Metabolism of Phospholipids in Peripheral Nerve from Rats with Chronic Streptozotocin-induced Diabetes Increased Turnover of Phosphatidylinositol-4,5-Bisphosphate

The effect of chronic streptozotocin-induced diabetes on phospholipid metabolism in rat sciatic nerve in vitro was investigated. In normal nerve incubated for 2 h in Krebs-Ringer-bicarbonate buffer containing [32P]orthophosphate, radioactivity was primarily incorporated into phosphatidylinositol-4,5-bisphosphate and phosphatidylcholine. Smaller amounts were present in phosphatidylinositol-4-phosphate, phosphatidylinositol, and phosphatidic acid. As compared to controls, phosphatidylinositol-4,5-bisphosphate in nerves from animals made diabetic 2, 10, and 20 weeks earlier accounted for 30-46% more of the isotope, expressed as a percentage, incorporated into all phospholipids. In contrast, the proportion of radioactivity in phosphatidylcholine decreased by 10-25%. When the results were expressed as the quantity of phosphorus incorporated into phospholipid, only phosphatidylinositol-4,5-bisphosphate displayed a change. The amount of isotope which entered this lipid increased 60% and 67% for 2- and 10-week diabetic animals, respectively. Increased phosphatidylinositol-4,5-bisphosphate labeling was observed when epineurial-free preparations were used or when the composition of the incubation medium was varied. Sciatic and caudal nerve conduction velocities were decreased after 10 and 20 weeks but were unchanged after 2 weeks. We conclude that an increase in the turnover of phosphatidylinositol-4,5-bisphosphate in sciatic nerve from streptozotocin-diabetic rats appears relatively early and persists throughout the course of the disease. This metabolic alteration may be related to a primary defect responsible for the accompanying deficient peripheral nerve function.     

Epidermal growth factor stimulates phosphatidylinositol turnover for ten hours in A431 cells without activation of protein kinase C

Epidermal Growth Factor stimulated phosphatidylinositol turnover in A431 cells for up to ten hours. There was an increase in phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate and phosphatidic acid at all time points. The effects on the inositol phosphates were variable. Despite the activation of the phosphatidylinositol cycle, we were unable to demonstrate activation of protein kinase C.     

An electron spin resonance study of the novel radical cation produced during the horseradish peroxidase-catalyzed oxidation of tetramethylhydrazine

Thrombin stimulation of [³²P]-prelabeled platelets induces a rapid decrease of the radioactivity from phosphatidylinositol-4,5-bisphosphate. No significant change is observed in phosphatidylinositol-4-monophosphate. The initial, thrombin-induced decrease of phosphatidylinositol-4,5-bisphosphate is not inhibited by cytochalasin D or by compounds that interfere with the mobilization of Ca²⁺ such as 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the calmodulin-antagonist, trifluoperazine, prostacyclin and cyclic AMP. Our information indicates that the rapid loss of phosphatidylinositol-4,5-bisphosphate is linked to receptor activation and insensitive to Ca²⁺-mobilization.     

Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid

The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [3H]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in [3H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of [3H]inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.     

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.     

IV. Metabolism of inositides and the activation of platelets

Platelet activation is associated with the active metabolism of inositide lipids. Phosphodiesteratic cleavage of phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate is a consequence of receptor-coupled mechanisms. Degredation of phosphatidylinostiol-4,5-biphosphate is Ca²⁺ -insensitive while that of phosphatidylinositol requires Ca²⁺. The phosphodiesteratic breakdown of these inositides induces the formation of 1,2-diacylglycerol which is rapidly phosphorylated to phosphatidic acid. These biochemical changes might be related to fundamental mechanisms of amplication involved in the process of platelet activation. Phosphatidic acid constitutes an ubiquitous marker for the action of a wide variety of platelet stimuli.     

Separation of phosphatidylinositols and other phospholipids by two-step one-dimensional thin-layer chromatography

A quick and efficient thin-layer chromatographic procedure is described for the separation of phosphatidylinositol-4,5-bisphosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol, phosphatidylcholine, phosphatidic acid, and phosphatidylethanolamine. The method involves development of the phospholipids successively in two different solvent systems but in the same direction. The method is simple, reproducible, and gives good resolution of the six different phospholipids tested. About 8-10 32P-labeled phospholipids isolated from rat hepatocytes were separated by this method; the six mentioned above were identified. Thus, the technique has potential application for both qualitative and quantitative analysis of phospholipid mixtures, such as the phosphoinositides, in cell or tissue extracts.     

Role of the bilayer in the shape of the isolated erythrocyte membrane

Summary The determinants of cell shape were explored in a study of the crenation (spiculation) of the isolated erythrocyte membrane. Standard ghosts prepared in 5mm NaP_i (pH 8) were plump, dimpled disks even when prepared from echinocytic (spiculated) red cells. These ghosts became crenated in the presence of isotonic saline, millimolar levels of divalent cations, 1mm 2,4-dinitrophenol or 0.1mm lysolecithin. Crenation was suppressed in ghosts generated under conditions of minimal osmotic stress, in ghosts from red cells partially depleted of cholesterol, and, paradoxically, in ghosts from red cells crenated by lysolecithin. The susceptibility of ghosts to crenation was lost with time; this process was potentiated by elevated temperature, low ionic strength, and traces of detergents or chlorpromazine.In that ghost shape was influenced by a variety of amphipaths, our results favor the premise that the bilayer and not the subjacent protein reticulum drives ghost crenation. The data also suggest that vigorous osmotic hemolysis induces a redistribution of lipids between the two leaflets of the bilayer which affects membrane contour through a bilayer couple mechanism. Subsequent relaxation of that metastable distribution could account for the observed loss of crenatability.     

Phosphatidic acid is a specific activator of phosphatidylinositol-4-phosphate kinase.

The lipid dependence of phosphatidylinositol-4-phosphate (PIP) kinase purified from bovine brain membranes was investigated. In the assay used, PIP-Triton X-100 micelles containing the lipid to be tested were presented to the enzyme. Under these conditions, phosphatidic acid (PA) stimulated the enzyme activity in a concentration-dependent manner up to 20-fold when an equal molar ratio of PA to PIP was attained. Stimulation by PA was highly specific; other lipids including lyso-PA and dicetylphosphate had a relatively small effect. The activation by PA was completely suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). To investigate the effect of PA on PIP kinase activity in natural membranes, endogenous PA was generated in rat brain synaptosomal plasma membranes by incubation with phospholipase D. Subsequent phosphorylation with [gamma-32P]ATP yielded an enhanced labeling of PIP2 but not of PIP in these membranes. These results suggest that PIP kinase activity may be under control of PA levels in membranes. This may have important implications for the regulation of cellular responses by agonist-induced phosphoinositide turnover.     

Anionic phospholipid gradients: an uncharacterized frontier of the plant endomembrane network

Anionic phospholipids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), and its phosphorylated derivatives the phosphoinositides (e.g. phosphatidylinositol-4-phosphate [PI4P] and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]). Although anionic phospholipids are low-abundant lipids, they are particularly important for membrane functions. In particular, anionic lipids act as biochemical and biophysical landmarks that contribute to the establishment of membrane identity, signaling activities, and compartment morphodynamics. Each anionic lipid accumulates in different endomembranes according to a unique subcellular pattern, where they locally provide docking platforms for proteins. As such, they are mostly believed to act in the compartments in which they accumulate. However, mounting evidence throughout eukaryotes suggests that anionic lipids are not as compartment-specific as initially thought and that they are instead organized as concentration gradients across different organelles. In this update, we review the evidence for the existence of anionic lipid gradients in plants. We then discuss the possible implication of these gradients in lipid dynamics and homeostasis, and also in coordinating subcellular activities. Finally, we introduce the notion that anionic lipid gradients at the cellular scale may translate into gradients at the tissue level, which could have implications for plant development.

One-sentence  summaryMounting evidence reveals that anionic lipids exist in gradients within the plant endomembrane network that may have implications on lipid homeostasis and membrane dynamics.     

Inositol lipids and cell stimulation in mammalian salivary gland

The rat parotid salivary gland shows marked alterations in phospholipid metabolism when stimulated by certain agonists. These agonists are those which cause cellular Ca mobilization by activation of muscarinic, alpha-adrenergic or peptidergic (substance P) receptors. The phospholipid changes apparently reflect the activation of a phosphoinositide-phosphatidic acid cycle, the precise pathways of which are not known with certainty. The observed effects include (1) an increased labelling by 32PO4 of phosphatidylinositol and phosphatidic acid, (2) net synthesis of phosphatidic acid, (3) net breakdown of phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate. These effects apparently do not require the presence of extracellular Ca or the release of internal Ca and cannot be produced by the artificial introduction of Ca into the cytosol with Ca ionophores. These findings are consistent with the view that a receptor-mediated alteration in phosphoinositide metabolism represents an early step in the stimulus-response pathway in the parotid acinar cell. It has been suggested that phosphatidic acid synthesis might be of central importance in mediating Ca influx and that PIP2 breakdown might play a role in activation of Ca release. Evidence for these latter ideas is for the present largely circumstantial.     

PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells

A phosphatidylinositol (PI) kinase activity associated with certain protein tyrosine kinases important in cell proliferation phosphorylates the 3' hydroxyl position of PI to produce phosphatidylinositol-3-phosphate (PI-3-P). Here we report that, in addition to PI-3' kinase activity, anti-phosphotyrosine (alpha-P-tyr) immunoprecipitates from platelet-derived growth factor (PDGF)-stimulated smooth muscle cells (SMC) contain lipid kinase activities that utilize the substrates phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). These activities are absent in alpha-P-tyr immunoprecipitates from quiescent SMC. The product of PI-4-P phosphorylation appears to be phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), a lipid not previously reported. The product of PI-4,5-P2 phosphorylation is phosphatidylinositol-trisphosphate (PIP3). PI-3-P was detected in quiescent SMC and increased only slightly in response to PDGF. PIP3 and the putative PI-3,4-P2 appeared only after the addition of mitogen. Both the temporal production of these novel phospholipids after PDGF stimulation and the observation of the enzymatic activities that produce them in alpha-P-tyr immunoprecipitates suggest that these phospholipids are excellent candidates for mediators of the PDGF mitogenic response.     

Activation of human platelets by ADP causes a rapid rise in cytosolic free calcium without hydrolysis of phosphatidylinositol-4,5-bisphosphate

Phosphatidylinositol-4,5-bisphosphate decreased 40% within 10 seconds after the addition of thrombin to platelets. This thrombin-induced loss was accompanied by a corresponding increase of inositol phosphates. In contrast, within the first 60 seconds after exposure of platelets to ADP there was no detectable change in the amounts of phosphatidylinositol-4,5-bisphosphate or inositol phosphates. Both thrombin and ADP, however, caused a very rapid rise of cytosolic free calcium, as measured by Quin-2. The magnitude of this rise of calcium was similar for the two agonists. These results suggest that in platelets, agonist stimulation may lead to increased cytosolic free calcium independently of phosphatidylinositol-4,5-bisphosphate degradation.     

Regulation of type II phosphatidylinositol phosphate kinase by tyrosine phosphorylation in bovine rod outer segments

Type II phosphatidylinositol phosphate kinase (PIPKII) is an enzyme responsible for the synthesis of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) from phosphatidylinositol-5-phosphate (PI-5-P). In this study, we demonstrate the presence of PIPKII alpha in bovine photoreceptor rod outer segments (ROS) and the involvement of tyrosine phosphorylation in the regulation of its activity. PIPKII activity in bovine ROS was verified by the preferential conversion of synthetic dipalmitoyl PI-5-P to PI-4,5-P(2), lack of effect of phosphatidic acid, inhibition by heparin, immunoreaction with an anti-PIPKII alpha antibody on Western blots, and immunocytochemical localization in bovine and rat ROS by anti-PIPKII alpha. Immunoprecipitates of bovine ROS with the anti-PIPKII alpha antibody possessed PIPK enzymatic activity and preferentially used PI-5-P as substrate for PI-4,5-P(2) biosynthesis. The activity of PIPKII was greatly increased under conditions favoring tyrosine phosphorylation in ROS, and PIPKII activity was immunoprecipitated with anti-phosphotyrosine (anti-PY) antibodies from tyrosine phosphorylated ROS. Preincubation of ROS with tyrosine kinase inhibitors almost abolished the kinase activity in the anti-PY immunoprecipitates. Immunoblot analysis showed that PIPKII alpha was present in anti-PY immunoprecipitates from phosphorylated ROS but not from nonphosphorylated controls. We conclude that PIPKII alpha is present in ROS and that its activity is regulated by tyrosine phosphorylation.     

Dioctanoylglycerol and phorbol diesters enhance phosphorylation of phosphoprotein B-50 in native synaptic plasma membranes

The short chain diacylglycerol, 1,2-dioctanoylglycerol, at concentrations of 100-300 microM stimulated phosphorylation of the nervous system-specific membrane protein B-50 (Mr 48 kDa, IEP 4.5) in isolated synaptic plasma membranes both in the presence and absence of exogenous protein kinase C. Comparable enhancement of histone phosphorylation by purified protein kinase C was achieved with 1 microM neutral lipid. Phorbol dibutyrate was 100 times more potent than the diacylglycerol in stimulating endogenous B-50 kinase in the membranes, whereas 4-alpha-phorbol was without effect. These results further confirm that B-50 is phosphorylated physiologically by a C kinase. Our data are consistent with a negative feedback mechanism in which generation of 1,2-diacylglycerol by enhanced phosphatidylinositol-4,5-bisphosphate hydrolysis could stimulate B-50 phosphorylation, thereby diminishing phosphatidylinositol-4-phosphate kinase activity and decreasing phosphatidylinositol-4,5-bisphosphate biosynthesis.     

Onset of neurophysin self-association upon neurophysin/neuropeptide hormone precursor biosynthesis

32P-Labelled washed rabbit platelets were incubated with 0.6 nM platelet activating factor (PAF-acether), giving a full aggregation and release response within 30-60 s. The major phospholipid changes observed under these conditions were: (1) An increased labelling of phosphatidic acid (PA) within 10 s and of phosphatidylinositol (MPI) at 30 s, reflecting the activation of the MPI cycle via the cytosolic phospholipase C; (2) an enhancement of phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI) labelling at later incubation times; (3) an early degradation of TPI with a counterbalancing formation of DPI. The latter changes suggest a receptor-mediated stimulation of TPI-phosphomonoesterase, the role of which in the mechanism of platelet activation is discussed.     

A role for diacylglycerol in annexin A7-mediated fusion of lung lamellar bodies

Lung surfactant secretion in alveolar type II cells occurs following lamellar body fusion with plasma membrane. Annexin A7 is a Ca2+-dependent membrane-binding protein that is postulated to promote membrane fusion during exocytosis in some cell types including type II cells. Since annexin A7 preferably binds to lamellar body membranes, we postulated that specific lipids could modify the mode of annexin A7 interaction with membranes and its membrane fusion activity. Initial studies with phospholipid vesicles containing phosphatidylserine and other lipids showed that certain lipids affected protein interaction with vesicle membranes as determined by change in protein tryptophan fluorescence, protein interaction with trans membranes, and by protein sensitivity to limited proteolysis. The presence of signaling lipids, diacylglycerol or phosphatidylinositol-4,5-bisphosphate, as minor components also modified the lipid vesicle effect on these characteristics and membrane fusion activity of annexin A7. In vitro incubation of lamellar bodies with diacylglycerol or phosphatidylinositol-4,5-bisphosphate caused their enrichment with either lipid, and increased the annexin A7 and Ca2+-mediated fusion of lamellar bodies. Treatment of isolated lung lamellar bodies with phosphatidylinositol- or phosphatidylcholine phospholipase C to increase diacylglycerol, without or with preincubation with phosphatidylinositol-4,5-bisphosphate, augmented the fusion activity of annexin A7. Thus, increased diacylglycerol in lamellar bodies following cell stimulation with secretagogues may enhance membrane fusion activity of annexin A7.     

On the shape of human red blood cells interacting with flat artificial surfaces--the 'glass effect'.

The morphology of the human red blood cell (RBC) contained between two flat artificial surfaces has been investigated. Shape transformation from the discocytic into various crenated (echinocytic) states was not caused solely by glass ('glass effect'). Various organic polymers, and mica, were effective, provided the distance (0.1 mm) between the two surfaces was carefully controlled. The discocytic state could only be preserved using moderately hydrophobic glass, extensive dimethylsilylation induced stomatocytes. With washed blood samples crenation occurred in a potassium chloride medium and in the presence of EDTA. Temperature-dependent transformation in the shape of human erythrocytes occurred between two glass surfaces 0.1 mm apart, e.g., in a hemacytometer. With cells in blood diluted directly 200-times with isotonic saline crenation appeared at 32-36 degrees C. A sphero-echinocytic state prevailed at 34 degrees C and outside the temperature range of 32-36 degrees C the RBCs retained the shape of a biconcave disk. Cells responding to the 'glass effect' even at temperatures below the transition region did not respond further at elevated temperatures. The 'glass effect' was found to be dependent on the RBC concentration (hematocrit). Raising this concentration reversibly decreased the degree of crenation. The amount of endogenous albumin present was estimated to be insufficient to cover the exposed glass surfaces with a protein monolayer. With washed cells over a wide concentration range, approximately the same total amount of albumin (serum) had to be present to avoid crenation, as long as observation was performed at a fixed low cell concentration. The effect of albumin was not abolished by gamma-globulin or anti-human albumin IgG. The discocyte-stabilizing influence of albumin is discussed.     

Phospholipase D.

Phospholipase D catalyses the hydrolysis of the phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a free headgroup. Phospholipase D activities have been detected in simple to complex organisms from viruses and bacteria to yeast, plants, and mammals. Although enzymes with broader selectivity are found in some of the lower organisms, the plant, yeast, and mammalian enzymes are selective for phosphatidylcholine. The two mammalian phospholipase D isoforms are regulated by protein kinases and GTP binding proteins of the ADP-ribosylation and Rho families. Mammalian and yeast phospholipases D are also potently stimulated by phosphatidylinositol 4,5-bisphosphate. This review discusses the identification, characterization, structure, and regulation of phospholipase D. Genetic and pharmacological approaches implicate phospholipase D in a diverse range of cellular processes that include receptor signaling, control of intracellular membrane transport, and reorganization of the actin cytoskeleton. Most ideas about phospholipase D function consider that the phosphatidic acid product is an intracellular lipid messenger. Candidate targets for phospholipase-D-generated phosphatidic acid include phosphatidylinositol 4-phosphate 5-kinases and the raf protein kinase. Phosphatidic acid can also be converted to two other lipid mediators, diacylglycerol and lyso phosphatidic acid. Coordinated activation of these phospholipase-D-dependent pathways likely accounts for the pleitropic roles for these enzymes in many aspects of cell regulation.