Phosphatidylcholine hydrolysis stimulated by phorbol myristate acetate is mediated principally by phospholipase D in endothelial cells (original) (raw)
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Biochimica et biophysica acta, 1987
The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position ...
FEBS Letters, 1992
The potent protein phosphatase inhibitor, okadaic acid, was used to determine the possible role of protein phosphorylation reaction(s) in phorbol ester‐induced synthesis and hydrolysis of phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts. Okadaic acid (2μM) was found to enhance the stimulatory effects of lower concentrations (2.5–25 nM) of phorbol 12‐myristate 13‐acetate (PMA) on PtdCho synthesis, but not an PtdCho hydrolysis, after treatments for 30–60 min. These data support a view that in fibroblasts PMA simulates only PtdCho synthesis, and not PtdCho hydrolysis, by a protein phosphorylation‐dependent mechanism.
The regulation and cellular functions of phosphatidylcholine hydrolysis
Biochemical Journal, 1990
Mammalian phospholipase C Cell-free preparations from a variety of rat tissues exhibit phospholipase C activities that degrade PtdCho [22-26]. Phospholipase C activities of lysosomal origin with acid pH optima also hydrolyse phosphatidylethanolamine (PtdEtn), phosphatidylinositol and phosphatidylglycerol [22]. PtdChocleaving phospholipases C with alkaline pH optima have been detected in rat brain cytosol [23] and rat liver membranes [24]. Cell-free preparations from endothelial cells also degrade exogenous PtdCho [25,26]. More recently, phospholipases C that utilize PtdCho as substrate have been partially purified from dog heart cytosol [27], bull seminal plasma [28] and promonocytic U937 cells [29]. Utilizing exogenous phospholipids as substrates, these activities exhibit neutral pH optima and do not hydrolyse Vol.
Biochimica Et Biophysica Acta-molecular and Cell Biology of Lipids, 2003
Phosphatidylalcohols, such as phosphatidylethanol (PEth), are formed from phosphatidylcholine in the presence of a primary alcohol (e.g., ethanol). This 'transphosphatidylation' reaction is used as specific phospholipase D (PLD) assay. Accumulation of PEth in tissues is recognized as a reliable measure of PLD activity, as PEth is allegedly metabolically stable. The general validity of this assumption was reinvestigated in isolated rat heart, small intestine and brain slices. The half-times of 3 H-PEth degradation (labelled with 3 H-myristic acid and preformed by ethanol exposure for 30 min) were about 1 h in heart and small intestine, but 17 h in brain. As the formation of PEth is superimposed by simultaneous degradation, a mathematical model was established to calculate the differences between 'true' and 'apparent' PEth formation. As expected, this difference was relevant in heart and intestine, but not in brain tissue. For example, ischemia in the perfused heart for 30 min reversibly blocked PEth degradation and seemingly enhanced PEth formation; the block was reversed by ischemic preconditioning (IPC) and by pretreatment with diazoxide, an opener of mitochondrial K ATP channels. In conclusion, PEth degradation in heart was energy-dependent and rapid, which, when ignored, may lead to misinterpretation of PEth values with respect to PLD activity. D
2010
Ten to fifty micromoles of palmitoyl L carnitine (PC) or myristoyl D,L carnitine (MC) evoke a high amplitude elevation of cytosolic calcium level ([Ca 2+ ] i ), hypercontraction and cell death in the primary culture of rat ventricular myocytes. The lag period of this effect varies within 2-8 min and depends on the mitochondrial capacity to accumulate Ca 2+ . Maximal level of Ca 2+ , attainable at the end of the lag period, depends on calcium concentration in the external medium and is mediated by plasma membrane nonspecific permeability. Preincubation of cardiomyocytes with the inhibitors of phospholipase C, cytosolic phospholi pase A 2 and/or Ca 2+ /calmodulin dependent protein kinase II prevents cell death, increases lag period dura tion and reduces maximal [Ca 2+ ] i . Both PC and MC, even at low concentrations (1-5 µM), dramatically increase the frequency of Ca 2+ sparks and Ca 2+ waves in cardiomyocytes and promote the formation of sus tained microdomains with elevated calcium concentration. We discuss possible mechanisms of Ca 2+ -micro domain formation, where the "vicious circle" of Ca 2+ dependent phospholipases activation may arise. The "vicious circle" with combined autocatalytic action of Ca 2+ dependent phospholipases may be implicated in hydrolysis of membrane phosphatidylcholine and subsequent induction of nonselective permeability for Na + and Ca 2+ (lipid pore).
Biochemical Journal, 1993
The antagonization of phorbol 12-myristate 13-acetate (PMA)-stimulated phosphatidylcholine (PtdCho) biosynthesis by the phospholipid analogue hexadecylphosphocholine (HePC) in MDCK cells was investigated and compared with the corresponding influence in HeLa cells. In both cell lines, PMA-stimulated PtdCho biosynthesis was antagonized by 50 microM HePC. However, subsequent experiments provided evidence that PMA enhances PtdCho biosynthesis by at least two mechanisms: (i) by stimulation of choline uptake and (ii) by translocation of CTP:choline phosphate cytidylyltransferase to membranes. In MDCK cells, 5 nM PMA caused a 4-fold increase in [methyl-3H]choline incorporation into PtdCho, which was paralleled by an approx. 2-fold stimulation of choline uptake. These data indicate that choline uptake might play an important role in the regulation of PtdCho biosynthesis in this cell line, especially since we could not detect any significant increase in membrane-bound cytidyltransferase acti...
The Biochemical journal, 1992
Lysophosphatidic acid (LPA) is a simple phospholipid that possesses hormone- and growth-factor-like properties. LPA initiates its action by inducing GTP-dependent phosphoinositide hydrolysis and inhibiting adenylate cyclase [van Corven, Groenink, Jalink, Eichholtz & Moolenaar (1989) Cell 59, 45-54]. Here we show that LPA stimulates rapid breakdown of phosphatidylcholine (PC) in Rat-1 fibroblasts. LPA-induced PC breakdown occurs through activation of phospholipase D (PLD), as measured by the formation of free choline and phosphatidic acid and by transphosphatidylation in the presence of butan-1-ol. LPA also stimulates generation of diacylglycerol, but there is no detectable formation of phosphocholine, suggesting that a PC-specific phospholipase C (PLC) is not involved. The response to LPA was compared with that to endothelin, a potent inducer of phospholipid hydrolysis but a poor mitogen for Rat-1 cells. Our results indicate that: (1) LPA is less efficient than endothelin in inducin...
Journal of Neuroscience Research, 1992
It was shown that in LA-N-2 cells prelabeled with [3H-methyl]choline for 24 hr (Singh et al.: Mol Chem Neuropathol 14:53-66, 1991) the major intracellular and extracellular hydrophilic compound was phosphorylcholine. LA-N-2 cells were labeled with [14Cmethyllcholine for 24 hr, harvested, and incubated in HepeslBSAlsaline buffer for varying periods of time. The radioactive compound present in the cytosol and released into Hepes/BSA/saline buffer medium in the presence or absence of TPA was phosphorylcholine. There was a gradual increase in the appearance of radioactivity in the medium and this corresponded to a gradual decline in the radioactivity present in the cytosolic compartment with a statistically significant P value of <.005. Identical results were obtained with prelabeled cells subsequently incubated with TPA. There was no significant change in the amount of radioactivity associated with lipid suggesting that the phosphorylcholine may be released directly from the cytosolic compartment into the medium rather than originating through a phospholipase-C catalyzed hydrolysis of phosphatidylcholine. This possibility received support from experiments in which cells were electropermeabilized in the presence of radioactive phosphorylcholine. It was found that the introduced ['4C]phosphorylcholine was released intact into the incubation medium from the cytosolic compartment.