scholarly journals Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with N-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate

1992 ◽  
Vol 286 (3) ◽  
pp. 693-699 ◽  
Author(s):  
M C Chabot ◽  
L C McPhail ◽  
R L Wykle ◽  
D A Kennerly ◽  
C E McCall
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Water Soluble ◽  
Incubation Mixture ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Agonist Stimulation

The turnover of choline-containing phosphoglycerides (PC) in response to agonist stimulation is well documented in human neutrophils. We have now compared the enzymic pathways of N-formylmethionyl-leucylphenylalanine (fMLP)-, A23187- and phorbol-12-myristate 13-acetate (PMA)-induced diglyceride (DG) and phosphatidic acid (PA) generation in these cells. In order to distinguish between phospholipase C- and D-mediated PC breakdown, human neutrophils were radiolabelled with 1-O-[3H]alkyl-2-acyl-glycero-3-phosphocholine and stimulated in the presence of ethanol or propranolol. The addition of 0.5% ethanol to the incubation mixture resulted in the production of phosphatidylethanol, indicative of phospholipase D activation, in response to all three stimuli. Concomitant with phosphatidylethanol formation was a partial block of PA production. The production of DG was also partially blocked by addition of ethanol. Propranolol (200 microM) was also used to assess the contributions of phospholipases C and D toward DG generation. Inhibition of PA phosphohydrolase by propranolol resulted in the complete abolition of DG generation when neutrophils were stimulated with fMLP. In contrast, propranolol only partially inhibited DG generation in response to A23187 and PMA. These results suggested that DG production in response to fMLP stimulation is mediated via the activation of phospholipase D, whereas A23187- or PMA-induced DG generation may involve more than one pathway. However, examination of the water-soluble choline metabolites produced indicated that phospholipase D was responsible for the production of PA and DG in response to all three stimuli.

scholarly journals Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells

1990 ◽  
Vol 267 (2) ◽  
pp. 479-483 ◽  
Author(s):  
S P Halenda ◽  
A G Rehm
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Calf Serum ◽  
Platelet Activating Factor ◽  
Diacylglycerol Kinase ◽  
Sole Source ◽  
Phospholipid Metabolism ◽  
Ionophore A23187 ◽  
Hel Cells

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide

2011 ◽  
Vol 168 (6) ◽  
pp. 534-539 ◽  
Author(s):  
Nicolás Raho ◽  
Leonor Ramirez ◽  
M. Luciana Lanteri ◽  
Gabriela Gonorazky ◽  
Lorenzo Lamattina ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Acid Production ◽  
Diacylglycerol Kinase

scholarly journals Tyrosine phosphorylation is involved in receptor coupling to phospholipase D but not phospholipase C in the human neutrophil

1992 ◽  
Vol 281 (3) ◽  
pp. 597-600 ◽  
Author(s):  
I J Uings ◽  
N T Thompson ◽  
R W Randall ◽  
G D Spacey ◽  
R W Bonser ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Phospholipase D ◽  
Human Neutrophil ◽  
Human Neutrophils ◽  
Receptor Coupling ◽  
Kinase C

The tyrosine kinase inhibitors ST271, ST638 and erbstatin inhibited phospholipase D (PLD) activity in human neutrophils stimulated by fMet-Leu-Phe, platelet-activating factor and leukotriene B4. These compounds did not inhibit phorbol ester-stimulated PLD, indicating that they do not inhibit PLD per se, but probably act at a site between the receptor and the phospholipase. In contrast, the protein kinase C inhibitor Ro-31-8220 inhibited phorbol 12,13-dibutyrate- but not fMet-Leu-Phe-stimulated PLD activity, arguing against the involvement of protein kinase C in the receptor-mediated activation of PLD. ST271 did not inhibit Ins(1,4,5)P3 generation, but did inhibit protein tyrosine phosphorylation stimulated by fMet-Leu-Phe. The phosphotyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation and stimulated PLD. These results suggest that tyrosine kinase activity is involved in receptor coupling to PLD but not to PtdIns(4,5)P2-specific phospholipase C in the human neutrophil.

scholarly journals Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1676-1683 ◽  
Author(s):  
X Yang ◽  
L Sun ◽  
S Ghosh ◽  
AK Rao
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Platelet Activating Factor ◽  
Ionophore A23187 ◽  
Serotonin Secretion ◽  
Platelet Signaling ◽  
Intracellular Mediators ◽  
Gamma S ◽  
Hydrolysis Of ◽  
Pkc Activation

Signal transduction on platelet activation involves phosphoinositide- specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet- activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5′-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate- induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.

scholarly journals Phosphatidylinositol phosphodiesterase in higher plants

1980 ◽  
Vol 192 (1) ◽  
pp. 279-283 ◽  
Author(s):  
R F Irvine ◽  
A J Letcher ◽  
R M C Dawson
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Higher Plants ◽  
Water Soluble ◽  
Apium Graveolens ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Detectable Activity ◽  
Ph Range

1. The lower regions of the stem of celery (Apium graveolens L.) contain a soluble enzyme that hydrolyses phosphatidylinositol. 2. The lipoidal product of hydrolysis is diacylglycerol, and the water-soluble products are 1:2-cyclic phosphoinositol and phosphoinositol in the approximate proportions of 60% and 40% respectively: this indicates that a phosphodiesterase (phospholipase C-like) activity is cleaving the phosphatidylinositol. 3. The enzyme requires a bivalent cation, Ca2+ being the most effective activator. 4. The enzyme has a pH optimum, depending on conditions of assay, of pH 5.9-6.6 and in this pH range shows no detectable activity against phosphatidylcholine or phosphatidylethanolamine. 5. The activity is stimulated by phosphatidic acid and slightly inhibited (30% at concentrations equimolar with phosphatidylinositol) by phosphatidylcholine. 6. The phosphodiesterase was also detected (but not quantified) in the tips of the flowers in cauliflowers, in outer leaves of onion and in the elongating stem of daffodils. 7. The enzyme's properties are compared with equivalent mammalian enzymes, and its possible role in the catabolism of phosphatidylinositol in higher plants is discussed.

scholarly journals Assessment of receptor-dependent activation of phosphatidylcholine hydrolysis by both phospholipase D and phospholipase C.

1991 ◽  
Vol 2 (4) ◽  
pp. 299-309 ◽  
Author(s):  
T T Dinh ◽  
D A Kennerly
Keyword(s):  
Mast Cells ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Cell Activation ◽  
Immunoglobulin E ◽  
Water Soluble ◽  
Extracellular Medium ◽  
Acid Accumulation ◽  
The Masses ◽  
Hydrolysis Of

Enhancement of cellular phospholipase D (PLD)-1 and phospholipase C (PLC)-mediated hydrolysis of endogenous phosphatidylcholine (PC) during receptor-mediated cell activation has received increasing attention inasmuch as both enzymes can result in the formation of 1,2-diacylglycerol (DAG). The activities of PLD and PLC were examined in purified mast cells by quantitating the mass of the water-soluble hydrolysis products choline and phosphorylcholine, respectively. Using an assay based on choline kinase-mediated phosphorylation of choline that is capable of measuring choline and phosphorylcholine in the low picomole range, we quantitated the masses of both cell-associated and extracellular choline and phosphorylcholine. Activating mast cells by crosslinking its immunoglobulin E receptor (Fc epsilon-RI) resulted in an increase in cellular choline from 13.1 +/- 1.2 pmol/10(6) mast cells (mean +/- SE in unstimulated cells) to levels 5- to 10-fold higher, peaking 20 s after stimulation and rapidly returning toward baseline. The increase in cellular choline mass paralleled the increase in labeled phosphatidic acid accumulation detected in stimulated cells prelabeled with [3H]palmitic acid and preceded the increase in labeled DAG. Although intracellular phosphorylcholine levels were approximately 15-fold greater than choline in unstimulated cells (182 +/- 19 pmol/10(6) mast cells), stimulation resulted in a significant fall in phosphorylcholine levels shortly after stimulation. Pulse chase experiments demonstrated that the receptor-dependent increase in intracellular choline and the fall in phosphorylcholine were not due to hydrolysis of intracellular phosphorylcholine and suggested a receptor-dependent increase in PC resynthesis. When the extracellular medium was examined for the presence of water-soluble products of PC hydrolysis, receptor-dependent increases in the mass of both choline and phosphorylcholine were observed. Labeling studies demonstrated that these extracellular increases were not the result of leakage of these compounds from the cytosol. Taken together, these data lend support for a quantitatively greater role for receptor-mediated PC-PLD compared with PC-PLC during activation of mast cells.

scholarly journals Isolation and enzymic assay of choline and phosphocholine present in cell extracts with picomole sensitivity

1990 ◽  
Vol 270 (1) ◽  
pp. 63-68 ◽  
Author(s):  
J J Murray ◽  
T T Dinh ◽  
A P Truett ◽  
D A Kennerly
Keyword(s):  
Phospholipase C ◽  
Phospholipase D ◽  
Tissue Sample ◽  
Water Soluble ◽  
Choline Kinase ◽  
Simple Analysis ◽  
Cell Extracts ◽  
Sodium Tetraphenylboron ◽  
Hydrolysis Of ◽  
Enzymic Assay

Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.

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