guanosine-triphosphate and phosphatidylethanol

An oleate dependent form of phospholipase D is present in rat brain neuronal nuclei and both the hydrolytic and transphosphatidylation activities measured. Several acidic phospholipids were found to inhibit this activity in a dose dependent manner. The IC50 values varied from 3.5 microM for PIP2 to 200 microM for phosphatidic acid. The hydrolysis of PIP2 by phospholipase C would be expected to result in the disinhibition of the oleate dependent phospholipase D activity.

Topics: Adenosine Triphosphate; Animals; Brain; Cardiolipins; Cell Nucleus; Glycerophospholipids; Guanosine Triphosphate; Isoenzymes; Neurons; Oleic Acid; Oleic Acids; Phosphatidic Acids; Phosphatidylglycerols; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylserines; Phospholipase D; Phospholipids; Rats; Type C Phospholipases

ADP-ribosylation factor (ARF), a small GTPase required for vesicle formation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [14C]acetate for 72 h and, after disruption, fractionated on a linear sucrose gradient. ARF1-regulated PLD activity in each fraction was assessed by measurement of phosphatidylethanol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and plasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-dependent PLD activity in endomembranes and plasma membrane, and also identified an additional activity in the cytosol. In formyl-Met-Leu-Phe-stimulated cells, PLD activity as assessed by phosphatidylethanol formation was also associated with both the plasma membrane and endomembranes. Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), the distributions of inositol lipids and the kinases responsible for lipid phosphorylation were examined. PIP2 was highly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 synthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membrane as the major site of PIP2 production. However, endomembranes possessed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that:(1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments to function as a cofactor for ARF-regulated PLD, and (3) in intact cells, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.

Topics: 1-Phosphatidylinositol 4-Kinase; Acetates; ADP-Ribosylation Factor 1; ADP-Ribosylation Factors; Biomarkers; Cell Fractionation; Cell Membrane; Cells, Cultured; Centrifugation, Density Gradient; Chromatography, Thin Layer; Electrophoresis, Polyacrylamide Gel; Glycerophospholipids; Golgi Apparatus; GTP-Binding Proteins; Guanosine Triphosphate; N-Formylmethionine Leucyl-Phenylalanine; Neomycin; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylinositols; Phospholipase D; Phospholipids; Phosphotransferases (Alcohol Group Acceptor)

In dibutyryl-cAMP-differentiated HL-60 cells, histamine H1 and formyl peptide receptors mediate increases in the cytosolic Ca2+ concentration ([Ca2+]i) via pertussis toxin-sensitive G proteins of the Gi family. We compared the effects of 2-(3-chlorophenyl)-histamine (CPH) [2-[2-(3-chlorophenyl)-1H-imidazol-4-yl] ethanamine], one of the most potent and selective H1 receptor agonists presently available, with those of histamine and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in these cells. CPH increased [Ca2+]i through Ca2+ mobilization and Ca2+ influx. Unlike histamine-induced rises in [Ca2+]i, those induced by CPH were not desensitized in a homologous manner, and there was no cross-desensitization between CPH and histamine. Like fMLP, CPH activated phospholipases C and D, tyrosine phosphorylation, superoxide anion formation, and azurophilic granule release. The effects of CPH on [Ca2+]i, phospholipase D, and superoxide anion formation were inhibited by pertussis toxin. CPH and fMLP stimulated high affinity GTP hydrolysis by Gi proteins in HL-60 membranes. They also enhanced binding of guanosine-5'-O-(3-thio)triphosphate and GTP azidoanilide to, and cholera toxin-catalyzed ADP-ribosylation of, Gi protein alpha subunits. Histamine receptor antagonists did not inhibit the stimulatory effects of CPH, and CPH did not reduce fMLP binding in HL-60 membranes. Our data suggest that CPH activates Gi proteins in HL-60 cells through a receptor agonist-like mechanism that is, however, independent of known histamine receptor subtypes and formyl peptide receptors. CPH may be an agonist at an as yet unknown histamine receptor subtype or, by analogy with other cationic-amphiphilic substances, may activate G proteins directly. Future studies will have to take into consideration the fact that CPH, in addition to activating H1 receptors, may show other, most unexpected, stimulatory effects on G protein-mediated signal transduction processes.

Topics: Calcium; Cell Membrane; Enzyme Activation; Glucuronidase; Glycerophospholipids; GTP-Binding Proteins; Guanosine Triphosphate; Histamine; Histamine Agonists; Humans; In Vitro Techniques; L-Lactate Dehydrogenase; Manganese; N-Formylmethionine Leucyl-Phenylalanine; Pertussis Toxin; Phosphatidic Acids; Phosphatidylinositols; Phosphotyrosine; Receptors, Histamine H1; Respiratory Burst; Superoxides; Tumor Cells, Cultured; Tyrosine; Virulence Factors, Bordetella

Occupancy of chemotactic peptide receptors leads to rapid initiation of phospholipase D (PLD) activity in intact dimethylsulfoxide-differentiated HL-60 granulocytes (Pai, J.-K, Siegel, M.I., Egan, R.W., and Billah, M.M. (1988) J. Biol. Chem. 263, 12472). To gain further insight into the activation mechanisms, PLD has been studied in cell lysates from HL-60 granulocytes, using 1-0-alkyl-2-oleoyl-[32P]phosphatidylcholine (alkyl-[32P]PC), 1-0-[3H]alkyl-2-oleoyl-phosphatidylcholine [( 3H]alkyl-PC) and [14C]arachidonyl-phospholipids as substrates. In the presence of Ca2+ and GTP gamma S, post-nuclear homogenates degrade alkyl-[32P]PC to produce 1-0-alkyl-[32P]phosphatidic acid (alkyl-[32P]-PA), and in the presence of ethanol, also 1-0-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). By comparing the 3H/32P ratios of PA and PEt to that of PC, it is concluded that PA and PEt are formed exclusively by a PLD that catalyzes both hydrolysis and transphosphatidylation between PC and ethanol. Furthermore, PC containing either ester- or ether-linkage at the sn-1 position is degraded in preference to phosphatidylethanolamine and phosphatidylinositol by PLD in HL-60 cell homogenates. It is concluded that HL-60 granulocytes contain a PC-specific PLD that requires both Ca2+ and GTP for activation.

Topics: Calcium; Glycerophospholipids; Granulocytes; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Phosphatidic Acids; Phosphatidylcholines; Phospholipase D; Phospholipases; Substrate Specificity; Thionucleotides; Tumor Cells, Cultured

A cell-free system for the synthesis of phosphatidylalcohols was developed in sonicates of HL-60 cells. With [32P]phosphatidylcholine as the exogenous substrate, both phosphatidylethanol and phosphatidylglycerol were formed through a phospholipase D-catalyzed transphosphatidylation of ethanol and glycerol, respectively. The transphosphatidylation by phospholipase D was stimulated in vitro by 12-O-tetradecanoylphorbol-13-acetate (TPA) and required the addition of ATP for an optimal response. GTP-gamma-S, an activator of G protein systems, also stimulated the process by an independent mechanism. It is postulated that the stimulation of phospholipid metabolism through phospholipase D activation represents an important mechanism whereby TPA might modulate intracellular signal generating systems or influence the activity of membrane-bound proteins by altering their lipid environment.

Topics: Cell Fractionation; Cell Line; Enzyme Activation; Glycerophospholipids; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Phosphatidic Acids; Phosphatidylglycerols; Phospholipase D; Phospholipases; Tetradecanoylphorbol Acetate; Thionucleotides; Ultracentrifugation

Vasopressin, angiotensin II and epinephrine elicited the accumulation of phosphatidylethanol in rat hepatocytes exposed to ethanol and of phosphatidate in the absence of ethanol. When isolated liver plasma membranes were exposed to ethanol, GTP gamma S stimulated the production of phosphatidylethanol whereas phosphatidate was formed in the absence of ethanol. With increasing ethanol concentrations, phosphatidate formation declined whereas phosphatidylethanol production increased. These findings suggest that rat hepatocytes possess a hormone-dependent phospholipase D activity that can also catalyze the formation of phosphatidylethanol.

Topics: Angiotensin II; Animals; Calcium; Cell Membrane; Enzyme Activation; Epinephrine; Ethanol; Glycerophospholipids; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Liver; Male; Phosphatidic Acids; Phospholipase D; Phospholipases; Rats; Thionucleotides; Vasopressins