Regulation of phospholipase D in HL-60 granulocytes. Activation by phorbol esters, diglyceride, and calcium ionophore via protein kinase- independent mechanisms

It has recently been demonstrated that the chemotactic peptide N-formyl-Met-Leu-Phe activates phospholipase D (PLD) in dimethyl sulfoxide-differentiated HL-60 granulocytes to produce phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) (Pai, J.-K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) J. Biol. Chem. 263, 12472-12477). We now report that biologically active phorbol esters, a cell-permeable diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and calcium ionophore A23187 are also potent inducers of PLD in these HL-60 granulocytes. HL-60 granulocytes have been selectively labeled in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P by incubating the cells with alkyl-[32P]lyso-phosphatidylcholine (PC). When these labeled cells are treated with phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate, OAG, or A23187, alkyl-[32P]PA is formed. Because cellular ATP has not been labeled with 32P, the formation of alkyl-[32P]PA conclusively demonstrates PLD activation by these agents. In the presence of 0.5% ethanol, phorbol esters, OAG, and A23187 also induce formation of alkyl-[32P]PEt, demonstrating that the activated PLD catalyzes transphosphatidylation between the phosphatidyl moiety of the alkyl-[32P]PC and ethanol. Formation of alkyl-[32P]PA and alkyl-[32P]PEt in response to these various agents occurs in a time- and dose-dependent manner and exhibits differential Ca2+ requirements. Based on experiments with both [3H]alkyl-PC and alkyl-[32P]PC, it is concluded that alkyl-PA and alkyl-PEt formed in response to PMA, OAG, or A23187 are derived exclusively from PLD action on alkyl-PC. Furthermore, subthreshold concentrations of PMA (0.5-2.0 nM) or OAG (1.0-25 microM) combined with subthreshold levels of A23187 (15-60 nM) induce the formation of alkyl-[32P]PA and alkyl-[32P]PEt, suggesting that receptor-mediated activation of PLD might involve cooperative interactions between Ca2+ and diglyceride. Although PLD is activated by agents that also activate protein kinase C, the protein kinase C inhibitor, K252a, inhibits PMA-induced protein phosphorylation but causes only partial inhibition of PLD activation. We conclude that phorbol esters, OAG, and A23187 activate PLD in HL-60 granulocytes via protein kinase-independent as well as protein kinase-dependent mechanisms.     

Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes

There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-[32P] lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-[32P]PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-[32P]PA must be formed via PLD-catalyzed hydrolysis of alkyl-[32P]PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Formation of alkyl-[32P]PEt parallels that of alkyl-[32P]PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration. These results strongly support the hypothesis that in HL-60 granulocytes, PEt is formed via PLD-catalyzed transphosphatidylation. Moreover, using HL-60 granulocytes double-labeled by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC, it has been established that the early (30 s) appearance of alkyl-PA is due primarily to PLD, not phospholipase C as previously thought, and that alkyl-PEt is formed exclusively by PLD. These results constitute the first direct evidence for receptor-linked activation of PLD, leading to the generation of PA and PEt in an intact cell system.     

Activation of phospholipase D by chemotactic peptide in HL-60 granulocytes

Activation of phospholipase D (PLD) has been investigated in dimethylsulfoxide differentiated HL-60 granulocytes labeled in endogenous 1-0-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) by incubation with [3H]alkyl-lysoPC. Stimulation of these labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), induces rapid generation of [3H]phosphatidic acid (PA) and slower formation of [3H]diglyceride, suggesting hydrolysis of alkyl-PC by PLD. A unique feature of PLD is its ability to transfer the phosphatidyl moiety of phospholipids to alcohols (transphosphatidylation). This characteristic has been exploited to identify PLD activity. For example, when ethanol is present during stimulation of the HL-60 cells, [3H]phosphatidylethanol (PEt) is formed with a concomitant decrease in [3H]PA. Cells incubated with [32P]orthophosphate to label the terminal phosphate of ATP do not incorporate 32P into PEt, consistent with the [3H]PEt not being synthesized from [3H]diglyceride. In contrast, [3H]PA arises from both PLD and diglyceride kinase activities. Furthermore, PEt synthesis closely parallels PA formation and both are inhibited by an fMLP receptor antagonist, suggesting that both PA and PEt are derived from agonist-stimulated PLD action. These observations are consistent with phospholipase D-catalyzed breakdown of alkyl-PC in fMLP- stimulated granulocytes.     

Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in signal transduction

Human neutrophils have been labeled in 1-O-alkyl-phosphatidylcholine (alkyl-PC) with 32P by incubation with alkyl-[32P]lysoPC. Upon stimulation with the chemotactic peptide, formylMet-Leu-Phe (fMLP), these 32P-labeled cells produce 1-O-alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) and, in the presence of ethanol, 1-O-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Because the cellular ATP contains no 32P, alkyl-[32P]PA and alkyl-[32P]PEt must be formed from alkyl-[32P]PC by phospholipase D (PLD)-catalyzed hydrolysis and transphosphatidylation, respectively. Analyses of the sn-1 bonds by selective hydrolysis and mass measurements reveal that the PA and PEt formed during stimulation contain both ester and ether bonds with distributions similar to that in the endogenous PC. Furthermore, in neutrophils labeled in alkyl-[32P]PC, the specific activities of the diradyl-PA and diradyl-PEt formed during stimulation are similar to that of diradyl-PC. These results demonstrate that the fMLP-induced PLD utilizes diradyl-PC as the major substrate. It is further concluded that, at early times (30 s), PA and PEt are both formed almost exclusively by PLD. Following stimulation with fMLP, neutrophils double-labeled in alkyl-PC by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC generate [3H]alkyl-DG and [32P]orthophosphate [( 32P]PO4) with superimposable kinetics, indicating degradation of PA by a phosphohydrolase. Generation of [3H]alkyl-DG and [32P]PO4 lags behind PA formation and parallels the decline in PA accumulation. In addition, generation of both [3H]alkyl-PA and [3H]alkyl-DG requires extracellular Ca2+ and cytochalasin B. Furthermore, the phosphohydrolase inhibitor, propranolol, decreases both [3H]alkyl-DG and [32P]PO4 while increasing [3H]alkyl-PA and not altering [3H]alkyl-PEt. Moreover, the decreases in DG are accounted for by increases in PA. These results demonstrate that PLD-derived alkyl-PA is degraded by a phosphohydrolase to produce alkyl-DG. DG formed during stimulation contains both ester and ether-linked species and this DG formation is inhibited completely by propranolol. Upon stimulation, alkyl-[32P]PC-labeled neutrophils do not produce [32P]phosphocholine, suggesting that PC is not hydrolyzed by phospholipase C. In addition, PA is formed in amounts sufficient to account for all of the DG formed during stimulation. It is concluded that the DG formed during fMLP stimulation is derived almost exclusively from PC via the PLD/PA phosphohydrolase pathway.     

Activation of phospholipase D in normodense human eosinophils

Normodense human eosinophils have been labeled in 1-0-alkyl-phosphatidylcholine (alkyl-PC) with 32P by incubating isolated cells with alkyl-[32P]lysoPC. Stimulation of these 32P-labeled cells with C5a, A23187 or PMA in the presence of 0.5% ethanol resulted in time- and dose-dependent formation of alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) and alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Because cellular ATP does not contain 32P, alkyl-[32P]PA must have been formed by the hydrolytic action of phospholipase D (PLD) and not by the combined actions of phospholipase C and DG kinase. Regardless of the stimulating agent, alkyl-[32P]PEt formation paralleled that of alkyl-[32P]PA, suggesting that alkyl-PEt was the result of a PLD-catalyzed transphosphatidylation reaction between alkyl-PC and ethanol. These data provide the first definitive proof of receptor- and nonreceptor-mediated activation of PLD in normodense eosinophils derived from human blood.     

Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.     

Phorbol-12-myristate-13-acetate activation of phospholipase D in human neutrophils leads to the production of phosphatides and diglycerides

The contribution of phospholipase D (PLD) to the production of phosphatides (PA) and diglycerides (DG) in phorbol-12-myristate-13-acetate (PMA)-stimulated human neutrophils was studied. Neutrophils were double labeled with 1-O-[3H]alkyl-phosphatidylcholine [( 3H]alkyl-PC) and alkyl-[32P]PC. Upon stimulation with PMA, these cells produced 1-O-alkyl-PA (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidylethanol (alkyl-PEt) both containing 3H and 32P. Lagging behind alkyl-PA and alkyl-PEt formation was the production of 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), suggesting dephosphorylation of alkyl-PA by PA phosphohydrolase (PPH). Furthermore, the PPH inhibitor, propranolol, inhibited the formation of both [3H]alkyl-DG and [32P]PO4, while increasing alkyl-PA levels (containing both 3H and 32P). PMA-induced DG mass accumulation was also inhibited by propranolol. The results of this study demonstrate that PMA activates PLD in neutrophils leading to the generation of PA and that the bulk of the DG mass accumulation is derived from the sequential actions of PLD and PPH on PC.     

Pertussis Toxin-Insensitive G Protein Mediates Carbachol Activation of Phospholipase D in Rat Pheochromocytoma PC12 Cells

In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid-labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]-PEt, which reached a plateau within 1 min, in a concentration-dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12-myristate 13-acetate (PMA), also caused an increase in [3H]-PEt content, which reached a plateau at 30-60 min after exposure, but an inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 microM), blocked PMA-induced [3H]PEt formation by 77%, it had no effect on the CCh-induced formation. These results suggest that mAChR-induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP-binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), also stimulated [3H]PEt formation in intact and digitonin-permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5'-O-(2-thiodiphosphate) significantly inhibited CCh- and GTP gamma S-induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 microM) had no effect on [3H]PEt formation by GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)     

An indirect pathway of receptor-mediated 1,2-diacylglycerol formation in mast cells. I. IgE receptor-mediated activation of phospholipase D

The current studies explore the role of phospholipase D (PLD) in mast cell activation. Although most investigators believe that receptor-mediated accumulation of 1,2-diacylglycerol (DAG) occurs by phospholipase C hydrolysis of phosphoinositides, our previous work indicated a modest role for these substrates and suggested that phosphatidylcholine (PC) is the more likely substrate. PLD cleaves the terminal phosphodiester bond of phospholipids to yield phosphatidic acid (PA), but in the presence of ethanol, it transfers the phosphatidyl moiety of the phospholipid substrate to ethanol producing phosphatidylethanol (PEt); a reaction termed transphosphatidylation. In purified rat mast cells prelabeled with [3H]arachidonic acid, [3H]palmitic acid, or 1-O-[3H]alkyl-lysoPC, a receptor-associated increase in PLD activity was initially suggested by the rapid accumulation of labeled PA, although other mechanisms might be involved. PLD activity was assessed more directly by the production of labeled PEt by PLD-mediated transphosphatidylation in the presence of ethanol. IgE receptor cross-linking resulted in a 3- to 10-fold increase in PLD activity during the 10 min after stimulation, approximately 50% of which occurred during the first two min. PEt formation was dependent on the concentration of ethanol and was maximal at 0.5%. At concentrations of ethanol greater than or equal to 0.2%, receptor-dependent formation of PA was reduced suggesting that the ethanol promoted transphosphatidylation at the expense of hydrolysis. The dose-related decline in PA accumulation seen in the presence of ethanol was similar to ethanol-mediated inhibition of exocytosis suggesting that receptor-mediated PA formation may be of regulatory importance. These observations indicate that PLD-mediated formation of PA occurs in stimulated mast cells and, in conjunction with separate findings of PA phosphohydrolase conversion of PA to DAG in mast cells, suggest that a major mechanism of DAG formation during mast cell activation is PC----PA----DAG.     

Phospholipase D activity in nontransformed and transformed fibroblasts.

Rat embryo fibroblasts (REF52 cells) and the simian virus 40 transformed derivative (WT6 Ag6) were employed to characterize phospholipase D (PLD) activity in normal and transformed cells. In cells prelabeled with [3H]myristic acid or [3H]glycerol and treated with 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 ng/ml medium) or vasopressin (VP, 100 ng/ml medium) in the presence of ethanol, the formation of labeled phosphatidylethanol (PEt) was 3- to 5-fold higher in REF52 cells than in the transformed cells. The transphosphatidylation of phosphatidylcholine (PC) to PEt was further examined in cell-free assay systems. Results demonstrated that the formation of PEt in the cell-free assays was dependent on the mode of substrate presentation and the source of the PC. With endogenous membrane-bound substrate, the formation of [3H]myristoyl-PEt was 5-fold higher in homogenates derived from normal cells as compared to transformed cell homogenates. In experiments using exogenous labeled PC isolated from either REF52 or transformed cells as substrate, cell-free PLD activity differed greatly with regard to the source of the PC. The formation of PEt from REF52-derived PC was approx. 4-fold higher as compared to PEt formed with PC derived from the transformed cells, irrespective of enzyme source. The results demonstrate that PLD in intact nontransformed fibroblasts is activatable by TPA and VP to a greater extent than in the transformed counterpart. The results from cell-free assays suggest that PLD activity is more dependent on the type of PC substrate than on the source of the enzyme.     

Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. Evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway and direct formation of diacylglycerol by a phospholipase C pathway

The effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on phospholipid degradation was investigated in three cell lines of dissimilar origin, Madin-Darby canine kidney cells (MDCK), rat aorta smooth muscle cells (RASM), and bovine pulmonary artery endothelial cells (BPAE). In cells prelabeled with [3H]myristic acid, which is predominantly incorporated into phosphatidylcholine (PC), TPA treatment (80 nM) in the absence or presence of ethanol (2%) in the culture medium resulted in either the rapid generation of [3H]phosphatidate (PA) or the sustained accumulation of [3H]phosphatidylethanol (PEt), respectively. Increases in [3H]PA and [3H]PEt were paralleled by quantitative decreases in cellular [3H]PC radioactivity. TPA-induced [3H]PEt formation occurred in a similar fashion, irrespective of the presence of Ca2+ in the culture medium. The experiments demonstrate that TPA elicits PC degradation by phospholipase D (PLD) in cells of diverse origin. Data from further experiments revealed a complex relationship between TPA-induced [3H]PA and [3H]diacylglycerol (DG) generation in the three cell lines that was suggestive of dual pathways for the generation of [3H]DG. Experiments to discern the pathways for TPA-induced, PC-derived DG were conducted by comparing the variation of [3H]PA and [3H]DG formation in the absence and in the presence of increasing ethanol concentrations in the culture medium. With increasing amounts of ethanol, the formation of [3H]PA decreased at the expense of [3H]PEt formation, and depending upon the pathway operable, the amount of [3H]DG formed was either decreased, indicative of indirect formation of DG via PA phosphohydrolase, or not modified, indicative of DG formation by a direct phospholipase C (PLC) pathway. Increasing the concentration of ethanol in the medium blocked TPA-induced [3H]DG generation in MDCK cells in a concentration-dependent manner, while the formation of [3H]PEt increased at the expense of [3H]PA formation. In BPAE cells the presence of ethanol likewise reduced TPA-elicited formation of DG. Conversely, in two smooth muscle cell lines, RASM and A-10, ethanol was without influence on TPA-induced formation of [3H]DG, although [3H]PEt was generated at the expense of [3H]PA. In RASM cells prelabeled with [3H]choline, TPA induced the release to the medium of [3H]choline and [3H]phosphocholine, indicative of both PLD and PLC activation. These results show that TPA elicits DG formation from PC in MDCK cells predominantly by an indirect pathway, whereas in arterial smooth muscle cells DG is formed in part by the direct action of PLC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complement C5a activation of phospholipase D in human neutrophils. A major route to the production of phosphatidates and diglycerides

The contribution of phospholipase D (PLD) to the production of phosphatidic acid (PA) and diglyceride (DG) by C5a-stimulated human neutrophils has been studied. Membrane-associated 1-O-alkyl-phosphatidylcholine (alkyl-PC) was double labeled with 3H and 32P by incubating neutrophils with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC. Upon stimulation with recombinant C5a, these labeled neutrophils produce 1-O-alkyl-phosphatidic acid (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidyl-ethanol (alkyl-PEt), containing both 3H and 32P. Formation of radiolabeled alkyl-PEt parallels that of radiolabeled alkyl-PA and requires both extracellular Ca2+ and cytochalasin B. Furthermore, the 3H/32P ratios of alkyl-PA and alkyl-PEt formed during stimulation are very similar to that of th substrate alkyl-PC. These results demonstrate that, in C5a-stimulated neutrophils, alkyl-PA and alkyl-PEt are formed from alkyl-PC almost exclusively by PLD-catalyzed hydrolysis and transphosphatidylation, respectively. Upon C5a stimulation, neutrophils labeled with 3H and 32P also produce 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), but not [32P]phosphocholine. [3H]Alkyl-DG and [32P]PO4 are formed in parallel, although temporally lagging behind alkyl-PA. Propranolol, a PA phosphohydrolase (PPH) inhibitor, decreases the formation of both [3H]alkyl-DG and [32P]PO4, although increasing alkyl-PA accumulation. These data support the conclusion that alkyl-DG is formed from alkyl-PC by the combined activities of PLD and PPH and not by phospholipase C (PLC). Furthermore, by using [3H]acyl-PC-labeled neutrophils, it is demonstrated that, like alkyl-PC, 1-acyl-PC is also degraded sequentially by PLD and PPH to 1-acyl-DG. Propranolol does not inhibit phosphoinositide-specific PLC and yet it causes almost complete inhibition of the total DG mass accumulation in C5a-stimulated neutrophils. We conclude that, in cytochalasin B-treated neutrophils stimulated with C5a, PLD-catalyzed hydrolysis of PC determines the levels of both PA and DG with potentially important ramifications for neutrophil-mediated defense functions.     

Phorbol ester-stimulated hydrolysis of phosphatidylcholine and phosphatidylethanolamine by phospholipase D in HeLa cells. Evidence that the basal turnover of phosphoglycerides does not involve phospholipase D

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated the release of [3H]ethanolamine from HeLa cells prelabeled with [3H]ethanolamine within 2 min, and of [3H]choline from cells prelabeled with [3H]choline after a lag of 10-20 min. This result suggests that TPA activates phospholipase D. Propranolol alone or propranolol plus TPA stimulated phosphatidic acid (PA) labeling in cells prelabeled with [3H]hexadecanol. In the presence of ethanol, TPA stimulated the accumulation of labeled phosphatidylethanol (PEth); no PEth was formed in the absence of TPA. TPA-dependent PEth accumulation was not observed in cells pretreated with TPA to down-regulate protein kinase C, whereas propranolol-induced accumulation of PA was unaffected by TPA pretreatment. Incubation of prelabeled cells with propranolol alone caused a rapid loss of label and phospholipid mass from both phosphatidylethanolamine and phosphatidylcholine (PC) together with an accumulation of PA and phosphatidylinositol plus phosphatidylserine. When [3H]hexadecanol-prelabeled cells were pulse labeled with 32P to label nucleotide pools, propranolol induced the accumulation of both 3H- and 32P-labeled PA. When cells were prelabeled with lyso-PC double labeled with 3H and 32P, and incubated with propranolol, only 3H-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of 32P from the lipid. These results suggest that the basal turnover of phosphatidylethanolamine and PC involves the sequential actions of phospholipase C, diglyceride kinase, and PA phosphohydrolase.     

Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils

Stimulation of human polymorphonuclear leukocytes (PMN) may result in the metabolism of phospholipids other than phosphoinositides to generate second-messenger intermediary metabolites. We investigated agonist-induced breakdown of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC), which constitutes almost half the diradyl-GPC fraction in human PMN (Mueller, H. W., O'Flaherty, J. T., Green, D. G., Samuel, M. P., and Wykle, R. L. (1984) J. Lipid Res. 25: 383-388), in cells prelabeled with 1-O-[3H] alkyl-2-acyl-GPC. We also utilized normal-phase high pressure liquid chromatography to quantitate the accumulation of diradylglycerols (1-O-alkyl-2-acylglycerols and diacylglycerols) in stimulated PMN. Phorbol-12-myristate-13-acetate (PMA), 1-oleoyl-2-acetyl-sn-glycerol-, calcium ionophore A23187-, and f-methionyl-leucyl-phenylalanine (fMLP) stimulation of PMN resulted in a time- and concentration-dependent hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA) and 1-O-[3H]alkyl-2-acylglycerol. In all cases formation of 1-O-[3H]alkyl-2-acyl-PA preceded that of 1-O-[3H]alkyl-2-acylglycerol. The times between addition of stimulus and appearance of 1-O-[3H] alkyl-2-acylglycerol varied for PMA (40 s at 1.6 microM), A23187 (5 min at 5 microM), and fMLP (30 sec at 1 microM). Preincubation of cells with 1 microgram/ml pertussis toxin (PT) inhibited the breakdown of 1-O-[3H]alkyl-2-acyl-GPC in cells stimulated with 1 microM fMLP, indicating a role for a PT-sensitive G protein with this stimulus. Quantitation of diglycerides as diradylglycerobenzoates in PMN stimulated with PMA (10 min), A23187 (10 min), or fMLP demonstrated marked accumulation of both 1-O-alkyl-2-acylglycerols and diacylglycerols. The highest increases over controls were observed for fMLP (33-fold for 1-O-alkyl-2-acylglycerols and 17-fold for diacylglycerols). In stimulated PMN prelabeled with 1-O-[3H]hexadecyl-2-acyl-GPC and 1-O-alkyl-2-acyl-sn-glycero-3-[32P]phosphocholine, the ratio of 3H to 32P in 1-O-alkyl-2-acyl-PA compared to 1-O-alkyl-2-acyl-GPC suggested the involvement of a phospholipase D in the hydrolysis of 1-O-[3H]-alkyl-2-acyl-GPC. Thus, stimulation of human PMN results in the hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC to yield 1-O-[3H] alkyl-2-acyl-PA and 1-O-[3H]alkyl-2-acylglycerol possibly initiated by activation of a phospholipase D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of phosphatidylcholine-selective and phosphatidylinositol-selective phospholipases D in Madin-Darby canine kidney cells.

Intact cells and cell-free systems were employed to characterize phospholipase D (PLD) activity in Madin-Darby canine kidney (MDCK) cells. In cells prelabeled with [3H]glycerol, 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited phosphatidylcholine (PC) hydrolysis by PLD, as shown by the prolonged formation of [3H]phosphatidylethanol (PEt) and an accompanying decrease in [3H]PC. In contrast, bradykinin elicited rapid formation of [3H]PEt (approximately 1 min) accompanied by a decrease in [3H]phosphatidylinositol (PI). When the agonists were administered simultaneously, [3H]PEt formation was biphasic. In cells prelabeled with [3H] choline, at times less than 1 min, bradykinin failed to induce significant change in [3H]choline release. Bradykinin-induced formation of [3H]PEt in the [3H]glycerol-labeled cells was strictly dependent on extracellular Ca2+, whereas TPA-induced formation of [3H]PEt did not require extracellular Ca2+. Cell-free assays for PLD were used to assess the enzyme location, substrate specificity, and cofactor requirements. The PC-PLD activity (PEt formation) against [3H]stearoyl-PC was primarily localized in the 440 x g pellet (membrane- and nuclear-associated), preferred PC as a substrate, required detergent, and was not influenced by Ca2+ at low concentrations but was inhibited by Ca2+ in excess of 0.5 mM. The PI-PLD activity against [3H]stearoyl-PI was found largely in the 100,000 x g supernatant (cytosol), was strictly Ca(2+)-dependent, and did not require detergent. From these data, we conclude that MDCK cells contain two PLD subtypes: 1) a membrane-associated, PC-selective enzyme that responds to TPA resulting in prolonged hydrolysis of PC (the PC-PLD is Ca(2+)-independent, but requires detergent); 2) a cytosolic, PI-selective enzyme that responds rapidly but transiently to bradykinin (the PI-PLD requires Ca2+ but not detergent).     

Calcium rather than protein kinase C is the major factor to activate phospholipase D in FMLP-stimulated rabbit peritoneal neutrophils. Possible involvement of calmodulin/myosin L chain kinase pathway.

In the present study, we first investigated which of the factors, protein kinase C (PKC) or Ca2+, plays an important role in activation of phospholipase D (PLD) of rabbit peritoneal neutrophils stimulated by the chemoattractant FMLP. PLD activity was assessed by measuring [3H]phosphatidylethanol ([3H]PEt), the unambiguous marker of PLD, generated by [3H]lyso platelet-activating factor-prelabeled neutrophils in the presence of ethanol. PKC inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl-2-methylpiperazine dihydrochloride, augmented the plateau level of [3H]PEt produced in FMLP-stimulated cells, although they had no effect on the initial rate of the formation. Furthermore, it was found that the FMLP-stimulated [3H]PEt formation was inhibited by pretreatment of cells with PMA, a PKC activator, and exposure of cells to staurosporine before PMA pretreatment moderately blocked the PMA inhibition. Ca2+ ionophore ionomycin, as well as FMLP, stimulated [3H]PEt formation, accompanied by a decrease in [3H]phosphatidylcholine, in a time- and concentration-dependent manner. Both FMLP and ionomycin absolutely required extracellular Ca2+ to increase [3H]PEt formation. These results imply that elevated intercellular Ca2+ by FMLP stimulation is the major factor for PLD activation and that PKC rather negatively regulates the enzyme activity. Interestingly, a calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide, and a myosin L chain kinase inhibitor, 1-(5-iodonaphthalene-1-sulfonyl)-1H-h exahydro-1,4-diazepine hydrochloride, both inhibited the ionomycin- and FMLP-stimulated [3H]PEt formation in a concentration-dependent manner. Results obtained in this study suggest that, in FMLP-stimulated rabbit peritoneal neutrophils, increased intracellular Ca2+ activates PLD through calmodulin/myosin L chain kinase pathway and, thereafter, the enzyme activation is turned off by simultaneously activated PKC.     

Lack of Phospholipase D Activity in Chromaffin Cells: Bradykinin-Stimulated Phosphatidic Acid Formation Involves Phospholipase C in Chromaffin Cells but Phospholipase D in PC12 Cells

The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.     

Conversion of alkylacetylglycerol to platelet-activating factor in HL-60 cells and subcellular localization of the mediator

A human promyelocytic leukemia (HL-60) cell line was used to investigate the conversion of 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) to platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by intact cells and in subcellular fractions in order to examine the fate of PAF synthesized de novo. Lipid extracts obtained from undifferentiated HL-60 cells incubated with [3H]alkylacetyl-G contained 2-4% of the label as [3H]PAF; several related metabolites were also detected. The yield of [3H]PAF could be dramatically increased by pretreating the cells with either oleic acid, an activator of CTP:phosphocholine cytidylyltransferase, or phenylmethylsulfonyl fluoride, an inhibitor of PAF acetylhydrolase. These results, together with a kinetic study of [3H]alkylacetyl-G metabolism, indicate the sequential participation of a cholinephosphotransferase for the conversion of [3H]-alkylacetyl-G to PAF and acetylhydrolase and transacylase activities in the remodeling pathway that metabolize the newly formed [3H]PAF to 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3-phosphocholine. The dithiothreitol-insensitive cholinephosphotransferase activity capable of converting alkylacetyl-G to PAF was localized in subcellular fractions that contain CDP-choline:1,2-dioleoyl-sn-glycerol cholinephosphotransferase (dithiothreitol-sensitive), as well as marker enzyme activities for the endoplasmic reticulum and Golgi membranes. Subcellular localization analyses also indicated that the majority of newly formed [3H]PAF and a large portion of its deacetylated metabolite were associated with the plasma membrane-containing fractions, whereas most of the 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3- phosphocholine was present in the intracellular organelles. Incubations of HL-60 cells with exogenous [3H]PAF produced a similar subcellular distribution of metabolites. Very little (less than 10%) of the [3H]PAF produced from [3H]alkylacetyl-G was released from intact cells under a variety of incubation conditions but 50% of the de novo-derived mediator was recovered in the medium of cells that were permeabilized with saponin. Our results indicate that PAF is rapidly translocated from its intracellular site of enzymatic synthesis to the plasma membrane where it is apparently sequestered in a pool that is not accessible to extracellular acceptors in contact with intact cells.     

Enhancing effect of wortmannin on muscarinic stimulation of phospholipase D in rat pheochromocytoma PC12 cells.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK), enhanced carbachol-induced formation of [3H]phosphatidylethanol ([3H]PEt), a marker of phospholipase D (PLD) activity, in [3H]palmitic acid-labeled PC12 cells. The apparent EC50 value was 1.5 microM, and the effect was maximal at 3 microM and slightly attenuated at higher concentration. Wortmannin alone had no significant effect on [3H]PEt formation. The enhancing effect of wortmannin was observed at the initial increasing phase of [3H]PEt formation but not at the subsequent plateau phase. Wortmannin enhanced also phorbol ester-induced PLD activation. Although the precise mechanism remains to be clarified, these results suggest that MLCK may be involved in PLD regulation in PC12 cells.     

Differential activation by fMet-Leu-Phe and phorbol ester of a plasma membrane phosphatidylcholine-specific phospholipase D in human neutrophil

Signal transduction involving phosphatidylcholine hydrolysis has been investigated in human neutrophils (PMN) after in situ generation of [3H]alkylacyl-sn-glycero-3-phosphocholine ([3H]alkylacyl-GPC) by cell incubation with [3H]alkylacetyl-GPC. When PMN were stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe(fMLP) or phorbol myristate acetate (PMA) in the presence of cytochalasin B, both 1-O-alkyl-2-acyl-sn-glycero-3-phosphate (PA) and 1-O-alkyl-2-acyl-sn-glycerol (AAG) were generated. On addition of the agonists in the presence of ethanol, phosphatidylethanol (PEt) [corrected] was formed with a concomitant decrease in PA and AAG. These results indicate the presence of a phospholipase D (PLD) acting on phosphatidylcholine in human PMN. The kinetics of hydrolysis were quite different according to the stimulus. Whereas fMLP induced a maximum rise in PA and AAG at 30-45 s, these products began to appear only after 1 min upon cell incubation with PMA. Similar amounts of products were formed at 1 min with fMLP and only at 5 min with PMA. Although similar time courses of PA generation were obtained in the absence of cytochalasin B, AAG were no longer involved and therefore cannot account for intracellular second messenger under physiological conditions. Subcellular distribution studies demonstrated the exclusive location of PA and PEt [corrected] in the plasma membrane. The possible involvement of PA in respiratory burst activation is discussed.