Inositol lipids, phosphatidate and diacylglycerol share stearoylarachidonoylglycerol as a common backbone in thrombin-stimulated human platelets.

Gel-filtered human platelets were stimulated with 5i.u. of thrombin/ml for times up to 1 min. The fatty acid composition of inositol-containing phospholipids, phosphatidic acid and diacylglycerol was determined by g.l.c. in control and thrombin-stimulated platelet suspensions. Inositol phospholipids were found to have similar proportions of stearic and arachidonic acids, the sum of these representing 86.6% of the total fatty acids in phosphatidylinositol (PtdIns), 76.9% in phosphatidylinositol 4-phosphate (PtdIns4P) and 85.4% in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. However, arachidonic and stearic acids were less abundant in phosphatidic acid (PtdA) and diacylglycerols in non-stimulated platelets. A transient decrease in the mass of PtdIns(4,5)P2 was observed after 5-10s of thrombin stimulation, followed by an increase after 30s. The amounts of PtdIns4P and PtdIns decreased throughout the experiment. A transient accumulation of stearoylarachidonoylglycerol was observed at 5s, whereas stearoylarachidonoylglycerol 3-phosphate (PtdA) was produced in increasing amounts throughout the experiment. The decrease in inositol-containing phospholipids was not fully compensated for by the production of diacylglycerol or PtdA [or PtdIns(4,5)P2] at 1 min. All the changes in inositol phospholipids, as well as those observed in diacylglycerols and PtdA, were due to a parallel reduction or increase in the contents of stearic and arachidonic acids, with a stoichiometry equal to 1. Taken together, this suggests an interconversion of all these lipids with the utilization of a common backbone, stearoylarachidonoylglycerol. The deacylation of this diacylglycerol could account for up to 4-5nmol of arachidonate/10(9) platelets after 1 min stimulation by thrombin.     

Regulation fo protein kinase C activity by various lipids

Protein kinase C has recently attracted considerable attention because of its importance in the control of cell division, cell differentiation, and signal transduction across the cell membrane. The activity of this enzyme is altered by several lipids such as diacylglycerol, free fatty acids, lipoxins, gangliosides, and sulfatides. These lipids may interact with protein kinase C either directly or through calcium ions and produce their regulatory effect (activation or inhibition) on the activities of the enzymes phosphorylated by this kinase. These processes widen our perspective of the regulation of intercellular and intracelluular communication.Abbreviations used (PK-C) Protein kinase C - (cAMP-PK) cAMP dependent protein kinase - (DAG) diacylglycerol - (PtdSer) phosphatidylserine - (InsP ₃) inositol 1,4,5-trisphosphate - (PtdIns 4,5-P₂) inositol 4,5 bisphosphate - (FFA) free fatty acid - (MBP) myelin basic protein - (ATP) adenosine triphosphate - (GTP) guanine triphosphate - (TPA) 12-tetradecanoylphorbol-13-acetate - (EGF) epidermal growth factor - (PDGF) platelet derived growth factor - (NeuNAc) and N-acetylneuraminic acid     

Protein kinase C stimulates PtdIns-4,5-P2-phospholipase C activity.

The tumour promoter, phorbol ester 12,13-dibutyrate (PDBu), acts on rectal palisadic epithelial cells and mimics the effects of neuroparsin, an antidiuretic neuronal hormone isolated from nervous lobes of the African locust corpora cardiaca. PDBu stimulated Ca2+-dependent phospholipase C (PLC) activity resulting in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production, increased cytosolic free calcium (monitored with the probe indo-1) and rectal fluid resorption. A 15-min pre-treatment with polymyxin B (PMXB), a protein kinase C (PKC) inhibitor acting at the phosphatidylserine (PS) binding site, suppressed PDBu stimulatory effects on free calcium entry and fluid resorption but not on phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) breakdown. On the contrary, bisindolylmaleimide Ro 32-0432 (which inhibits PKC at its ATP binding site) abolished entirely PDBu-stimulated PLC activity. It was concluded that two PKC are involved in transduction of the antidiuretic signal of neuroparsin. One PKC is PMXB sensitive and stimulates biological response after cytosolic free Ca2+ increase, while another PKC, insensitive to the PKC inhibitor, regulates the processes induced by the former PKC. Since PMXB-insensitive PKC exerts a stimulatory effect on PtdIns-4,5-P2-PLC production, this original mechanism may be considered as a new signalling pathway under control of PKC.     

Phosphoinositide hydrolysis by guanosine 5''-[gamma-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes.

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] of turkey erythrocytes were labelled by using either [32P]Pi or [3H]inositol. Although there was little basal release of inositol phosphates from membranes purified from labelled cells, in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) the rate of accumulation of inositol bis-, tris- and tetrakis-phosphate (InsP2, InsP3 and InsP4) was increased 20-50-fold. The enhanced rate of accumulation of 3H-labelled inositol phosphates was linear for up to 20 min; owing to decreases in 32P specific radioactivity of phosphoinositides during incubation of membranes with unlabelled ATP, the accumulation of 32P-labelled inositol phosphates was linear for only 5 min. In the absence of ATP and a nucleotide-regenerating system, no InsP4 was formed, and the overall inositol phosphate response to GTP[S] was decreased. Analyses of phosphoinositides during incubation with ATP indicated that interconversions of PtdIns to PtdIns4P and PtdIns4P to PtdIns(4,5)P2 occurred to maintain PtdIns(4,5)P2 concentrations; GTP[S]-induced inositol phosphate formation was accompanied by a corresponding decrease in 32P- and 3H-labelled PtdIns, PtdIns4P and PtdIns(4,5)P2. In the absence of ATP, only GTP[S]-induced decreases in PtdIns(4,5)P2 occurred. Since inositol monophosphate was not formed under any condition, PtdIns is not a substrate for the phospholipase C. The production of InsP2 was decreased markedly, but not blocked, under conditions where Ins(1,4,5)P3 5-phosphomonoesterase activity in the preparation was inhibited. Thus the predominant substrate of the GTP[S]-activated phospholipase C of turkey erythrocyte membranes is PtdIns(4,5)P2. Ins(1,4,5)P3 was the major product of this reaction; only a small amount of Ins(1:2-cyclic, 4,5)P3 was released. The effects of ATP on inositol phosphate formation apparently involve the contributions of two phenomena. First, the P2-receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) greatly increased inositol phosphate formation and decreased [3H]PtdIns4P and [3H]PtdIns(4,5)P2 in the presence of a low (0.1 microM) concentration of GTP[S]. ATP over the concentration range 0-100 microM produced effects in the presence of 0.1 microM-GTP[S] essentially identical with those observed with 2MeSATP, suggesting that the effects of low concentrations of ATP are also explained by a stimulation of P2-receptors. Higher concentrations of ATP also increase inositol phosphate formation, apparently by supporting the synthesis of substrate phospholipids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Occurrence and functions of the phosphatidylinositol cycle in the myocardium

In the last decade a great deal of attention was awarded to a signal transduction pathway which is utilized primarily by Ca²⁺ mobilizing signal molecules and which involves the hydrolysis of a quantitatively minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) by a PtdIns-specific phospholipase C (PLC). The evidence for the existence of receptor-mediated GTP binding protein-coupled PLC in myocardium and its possible functions are briefly summarized. The minireview is concentrated on the following aspects: 1) cellular localization and synthesis of polyphospho-PtdIns from PtdIns, 2) desensitization of the ₁-adrenergic agonist and endothelin-1 mediated PtdIns responses, 3) oscillatory Ca²⁺ transients initiated by Ptdlns(4,5)P₂ hydrolysis, 4) polyunsaturated fatty acids as constituents of polyphospho-PtdIns and of the protein kinase C activator 1,2-diacylglycerol (DAG), 5) source other than Ptdlns(4,5)P₂ contributing to the stimulated DAG, 6) role of the PtdIns pathway in cardiomyocyte growth and gene expression during the hypertrophic response. (Mol Cell Biochem116: 59–67, 1992)Abbreviations Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P₂ - Phosphatidylinositol 4-monophosphate PtdIns(4)P₄ - Phosphatidylinositol PtdIns - Inositol 1,4,5-triphosphate Ins(1,4,5)P₃ - Inositol 1,3,4,5-tetrakisphosphate Ins(1,3,4,5)P₄ - Inositol 1-monophosphate Ins(1)P - Inositol 1,4-bisphosphate Ins(1,4)P₂ - Inositol Ins - Inositolphosphates InsP_n - Guanine 5'-triphosphate GTP - GTP binding protein G-protein - Phosphatidylinositolspecific phospholipase C PLC - Protein kinase C PKC - 1,2-Diacylglycerol DAG - Monoacylglycerol MAG - cytidyldiphoshate-diacylglycerol CDP-DAG - Sarcolemma SL - Sarcoplasmic reticulum SR - Stearic acid 18:0 - Polyunsaturated fatty acids PUFA - Arachidonic acid 20:4n-6 - Linoleic acid 18:2n-6 - Eicosapentaenoic acid 20:5n-3 - Docosahexaenoic acid 22:6n-3 - Phosphatidic acid PtdOH - Phospholipase D PLD - Phosphatidylcholine PtdChol     

Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements.

The effect of the GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with adenosine 5'-[gamma-thio]triphosphate, and was inhibited by guanosine 5'-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.     

Effect of insulin and insulin-like growth factors I and II on phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate breakdown in liver from humans with and without type II diabetes

We have characterized a plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and a cytosolic phosphatidylinositol (PI)-specific PLC in human liver. Epinephrine, 1 x 10(-5) M, and vasopressin, 1 x 10(-8) M, stimulated PIP2-PLC which was enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). PI-PLC stimulation was not observed by these agents. Insulin and insulin-like growth factors (IGF-I and IGF-II) in the presence and absence of GTP gamma S did not stimulate PIP2-PLC or PI-PLC in plasma membranes and cytosol preparations nor phosphoinositide breakdown in isolated human hepatocytes. Furthermore, serendipitly we found that PIP2-PLC activity was increased in liver membranes from obese patients with type II diabetes when compared to obese and lean controls. We conclude that in human liver, insulin and IGFs are not members of the family of hormones generating inositol trisphosphate (IP3) as a second messenger. Furthermore, the increased PIP2-PLC in diabetic liver may result in: (a) increased intracellular concentrations of IP3 and thus increased Ca2+, which has been postulated to induce insulin resistance; and (b) increased diacylglycerol and thus increased protein kinase C which phosphorylates the insulin receptor at serine residues inactivating the insulin receptor kinase. While the mechanism of increased PIP2-PLC activity in diabetes is unknown, it may initiate a cascade of events that result in insulin resistance.     

Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.

The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.     

Role of a guanine nucleotide-binding regulatory protein in the hydrolysis of phosphatidylinositol 4,5-bisphosphate in a human T cell line

The CD3(T3)/antigen receptor complex appears to function by transducing an antigen signal presented by macrophages into the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. In order to find out how the CD3/antigen receptor complex regulates the hydrolysis of PtdIns(4,5)P2 to diacylglycerol and inositol trisphosphate, we investigated the possible role played by a guanine nucleotide-binding regulatory protein in PtdIns(4,5)P2 hydrolysis in a human T cell leukemia line, JURKAT. JURKAT cells were made permeable to Al3+, F-, GTP, and a nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), by treatment with pseudomonal cytotoxin. In the presence of AlCl3 NaF stimulated the release of inositol phosphates in the cytotoxin-treated JURKAT cells. NaF plus AlCl3 induced increases in inositol tris-, bis-, and mono-phosphates and decreases in PtdIns(4,5)P2, phosphatidylinositol 4-phosphate, and phosphatidylinositol within 5 min after addition to the cytotoxin-treated cells at 37 C. GTP gamma S stimulated, to some extent, polyphosphoinositide hydrolysis in the cytotoxin-treated JURKAT. The cytotoxin-treated JURKAT cells retained the ability to respond to anti-Leu-4 with polyphosphoinositide hydrolysis. It has been shown that Al3+ in the presence of F- modulates the activity of various guanine nucleotide-binding regulatory proteins. Therefore, the results obtained in this study indicate that a guanine nucleotide-binding regulatory protein regulates the polyphosphoinositide breakdown in JURKAT cells by influencing phosphodiesterase activity.     

Regulation of Phospholipase C Activity by Calcium Ions and Guanine Nucleotide in the Normoxic Cat Carotid Body

The carotid bodies (CB) are a paired chemoreceptor organ located at the bifurcation of the common carotid arteries. High O₂ tension suppresses while low tension activates afferent carotid chemoreceptor activity and the chemoreflex ventilatory response in the cat. The intracellular mechanism of chemotransduction is till now unknown. Previously we have shown different activities of phospholipase C (PLC) in normoxic, hypoxic and hyperoxic cat carotid body. Now we have addressed the question whether calcium ions and G-protein could be regulators of the formation of lipid derived messenger molecules in the cat carotid body. To answer this question, the PLC acting against [³H] inositol-phosphatidylinositol (PtdIns) and [³H] inositol-phosphatidylinositol-4, 5-bisphosphate [PtdIns(4,5)P₂] in the cat CB were investigated using labelled phospholipids as a source of the substrate. CB homogenate was used as a source of the enzyme. The results indicate that PLC acting on PtdIns is Ca²⁺-dependent, in contrary to that acting on PtdIns(4,5)P₂ which remains active in the presence of 10 mM EGTA. PtdIns(4,5)P₂-PLC is stimulated by GTPS. In the presence of Ca²⁺, GTPS has a synergistic stimulatory effect. PLC acting on PtdIns is not activated by GTPS. In the presence of calcium ions dopamine and a nonhydrozylable analogue of acetylocholine, carbachol, have a small stimulatory effect of about 30 % on PLC acting on PtdIns(4,5)P₂. GTPS enhances this effect. These results allow us to suggest that there are two pathways of phosphoinositides degradation in the CB, one of them is regulated by calcium ions/PtdIns-PLC/, the other one by G-protein/PtdIns(4,5)P₂-PLC/.     

A rapid effect of thyroxine on accumulation of diacylglycerols and on activation of protein kinase C in liver cells

L-Thyroxine rapidly stimulated the accumulation of diacylglycerols in isolated hepatocytes and in liver when lipids were prelabeled with [14C]oleic acid or with [14C]CH3COONa. Perfusion of the liver of hypothyroid animals with L-thyroxine-containing solution or incubation of liver fragments with the hormone increased the content of diacylglycerols in the liver cells. The increase in [14C]diacylglycerol level in the liver cells was accompanied by a decrease in the level of [14C]phosphatidylcholine, whereas contents of other 14C-labeled phospholipids, such as phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns4P), and phosphatidylinositol-4,5-bis-phosphate (PtdIns(4,5)P2), and of 14C-labeled fatty acids were the same as in the control. The L-thyroxine-induced accumulation of diacylglycerols in hepatocytes was not affected by neomycin but was inhibited by propranolol. Incubation of hepatocytes prelabeled with [14C]oleic acid with L-thyroxine and ethanol (300 mM) was accompanied by generation and accumulation of [14C]phosphatidylethanol that was partially suppressed by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). L-Thyroxine was responsible for the translocation of protein kinase C from the cytosol into the membrane fraction and for a many-fold activation of the membrane-bound enzyme. D-Thyroxine failed to affect the generation of diacylglycerols in hepatocytes and the activity of protein kinase C.     

Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates.

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alternative metabolic fates of phosphatidylinositol produced by phosphatidylinositol synthase isoforms in Arabidopsis thaliana

PtdIns is an important precursor for inositol-containing lipids, including polyphosphoinositides, which have multiple essential functions in eukaryotic cells. It was previously proposed that different regulatory functions of inositol-containing lipids may be performed by independent lipid pools; however, it remains unclear how such subcellular pools are established and maintained. In the present paper, a previously uncharacterized Arabidopsis gene product with similarity to the known Arabidopsis PIS (PtdIns synthase), PIS1, is shown to be an active enzyme, PIS2, capable of producing PtdIns in vitro. PIS1 and PIS2 diverged slightly in substrate preferences for CDP-DAG [cytidinediphospho-DAG (diacylglycerol)] species differing in fatty acid composition, PIS2 preferring unsaturated substrates in vitro. Transient expression of fluorescently tagged PIS1 or PIS2 in onion epidermal cells indicates localization of both enzymes in the ER (endoplasmic reticulum) and, possibly, Golgi, as was reported previously for fungal and mammalian homologues. Constitutive ectopic overexpression of PIS1 or PIS2 in Arabidopsis plants resulted in elevated levels of PtdIns in leaves. PIS2-overexpressors additionally exhibited significantly elevated levels of PtdIns(4)P and PtdIns(4,5)P(2), whereas polyphosphoinositides were not elevated in plants overexpressing PIS1. In contrast, PIS1-overexpressors contained significantly elevated levels of DAG and PtdEtn (phosphatidylethanolamine), an effect not observed in plants overexpressing PIS2. Biochemical analysis of transgenic plants with regards to fatty acids associated with relevant lipids indicates that lipids increasing with PIS1 overexpression were enriched in saturated or monounsaturated fatty acids, whereas lipids increasing with PIS2 overexpression, including polyphosphoinositides, contained more unsaturated fatty acids. The results indicate that PtdIns populations originating from different PIS isoforms may enter alternative routes of metabolic conversion, possibly based on specificity and immediate metabolic context of the biosynthetic enzymes.     

Pathway for the formation of D-3 phosphate containing inositol phospholipids in intact human platelets

We have identified the structure of phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in human platelets. These lipids accounted for less than 2% of the total 32P incorporated into inositol phospholipids in the platelets. All three lipids were labeled in unstimulated platelets, but incorporation of 32P changed rapidly by 15 s after thrombin stimulation, suggesting that they are important in platelet activation. Specific inositol polyphosphate phosphatases were used to both identify the lipid structures and to determine the route of synthesis of these lipids. During 32P labeling and after thrombin stimulation of human platelets, as much as 60% of the total radioactivity present in PtdIns(3,4)P2 was found in the D-4 phosphate and only 35% in the D-3 phosphate indicating that PtdIns(3)P is the precursor of PtdIns(3,4)P2. In addition, the D-5 and D-4 phosphates of PtdIns(3,4,5)P3 each contained 35-40% of the total radioactivity in the molecule compared with only 18-28% in the D-3 position, suggesting that PtdIns(3,4)P2 and not PtdIns(4,5)P2 is the major precursor of this lipid. These results define the predominant pathway for synthesis of these lipids in platelets as PtdIns----PtdIns(3)P----PtdIns(3,4)P2----PtdIns(3,4,5)P3.     

Subcellular localization of inositol lipids in blood platelets as deduced from the use of labelled precursors.

1. By rapid fractionation of blood platelet lysates on Percoll density gradients at alkaline pH (9.6), a very pure plasma-membrane fraction was obtained, as well as discrimination between endoplasmic reticulum and lysosomes. 2. Labelling of intact platelets with [32P]Pi followed by subcellular fractionation showed an exclusive localization of all inositol lipids in the plasma membrane. 3. Preincubation of whole platelets with myo-[3H]inositol in a buffer containing 1 mM-MnCl2 allowed incorporation of the label into PtdIns (phosphatidylinositol) of both plasma and endoplasmic-reticulum membrane, whereas [3H]PtdIns4P (phosphatidylinositol 4-phosphate) and [3H]PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) were exclusively found on the plasma membrane. 4. It is concluded that PtdIns4P and PtdIns(4,5)P2 are exclusively localized in the plasma membrane, whereas PtdIns is present in both plasma and endoplasmic-reticulum membranes. This could provide an explanation for previously reported data on hormone-sensitive and -insensitive inositol lipid pools.     

The substrate specificity of phosphoinositide phospholipase C in rat heart sarcolemma

In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [³H-]phosphatidylinositol 4,5-biphosphate ([³H-]PtdIns(4,5)P ₂) in an optimized assay mixture containing 15 leg SL protein, 100 mM NaCl, 1 mM free Ca²⁺,14 mM Na-cholate and 20 AM [³H-]PtdIns (4,5)P ₂ (400–500 dpm/gm-l) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14±0.55 nmol-mg^–1·2.5 min^–1. The addition of Mg²⁺ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na⁺ and at a low Ca²⁺ concentration (0.3 M), Mg²⁺ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC₅₀0.07 mM). PtdIns4P seemd to be a good substrate for the rat SL PLC (23.07 ± 1.57 nmol·mg^–1·2.5 min^–1) whereas PtdIns was hydrolysed at a very low rate (0.36 ± 0.08 nmol·mg^–1·2.5 min^–1). Unlike PtdIns(4,5)P ₂, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca²⁺ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed. (Mol Cell Biochem116: 27–31, 1992).Abbreviations DAG sn-1,2-diacylglycerol - EGTA ethyleneglycol-O,O-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - Ins(1,4,5)P ₃ inositol 1,4,5-trisphosphate - InsP inositol monophosphate (unidentified isomer) - InsP ₂ inositol bisphosphate (unidentified isomer) - InsP ₃ inositol trisphosphate (unidentified isomer) - InsP _x any inositol phosphate - PLC phospholipase C - PtdIns phosphatidylinositol - PtdIns(4,5)P ₂ phosphatidylinositol 4,5-bisphosphate - PtdIns4P phosphatidylinositol 4-monophosphate - SL sarcolemma     

The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all-trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.     

The haemopoietic growth factors interleukin 3 and colony stimulating factor-1 stimulate proliferation but do not induce inositol lipid breakdown in murine bone-marrow-derived macrophages.

The haemopoietic growth factors interleukin 3 (IL-3) and colony stimulating factor-1 (CSF-1) stimulate the survival and proliferation of murine normal bone-marrow-derived macrophages. To establish whether these growth factors elicit their effects via the hydrolysis of phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] to form the second messengers inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] and diacylglycerol, macrophages were labelled with tracer quantities of [3H]inositol. Treatment of these cells with either IL-3 or CSF-1 did not alter the levels of PtdIns(4,5)P2 or Ins(1,4,5)P3. However, addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) which does not stimulate proliferation in macrophages caused a marked and rapid increase in the levels of Ins(1,4,5)P3, inositol bisphosphate and inositol monophosphate, and a decrease in the amount of PtdIns(4,5)P2. FMLP also evoked a rapid increase in intracellular cytosolic Ca2+ levels, as measured with quin 2 the Ca2+-sensitive fluorescent probe, whereas IL-3 and CSF-1 had no such effect. These results suggest that FMLP stimulates the hydrolysis of PtdIns(4,5)P2 to form the second messenger Ins(1,4,5)P3 which acts to increase the levels of cytosolic Ca2+, and that IL-3- and CSF-1-stimulated proliferation in macrophages is not associated with the formation of PtdIns(4,5)P2-derived second messengers.     

A guanine nucleotide-dependent phosphatidylinositol 4,5-diphosphate phospholipase C in cells transformed by the v-fms and v-fes oncogenes

The metabolism of phosphatidylinositol (PtdIns) was studied in a mink lung epithelial cell line and its subclones transformed by feline sarcoma viruses containing either the v-fms or v-fes oncogenes. The transformed cell lines had a higher rate of PtdIns turnover but did not have elevated levels of phosphorylated PtdIns species or PtdIns kinase activity. Significantly higher specific activities of a guanine nucleotide-activated PtdIns-4,5-diphosphate phospholipase C were detected in both transformed cell lines (F3CL7(v-fes), 55 pmol/min/mg of protein and G2M(v-fms), 18 pmol/min/mg of protein) as compared to the nontransformed parental cell line (CCL64, 2 pmol/min/mg of protein). The guanine nucleotide-stimulated phospholipase C activity was specific for PtdIns-4,5-diphosphate, and the water-soluble hydrolysis product was inositol 1,4,5-triphosphate. Both GTP and nonhydrolyzable GTP analogs activated the phospholipase C, whereas ATP was weakly effective and GDP was inactive. The phospholipase C activity was maximally active in the presence of 9 mM sodium cholate, had a sharp pH optimum of pH 6.5, and was not activated by calcium although hydrolysis was inhibited by high concentrations of EDTA. These data point to enhanced production of diacylglycerol and inositol 1,4,5-triphosphate second messengers in transformed cells due to the activation of guanine nucleotide-dependent PtdIns-4,5-diphosphate-specific phospholipase C and suggest that the generation of aberrant hormonally independent signals is associated with cell transformation by oncogenes encoding tyrosine-specific protein kinases.