Pertussis Toxin‐Insensitive G Protein Mediates Carbachol Activation of Phospholipase D in Rat Pheochromocytoma PC12 Cells
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Abstract: In the present study, an activation mechanism for phospholipase D (PLD) in [ 3 H]palmitic acid‐labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [ 3 H]phosphatidylethanol ([ 3 H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [ 3 H]PEt, which reached a plateau within 1 min, in a concentration‐dependent manner. The [ 3 H]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 [ 3 H]PEt content, which reached a plateau at 30–60 min after exposure, but an inactive phorbol ester, 4a‐phorbol 12,13‐didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 μM), blocked PMA‐induced [ 3 H]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) (OTPGmS), also stimulated [ 3 H]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 GTPΓS‐ induced [ 3 H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 μM) had no effect on [ 3 H]PEt formation by GTPγS. Pretreatment of cells with pertussis toxin (10–200 ng/ml) for 15 h failed to suppress CCh‐induced [ 3 H]PEt formation, although the pertussis toxin‐sensitive G protein(s) in membranes was completely ADP‐ribosylated under the same conditions. From these results, we conclude that the mechanisms of PMA‐ and CCh‐stimulated PLD activation are different from each other and that CCh‐induced PLD activation is independent of PKC and mediated, at least in part, via a pertussis toxin‐insensitive G protein.Keywords:
Phorbol
Phosphatidylethanol
Phospholipase D
Staurosporine
Phosphatidylethanol
Phospholipase D
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Phospholipase D has been shown to be a key enzyme in the signal transduction systems involved in neutrophil activation. In the presence of ethanol, the enzyme catalyzes a transphosphatidylation reaction through which phosphatidylethanol is formed instead of the normal product phosphatidic acid. The effects of ethanol on the formation of phosphatidylethanol and phosphatidic acid was studied in neutrophils from human alcoholics in vitro. Neutrophils were isolated and cellular lipids were labeled with [3H]oleate, whereafter the cells were preincubated with cytochalasin B. Subsequently, cells were stimulated with the chemotactic peptide formyl-Met-Leu-Phe in the presence of ethanol concentration ranging from 0 to 200 mM. In the presence of ethanol, both neutrophils from alcoholics and controls produced phosphatidylethanol, with a concomitant reduction of the production of phosphatidic acid. The amounts of phosphatidyl-ethanol and phosphatidic acid formed were dependent on the concentration of ethanol. In neutrophils from alcoholics, a higher apparent Km for the phospholipase D-mediated transphosphatidylation reaction was noted (58 mM ethanol compared with 28 mM in controls). The in vivo mass of phosphatidylethanol in recently drinking alcoholics was also analyzed in neutrophils. Measurable phosphatidyl-ethanol levels (average 5.6 pmol/10(8) neutrophils) were found in alcoholics up to 23 hr after the last intake of ethanol. Thus, in addition to the ethanol-induced changes in the normal production of phosphatidic acid, phosphatidylethanol accumulated in vivo in alcoholics may be expected to influence neutrophil function.
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Abstract: The phorbol ester 12‐ O ‐tetradecanoylphorbol 13‐acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [ 3 H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 n M TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D‐catalyzed transphosphatidylation, was formed. The half‐maximum effective concentrations (EC 50 ) of TPA were 25 n M for PA increase as well as for Peth formation. The formation of Peth in ethanol‐treated cells was accompanied by an inhibition of the TPA‐induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [ 3 H]Peth and a decrease in [ 3 H]PA. A protein kinase C inhibitor, 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoylglycerol (100 μ M ) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol‐treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.
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Phosphatidylethanol is a unique phospholipid which is formed in cell membranes only in the presence of ethanol. The reaction is catalysed by phospholipase D, an enzyme that normally catalyses the hydrolysis of phospholipids leading to the formation of phosphatidic acid. However, phospholipase D also utilizes short-chain alcohols as substrates resulting in the formation of the corresponding phosphatidylalcohol. This is a specific mechanism through which ethanol may interact with cell function. Phospholipase D is activated by several different receptors and has during recent years been suggested to play a role in cellular signalling. Secretory processes as well as long-term changes of cell function have been associated with the activation of phospholipase D. Since ethanol competes with water as a substrate for this enzyme, phosphatidylethanol is formed at the expense of the normal lipid product, phosphatidic acid, in an ethanol concentration-dependent manner. Therefore, the phospholipase D-mediated signal transduction diverges from production of the normal signalling lipid in the presence of ethanol. However, phosphatidic acid may also be formed by other pathways and their relative contribution to the formation of this lipid depends on the cell and receptor type. Thus, it is important to identify the signalling systems where phospholipase D dominates the lipid messenger production since these may be especially vulnerable to ethanol. In addition to a change in phospholipase D-mediated signal transduction, accumulation of phosphatidylethanol in cell membranes may also induce disturbances in cell function. Significant amounts of this abnormal phospholipid have been detected after ethanol exposure in brain and other organs from rat, in cultured cells as well as in human blood cells. The degradation of phosphatidylethanol is relatively slow and it remains in the cells after ethanol has disappeared. It is possible that an abnormal phospholipid that accumulates in cell membranes affects membrane-associated processes. Phosphasidylethanol is a lipid with a small, anionic head group and its biophysical properties are different compared with other phospholipids. Moreover, this lipid has been demonstrated to influence membrane charactenstics, enzyme activities and levels of signalling molecules. Thus, both the inhibition of phospholipase D-mediated signal transduction and the accumulation of phosphatidylethanol represent possible pathways through which ethanol may disturb cell function.
Phosphatidylethanol
Phospholipase D
PLD2
Phosphoinositide phospholipase C
Second messenger system
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Abstract Treatment of [ 14 C]choline‐ or [ 14 C]ethanolamine‐labeled NIH 3T3 fibroblasts with Bacillus cereus phosphatidylcholine‐specific phospholipase C (PLC) enhanced phospholipase D (PLD)‐mediated hydrolysis of the respective 14 C‐labeled phospholipids. PLD activity was stimulated by 1.5 U/mL of POLC and by 100 nM of the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (PMA) to similar extents. Treatment of 14 C]palmitic acid‐labeled fibroblasts with PLC in the presence of ethanol also enhanced PLD‐mediated formation of phosphatidylethanol; the effects of PLC and PMA were nonadditive. PLC had no effect on PLD activity in fibroblasts in which PKC was down‐regulated by prolonged (24 h) treatment with 300 nM PMA. These data indicate that treatment of fibroblasts with exogenous PLC results in PKC‐dependent activation of PLD.
Phosphatidylethanol
Phospholipase D
Phorbol
Bisindolylmaleimide
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Phosphatidylethanol
Phospholipase D
PLD2
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Abstract Phospholipase D is an important enzyme in signal transduction in neuronal tissue. A variety of assays have been used to measure phospholipase D activity in vitro . The most typical measure of phospholipase D activity is the production of phosphatidylethanol in the presence of ethanol. Phosphatidylethanol is a product of transphosphatidylation activity that is considered a unique property of phospholipase D. To support transphosphatidylation activity, high concentrations of ethanol may be required. Furthermore, most assays in the literature utilize a detergent. These extreme conditions, detergent and ethanol, may alter phospholipase D and hinder the study of its regulation. In this manuscript we describe an assay that eliminates these potentially confounding conditions. It utilizes high specific activity [ 3 H]butanol as a nucleophilic receptor. This eliminates the need for high concentrations of alcohol. The substrate is an analog of phosphatidylcholine that contains short‐chain fatty acids, 1,2‐dioctanoyl‐ sn ‐glycero‐3‐phosphocholine. Phospholipase D readily hydrolyzes this substrate in the absence of detergent. This novel assay should be useful in the further characterization of phospholipase D.
Lipidology
Phospholipase D
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Chronic treatment of neuroblastoma X glioma hybrid cells (NG 108-15) with the muscarinic cholinergic agonist carbachol, which acutely inhibits adenylate cyclase, resulted in a 104 +/- 10% increase in PGE1-stimulated cAMP accumulation. Pretreatment of intact cells with pertussis toxin can structurally modify the inhibitory regulatory protein, Gi, by ADP-ribosylation and thus abolish the acute inhibition by carbachol. Pretreatment of the cells with pertussis toxin also resulted in a 27 +/- 8% increase in PGE1-stimulated cAMP accumulation. In the pertussis toxin-treated cells, chronic treatment with carbachol did not further enhance the PGE1 stimulation. These results suggest that functional Gi is required for the development of muscarinic cholinergic-induced enhancement of PGE1-stimulated cAMP accumulation.
ADP-ribosylation
Muscarinic agonist
Adenylate Cyclase Toxin
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Treatment of 1-O-[3H]alkyl-2-acyl-phosphatidylcholine-prelabeled human polymorphonuclear leukocytes (PMNs) with staurosporine (50 nM to 1 microM) induced a time- and concentration-dependent generation of tritiated phosphatidic acid (PA), reaching approximately 225% of the control value at 15-20 min. In the presence of ethanol, staurosporine induced a production of phosphatidylethanol (PEt) reaching, 250% of control values, and partial inhibition of PA production, consistent with PLD activation. The amount of ether-linked acylglycerol (EAG) was weakly enhanced (29%) after 5 min of PMN treatment; longer treatment resulted in no significant EAG production, suggesting a possible late inhibition of PA hydrolase activity. Staurosporine concentrations that induced an elevation in PA completely depressed protein kinase C (PKC) activity in both soluble and particulate cell fractions, suggesting that PLD activation may occur independently from PKC activation. PLD may thus represent a potential cellular target for staurosporine action.
Staurosporine
Phosphatidylethanol
Phospholipase D
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