Subcellular localization of phospholipid changes in response to muscarinic stimulation of perfused bovine adrenal medulla.

The effects of carbachol on catecholamine secretion and [32P]Pi incorporation into phospholipids was studied in perfused bovine adrenal medulla. After a labelling period, the gland was stimulated with carbachol in the absence of 32P. Subcellular fractions were then prepared from the medulla. Carbachol roughly halved the specific radioactivities of phosphatidylinositol and phosphatidate in microsomal, chromaffin-granule, mitochondrial and plasma-membrane fractions. With Ca2+-free perfusion medium, catecholamine secretion was abolished but the phospholipid changes remained. Stimulation of secretion by KCl was not accompanied by phospholipid changes. The results are not consistent with the theory relating phosphatidylinositol hydrolysis and Ca2+ gating.     

A comparison of the effects of phytohaemagglutinin and of calcium ionophore A23187 on the metabolism of glycerolipids in small lymphocytes.

1. The effects of phytohaemagglutinin and of a Ca2+ ionophore (A23187) on glycerolipid metabolism in lymphocytes from pig lymph nodes were compared (a) by studying the incorporation of [32P]Pi and [3H]glycerol, and (b) by following the redistribution of [3H]glycerol among the lipids caused by these agents in pulse-chase experiments. 2. Phytohaemagglutinin only stimulated 32P incorporation into phosphatidylinositol and, to a slight extent, phosphatidate. Removal of most of the extracellular Ca2+ somewhat decreased this response. 3. Ionophore A23187 stimulated the labelling of phosphatidate and phosphatidylinositol with 32P to a much greater extent than did phytohaemagglutinin: the increase in phosphatidate labelling, but not that of phosphatidylinositol, was almost abolished by the removal of extracellular Ca2+. 4. The combined effects of phytohaemagglutinin and ionophore appeared to be additive, rather than synergistic. 5. Treatment with ionophore A23187 somewhat decreased the total incorporation of [3H]glycerol into glycerolipids, possibly because it lowered cell ATP content. In these experiments di- and tri-acylglycerol behaved anomalously, triacylglycerol labelling being suppressed completely, whereas that of diacylglycerol was enhanced. The pulse-chase results revealed that triacylglycerol was converted into diacylglycerol in the ionophore-treated cells, and the availability of this diacylglycerol probably led to the enhanced labelling of phosphatidate and phosphatidylinositol in the these cells. 6. Thus an increase in intracellular Ca2+ concentration appeared to have three effects on glycerolipid metabolism: (a) slight inhibition of some metabolic step preceding phosphatidate synthesis, (b) inhibition of diacylglycerol acyltransferase and (c) activation of a triacylglycerol lipase. 7. In contrast, it seems likely that the only effect of phytohaemagglutinin is to stimulate phosphatidylinositol breakdown. 8. Pig polymorphonuclear leucocytes treated with ionophore A23187 showed metabolic changes that were similar to those demonstrated with lymphocytes. 9. A possible similarity is suggested between Ca2+-stimulated triacylglycerol lipase in lymphocytes and polymorphonuclear leucocytes and previous observations of enhanced triacylglycerol metabolism in stimulated cells whose metabolic functions involve membrane fusion.     

The effect of gluconeogenesis on phospholipid turnover in isolated chick proximal tubule cells

Previous work from this laboratory has shown that isolated chick renal proximal tubule cells possess an Na+-dependent Pi transport system and that Pi uptake is stimulated under gluconeogenic conditions. It is shown in the present paper that gluconeogenesis is associated with a rapid incorporation of Pi into membrane phospholipids, particularly phosphatidylinositol, and some evidence has been obtained for a change in the relative amounts of phosphatidylinositol polyphosphates under gluconeogenic conditions. There is no increase in the total phospholipid phosphate content however, suggesting that pyruvate-induced incorporation of Pi into phospholipids represents accelerated turnover rather than a net increase in synthesis. It is suggested that the stimulation of Na+-dependent Pi uptake by pyruvate is related to the increased rate of phospholipid turnover. Thus Pi transport may be a further example of a physiological system that is influenced by phosphatidylinositol metabolism. The role of phosphatidylinositol phosphates could be to stimulate transfer of transporter molecules from internal stores to the brush-border membrane of the cell.     

Phosphatidic acid and phosphatidylinositol labelling in adipose tissue. The role of endogenously formed adenosine.

Incorporation of [32P]Pi into phosphatidic acid and phosphatidylinositol of hamster epididymal adipocytes was partially inhibited by 3-isobutyl-1-methylxanthine. This effect of 3-isobutyl-1-methylxanthine was antagonized by isopropyl-N6-phenyladenosine but not by 2',5'-dideoxyadenosine, prostaglandin E1 or clonidine. N6-Phenylisopropyladenosine did not affect incorporation of [32P]Pi into phosphatidic acid or phosphatidylinositol when 3-isobutyl-1-methylxanthine was not present. In contrast with 3-isobutyl-1-methylxanthine inhibition of [32P]Pi incorporation into phospholipids, which was blocked only by N6-phenylisopropyladenosine, accelerated lipolysis was blocked by prostaglandin E1, clonidine and 2',5'-dideoxyadenosine as well as by N6-phenylisopropyladenosine. Phospholipid labelling was also decreased in the presence of adenosine deaminase, but not in the presence of isoprenaline (isoproterenol). The stimulatory effect of N6-phenylisopropyladenosine on [32P]Pi incorporation into phospholipids in cells exposed to 3-isobutyl-1-methylxanthine was evident as soon as 3 min after addition of the adenosine analogue and maximum 10 min after its addition. As observed by others, [32P]Pi incorporation into phospholipids was increased by the alpha 1-selective agonist methoxamine. The stimulatory effect of methoxamine occurred with a time course similar to that of N6-phenylisopropyladenosine and was present at nearly equal magnitude in the absence or presence of 3-isobutyl-1-methylxanthine. The inhibitory effects of 3-isobutyl-1-methylxanthine and adenosine deaminase on phospholipid labelling are attributed to blockade of the action, or to the enzymic removal, of adenosine formed in and released from the fat-cells during their incubation. Supporting this view is the selective reversal of the actions of 3-isobutyl-1-methylxanthine and of adenosine deaminase by N6-phenylisopropyladenosine. These findings suggest an important role for endogenous adenosine in regulation of phospholipid turnover in adipocytes.     

The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+.

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated 'second messenger' which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular 'messenger' for controlling metabolic processes during insulin action.     

Activation of (arachidonyl) phosphatidylinositol turnover in rabbit neutrophils by the calcium ionophore A23187.

The addition of the Ca2+ ionophore A23187 to rabbit neutrophils stimulated [14C]arachidonic acid incorporation into phosphatidylinositol and lysosomal enzyme secretion. A significant increase in phosphatidylinositol labelling was observed after a 2 min exposure to 0.1 microM-ionophore A23187. Maximum increases in rate of labelling were obtained with 1 microM-ionophore A23187 within 1 min, declining to basal rates after 15 min. Similarly, maximum rate of enzyme release occurred during the first 2 min of exposure to ionophore and release was essentially complete by 15 min. Threshold and peak ionophore A23187 concentrations for stimulating both processes were identical. In contrast with the specificity of phosphatidylinositol labelling induced by 1 microM-ionophore A23187 in the absence of cytochalasin B, ionophore also significantly stimulated labelling of phosphatidylserine and phosphatidylethanolamine in the presence of cytochalasin B. With a threshold ionophore concentration (0.1 microM), the enhanced incorporation of arachidonate was relatively specific for phosphatidylinositol in cytochalasin-treated cells. Ionophore A23187 did not accelerate labelling of phosphatidylinositol by [14C]acetate or [14C]glycerol, indicating that ionophore A23187 does not stimulate phosphatidylinositol synthesis de novo, although it did promote [14C]palmitate and [32P]Pi incorporation into neutrophil phosphatidylinositol. However, the increase in phosphatidylinositol labelling with these latter precursors was generally slower in onset and much more modest in magnitude than that observed with arachidonic acid. These results support the hypothesis that a Ca2+-dependent phospholipase, which acts on the arachidonate moiety of phosphatidylinositol, is responsible for initiating at least certain of the membrane events coupled to the release of secretory product from the neutrophil.     

The relationship of calcium to receptor-controlled stimulation of phosphatidylinositol turnover. Effects of acetylcholine, adrenaline, calcium ions, cinchocaine and a bivalent cation ionophore on rat parotid-gland fragments.

The possibility that Ca2+ ions are involved in the control of the increased phosphatidylinositol turnover which is provoked by alpha-adrenergic or muscarinic cholinergic stimulation of rat parotid-gland fragments has been investigated. Both types of stimulation provoked phosphatidylinositol breakdown, which was detected either chemically or radiochemically, and provoked a compensatory synthesis of the lipid, detected as an increased rate of incorporation of 32Pi into phosphatidylinositol. Acetylcholine had little effect on the incorporation of labelled glycerol, whereas adrenaline stimulated it significantly, but to a much lower extent than 32P incorporation: this suggests that the response to acetylcholine was entirely accounted for by renewal of the phosphorylinositol head-group of the lipid, but that some synthesis de novo was involved in the response to adrenaline. The responses to both types of stimulation, whether measured as phosphatidylinositol breakdown or as phosphatidylinositol labelling, occurred equally well in incubation media containing 2.5 mm-Ca2+ or 0.2 mm-EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid]. Incubation with a bivalent cation ionophore (A23187) led to a small and more variable increase in phosphatidylinositol labelling with 32Pi, which occurred whether or not Ca2+ was available in the extracellular medium: this was not accompanied by significant phosphatidylinositol breakdown. Cinchocaine, a local anaesthetic, produced parallel increases in the incorporation of Pi and glycerol into phosphatidylinositol. This is compatible with its known ability to inhibit phosphatidate phosphohydrolase (EC 3.1.3.4) and increase phosphatidylinositol synthesis de novo in other cells. These results indicate that the phosphatidylinositol turnover evoked by alpha-adrenergic or muscarinic cholinergic stimuli in rat parotid gland probably does not depend on an influx of Ca2+ into the cells in response to stimulation. This is in marked contrast with the K+ efflux from this tissue, which is controlled by the same receptors, but is strictly dependent on the presence of extracellular Ca2+. The Ca2+-independence of stimulated phosphatidylinositol metabolism may mean that it is controlled through a mode of receptor function different from that which controls other cell responses. Alternatively, it can be interpreted as indicating that stimulated phosphatidylinositol breakdown is intimately involved in the mechanisms of action of alpha-adrenergic and muscarinic cholinergic receptor systems.     

Possible role of phosphatidylinositol turnover in insulin release from isolated rat pancreatic islets

We have previously reported occurrence of Ca2+-activated, phospholipid-dependent protein kinase (referred as protein kinase C) in the rat pancreatic islets. It has been suggested that unsaturated diacylglycerol which results from hydrolysis of phosphatidylinositol by phospholipase C-like enzyme activates protein kinase C. Therefore, we studied the effect of exogenous phospholipase C on insulin release from isolated islets of rat pancreas. Bacterial phospholipase C enhanced insulin release induced by glucose in a dose dependent manner. The effect, however, was decreased in the islets pretreated with colchicine. Both phospholipase C and glucose caused an increase in 32p incorporation into phosphatidylinositol. These results indicate that phospholipid metabolism is linked to the insulin release mechanism.     

Effects of insulin and protein synthesis inhibitors on phospholipid metabolism, diacylglycerol levels, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes

BC3H-1 myocytes were cultured with 32PO4 for 3 days to label phospholipids to constant specific activity. Subsequent treatment with physiological concentrations of insulin provoked 40-70% increases in 32PO4 levels (reflecting increases in mass) in phosphatidic acid, phosphatidylinositol, and polyphosphoinositides, and, lesser, 20-25% increases in phosphatidylserine and the combined chromatographic area containing phosphatidylethanolamine plus phosphatidylcholine plus phosphatidylcholine. Insulin-induced increases in phospholipids were significant within 5 min and near-maximal at 15-30 min. Comparable rapid insulin-induced increases in [3H]phosphatidylinositol were observed in myocytes prelabeled with [3H]inositol. These insulin effects (as per prolonged pulse-chase experiments) were due to increase phospholipid synthesis rather than decreased phospholipid degradation. Cycloheximide (and puromycin) pretreatment prevented insulin-induced increases in phospholipids and rapidly reversed ongoing insulin effects on phospholipids and pyruvate dehydrogenase activity. Insulin also rapidly increased diacylglycerol levels. These findings suggest that: (a) insulin provokes rapid increases in de novo synthesis of phosphatidic acid and its derivatives, e.g. phosphoinositides and diacylglycerol; (b) protein synthesis inhibitors diminish phospholipid levels in insulin-treated (but not control) tissues by increasing phospholipid degradation (?phospholipase(s) activation); and (c) changes in phospholipids and diacylglycerol may be important for changes in pyruvate dehydrogenase and other enzymatic activities during treatment with insulin and/or protein synthesis inhibitors.     

Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin.

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.     

Anoxic injury accelerates phosphatidylcholine degradation in cultured cardiac myocytes by phospholipase C.

In neonatal cultured cardiac myocytes under normoxic conditions, 32Pi incorporation pattern into various phospholipids, and double-labeling experiments with 32Pi and [3H]methyl choline, suggest that phosphatidylcholine and phosphatidylinositol are turned over rapidly, whereas the turnover of phosphatidylethanolamine is probably much slower. While increased levels of the corresponding lysophospholipids were not found under anoxia, release of diacylglycerol and phosphorylcholine was observed. These data strongly suggest that phospholipase C, and not phospholipase A2, is involved in phospholipid degradation in cultured cardiomyocytes under anoxic conditions.     

Short-term stimulation by adenosine of basal and insulin-induced glycogen synthesis in rat adipose tissue

The effects of adenosine on glycogen metabolism have been studied in isolated fat-pads from epididymal adipose tissue. Adenosine caused a sustained short-term increase in the incorporation of [U-14C]glucose into glycogen, as well as a stimulation of both basal and insulin-induced [1-14C]glucose oxidation. Adenosine produced changes also in the activity of glycogen synthase and phosphorylase, these effects being apparent only when glucose was present in the incubation medium. The addition of adenosine prevented the depressed synthesis of glycogen observed in the presence of dibutyryl cyclic AMP. In the presence of adenosine deaminase, the stimulation by insulin of glycogen synthesis was markedly decreased. The results suggest that adenosine may have a regulatory role on glycogen synthesis by facilitating the glucose transport.     

Development of insulin sensitivity in white adipose tissue during the suckling-weaning transition in the rat. Involvement of glucose transport and lipogenesis.

The changes of insulin responsiveness of white adipose tissue during the suckling-weaning transition in the rat were investigated in vitro on isolated adipocytes. Insulin binding, glucose transport and glucose metabolism in adipocytes from suckling rats and from rats weaned on to a high-carbohydrate (HC) or a high-fat (HF) diet were compared. Despite similar insulin binding, insulin-stimulated glucose transport rate is lower in adipocytes from suckling rats and HF-weaned rats than in adipocytes from HC-weaned rats. Moreover, whereas insulin markedly stimulates glucose metabolism in adipocytes from HC-weaned rats, glucose metabolism is totally unresponsive to insulin in adipocytes from suckling and HF-weaned rats. This insulin resistance is associated with a very low rate of lipogenesis and low activities of acetyl-CoA carboxylase, fatty acid synthase and pyruvate dehydrogenase.     

Expression of C-Type Natriuretic Peptide in the Bovine Pineal Gland

Abstract: The effect of lithium on inositol phospholipid resynthesis in primary cultures of cerebellar granule cells was studied. During activation of phospholipase C by the combined action of a muscarinic agonist and mild depolarization, the levels of inositol phospholipids as well as the inositol phospholipid precursor CMP-phosphatidate appeared highly sensitive to lithium with half-maximal accumulation of CMP-phosphatidate attained at 0.5 m M LiCl, a concentration close to that in the plasma of patients subjected to lithium therapy. Under the same conditions, the effect of lithium on inositol phospholipid metabolism appeared to be mediated by depletion of cytoplasmic free inositol content. This was indicated by the observation that preincubation for 48 h in high extracellular inositol concentrations could decrease or delay the depletion of inositol phospholipids and the accumulation of CMP-phosphatidate induced by 10 m M LiCl. Because even relatively high concentrations of extracellular inositol (500 µ M ) only partially prevented inositol phospholipid depletion, cerebellar granule cells appear to have a comparatively low capacity to accumulate inositol intracellularly, in comparison with other brain cells in culture. The relationship between CMP-phosphatidate accumulation and phospholipase C activity has also been investigated using a range of agonists that have been reported to act on cerebellar granule cells.     

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.     

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.     

CHOLINERGIC STIMULATION OF PHOSPHOLIPID LABELLING FROM [32P]ORTHOPHOSPHATE IN GUINEA-PIG CORTEX SYNAPTOSOMES IN VITRO: SUBSYNAPTOSOMAL LOCALIZATION

Abstract— Subsynaptosomal localization of stimulation of phospholipid labelling by cholinergic agents was investigated. Synaptosomes prepared from guinea-pig cortex were incubated with [³²P]orthophosphate in the presence or absence of 10⁻³ m carbamylcholine. Following incubation and osmotic shock, lysed synaptosomes were subjected to density gradient fractionation. Subsynaptosomal fractions were examined by electron microscopy and analysed for enzyme activities and ³²P-labelled lipids.
In the absence of carbamylcholine, labelled phosphatidate and phosphatidylinositol were recovered in layers and interfaces A, B, C and D formed over 0.9, 1.1, 1.2 and 1.3 m sucrose, with highest amounts of label in fractions C and D for both lipids. Carbamylcholine induced the greatest increment in these two labelled lipids in fractions A and B. This distribution correlated with the presence of acetylcholinesterase activity and membrane ghosts. No correlation was found among the four fractions between the induced increase in labelling and succinic dehydrogenase activity or with the abundance of mitochondria, synaptic vesicles, or cytoplasmic fragments identified by electron microscopy. In contrast with the increases seen in phosphatidylinositol and phosphatidate labelling, carbamylcholine caused a decrease in ³²P-labelling of the polyphosphoinositides, and this effect was seen primarily in the heavier subsynaptosomal fractions, C and D.     

Phospholipid metabolism in Ehrlich ascites tumor cells. I. Turnover rate of phosphatidylinositol.

1. Radioactive precursors, 32 PI, [1-14C]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fraction of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate of [1-14C]glycerol, a higher rate of incorporation of 32Pi into phosphatidylinositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour. 2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection 32Pi and [2-3H]inositol. The incorporation of 32Pi into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32Pi) of phosphatidylinositol only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr. 3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.     

A significant increase of lysophosphatidylinositol 4-phosphate with insulin in isolated rat fat cells

We studied the effects of insulin on the incorporation of 32Pi into phospholipids in rat fat cells. When the cells were treated with insulin, a new radioactive phospholipid was detected on thin layer chromatography. The substance migrated slower than phosphatidylinositol 4,5-bisphosphate and was hardly detectable in the absence of insulin. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 120 microU/ml. Pretreatment of insulin with anti-insulin antibody or the cells with anti-insulin receptor antibody inhibited the effect of insulin. The product of phosphatidylinositol 4-phosphate hydrolyzed by phospholipase A2 was coincided with the substance on thin layer chromatography. Quinacrine inhibited the formation of the substance in a dose-dependent manner. These results suggested that insulin stimulates the generation of lysophosphatidylinositol 4-phosphate through the insulin-receptor interaction.     

Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism.

We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism.