and cold-acclimated rats

1. 32P-labelled inorganic phosphate incorporation into total and mitochondrial phospholipids was studied, in vitro, on brown adipose tissue (BAT) of control and cold-acclimated rats. 2. It was found that norepinephrine acts as in vivo, on BAT phospholipid metabolism via alpha 1 adrenergic receptors specifically increasing phosphatidic acid and phosphatidylinositol turnover with the same magnitude in both groups. 3. Cold-induced alpha 1 adrenergic desensitization is not as important as cold-induced beta adrenergic desensitization. 4. No specific effect of norepinephrine was seen in mitochondrial phospholipid turnover.     

THE EFFECT OF ELECTRICAL STIMULATION ON THE TURNOVER OF PHOSPHATIDIC ACID IN SYNAPTOSOMES FROM GUINEA-PIG BRAIN

Synaptosomes prepared from guinea-pig cerebral cortex were suspended in a medium containing [³²P]orthophosphate and subjected to electrical stimulation. When the synaptosomal phospholipids were subsequently separated, the most highly labelled was phosphatidic acid and electrical stimulation over a 10 min period increased incorporation of ³²P₁ into this lipid. Stimulated synaptosomes were osmotically lysed and subsynaptosomal fractions isolated. The electrically stimulated increase in phosphatidic acid labelling was localized in a fraction enriched in synaptic vesicles. This phospholipid effect was not merely a reflection of an increased specific radioactivity of synaptosomal ATP, due to the electrically stimulated increase in respiration. The time course of the phosphatidic acid effect suggests that it is synchronous with release of transmitter.     

Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown.

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.     

The subcellular distribution of 32P-labelled phospholipids, 32P-labelled ribonucleic acid and 125I-labelled iodoprotein in pig thyroid slices. Effect in vitro of thyrotrophic hormone and dibutyryl-3′,5′-(cyclic)-adenosine monophosphate

1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.     

The metabolism of phosphatidylinositol in the thyroid gland of the pig

1. The metabolism of phosphatidylinositol in pig thyroid has been investigated as a basis for understanding the specific stimulation of the synthesis of this phospholipid in the gland by thyrotropin. 2. The gland contained an active Ca(2+)-dependent phosphatidylinositol-splitting enzyme with an optimum pH of 5.3-5.5. 3. The major water-soluble product (65%) formed by this catabolic enzyme was not phosphorylinositol but a related compound, which may be a cyclic phosphorylinositol. Both this and phosphorylinositol (35%) were released simultaneously from the phosphatidylinositol substrate. 4. The phosphatidylinositol-splitting enzyme was found almost exclusively in the supernatant fraction obtained by homogenization of the gland. It was not present in the acid-phosphatase-containing particulate fraction. 5. The incorporation of [2-(3)H(1)]inositol into phosphatidylinositol in the presence of either CDP-diglyceride or CTP+ATP was most active in the microsomal fraction. 6. When thyroidal microsomes were labelled with [(3)H]inositol and (32)P, and then incubated with unlabelled inositol, there was a dramatic loss of (3)H labelling from the phosphatidylinositol, which was not accompanied by an equivalent loss of (32)P from the phosphate moiety. This turnover of the inositol moiety required nucleotide coenzymes. It is postulated that the phosphatidylinositol is split into inositol and a phosphorus-containing lipid precursor of the phospholipid that remains on the microsomal membrane and is recycled. 7. Isolated thyroidal mitochondria synthesized phosphatidylinositol from [2-(3)H(1)]inositol only because of their contaminating microsomal component. 8. Some evidence has been obtained of a rapid transfer of phosphatidylinositol molecules from thyroidal microsomes to mitochondria when these were incubated together in the presence of a supernatant fraction. 9. Both phosphatidylinositol breakdown by the supernatant fraction of the gland and synthesis by the microsomes were totally inhibited by 1mm-chlorpromazine. This drug is known to suppress thyrotrophin-induced stimulation of activity in thyroid slices.     

Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein

1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [(32)P]orthophosphate, [(14)C]choline and [(14)C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with (32)P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with (32)P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth hormone and tri-iodothyronine administered to hypophysectomized rats had additive effects in all the biosynthetic processes. The latent period of action of each hormone was maintained so that two waves of proliferation of endoplasmic reticulum occurred if the hormones were administered simultaneously. 7. It is concluded that there is some mechanism in the cell that tightly co-ordinates the formation of membranes, especially those of the endoplasmic reticulum, when an increased demand is made for protein synthesis.     

Phosphorylation of phospholipids in isolated guinea pig hearts stimulated with isoprenaline.

Phosphorylation of phospholipids was studied in Langendorff perfused guinea pig hearts subjected to beta-adrenergic stimulation. Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi and freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline. 32P incorporation into total phospholipids, individual phospholipids and polyphosphoinositides was analysed in whole tissue homogenates and membranes, enriched in sarcoplasmic reticulum, prepared from the same hearts. Isoprenaline stimulation of the hearts did not result in any significant changes in the levels of phosphate incorporation in the total phospholipid present in cardiac homogenates (11.6 +/- 0.4 nmol of 32P/g for control hearts and 12.4 +/- 0.5 nmol of 32P/g for isoprenaline-treated hearts; n = 6), although there was a significant increase in the degree of phospholipid phosphorylation in sarcoplasmic reticulum (3.5 +/- 0.3 nmol of 32P/mg for control hearts and 6.7 +/- 0.2 nmol of 32P/mg for isoprenaline-treated hearts; n = 6). Analysis of 32P incorporation into individual phospholipids and polyphosphoinositides revealed that isoprenaline stimulation of the hearts was associated with a 2-3-fold increase in the degree of phosphorylation of phosphatidylinositol monophosphate and bisphosphate as well as phosphatidic acid in both cardiac homogenates and sarcoplasmic reticulum membranes. In addition, there was increased phosphate incorporation into phosphatidylinositol in sarcoplasmic reticulum membranes. Thus, perfusion of guinea pig hearts with isoprenaline is associated with increased formation of polyphosphoinositides and these phospholipids may be involved, at least in part, in mediating the effects of beta-adrenergic agents in the mammalian heart.