Formation Of Choline Research Articles

Phospholipase C and phospholipase D are independently activated in rat hippocampal slices

In order to investigate a possible G-protein-mediated activation of phospholipase D (PLD) and its relationship to the activation of phosphoinositide-specific phospholipase C (PI-PLC), we measured the effects of aluminium fluoride and carbachol on choline release, the PLD-specific transphosphatidylation reaction (generation of phosphatidylpropanol) and the formation of inositol phosphates in rat hippocampal slices. Aluminium fluoride markedly enhanced the formation of choline and phosphatidylpropanol but failed to increase the formation of inositol phosphates. In contrast, the muscarinic agonist carbachol strongly stimulated PI-PLC but failed to activate PLD. We conclude that PLD in hippocampal slices is activated by a G-protein independently of phosphoinositide hydrolysis.

Extracellular ATP and UTP activation of phospholipase D is mediated by protein kinase C-epsilon in rat renal mesangial cells.

1. We have studied whether a nucleotide receptor mediates the effects of extracellular ATP and UTP on phosphatidylcholine metabolism in rat cultured glomerular mesangial cells. 2. ATP and UTP stimulated a biphasic 1,2-diacylglycerol (DAG) formation in [3H]-arachidonic acid-labelled mesangial cells. In contrast, in cells labelled with [3H]-myristic acid, a tracer that preferentially marks phosphatidylcholine, both nucleotides induced a delayed monophasic production of DAG with a concomitant increase in phosphatidic acid and choline formation. 3. A phospholipase D-mediated phosphatidylcholine hydrolysis was further suggested by the observation that ATP and UTP stimulate the accumulation of phosphatidylethanol, when ethanol was added to mesangial cells. 4. The rank order of potency of a series of nucleotide analogues for stimulation of phosphatidylethanol formation was UTP = ATP > ITP > ATP gamma S > beta gamma-imido-ATP = ADP > 2-methylthio-ATP = beta gamma-methylene-ATP = ADP beta S, while AMP, adenosine, CTP and GTP were inactive, indicating the presence of a nucleotide receptor. 5. Elevation of cytosolic free Ca2+ by the calcium ionophore A23187 (1 microM) or the Ca(2+)-ATPase inhibitor, thapsigargin (200 nM) slightly increased phosphatidylethanol formation. However, chelation of cytosolic Ca2+ with high concentrations of Quin 2 did not attenuate ATP- and UTP-induced phosphatidylethanol production, thus suggesting that Ca2+ is not crucially involved in agonist-stimulated phospholipase D activation. 6. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), but not the biologically inactive 4 alpha-phorbol 12,13-didecanoate, increased phospholipase D activity in mesangial cells, suggesting that PKC may mediate nucleotide-induced phosphatidylcholine hydrolysis. 7. Down-regulation of PKC-alpha and -delta isoenzymes by 8 h PMA treatment still resulted in full phospholipase D activation. In contrast, a 24 h treatment of mesangial cells with PMA, a regimen that also causes depletion of PKC-epsilon, markedly attenuated nucleotide-evoked phosphatidylethanol formation. In addition, the selective PKC inhibitor, calphostin C attenuated ATP- and UTP-induced phosphatidylethanol production.8. In summary, these data suggest that extracellular ATP and UTP use a common nucleotide receptor to activate phospholipase D-mediated phosphatidylcholine hydrolysis. Stimulation of phospholipase D appears to involve the PKC-epsilon isoenzyme, activated by DAG derived from phosphoinositide hydrolysis by phospholipase C.

Gas chromatographic determination of primary and secondary low-molecular-mass aliphatic amines in urine using derivatization with isobutyl chloroformate

A simple routine method for the gas chromatographic determination of methylamine, dimethylamine, ethylamine and methylethylamine in urine is presented. The method is based on a two-phase derivatization procedure with isobutyl chloroformate as reagent. The reaction is quantitative in 10 min. We found no artifact formation of either choline or trimethylamine (dietary amine compounds) or of dimethylethylamine or triethylamine (catalyst amines in the industrial setting). The chromatographic behaviour of the amine carbamates was excellent. The recoveries of methylamine, dimethylamine, ethylamine and methylethylamine in spiked urine samples were 82, 89, 100 and 96%, respectively, and the precision (the relative standard deviation) was 3.6, 1.8, 3.3 and 2.0%, respectively. The method was linear for the studied amine carbamates up to 250 mg/l. The endogenous amine concentrations in urine samples from ten normal subjects were: methylamine, 0.9 mg/l (mean; range 0.3–1.5); dimethylamine, 14.7 mg/l (mean; range 4.6–27.6); ethylamine, 0.8 mg/l (mean; range 0.2–2.3); methylethylamine, less than 0.02 mg/l.

Bradykinin and vasopressin activate phospholipase D in rat Leydig cells by a protein kinase C-dependent mechanism.

In the present study we report that bradykinin stimulated phospholipase D activity in rat Leydig cells. Bradykinin added for 8 min stimulated choline formation in a dose-dependent manner and, in the presence of ethanol, bradykinin (100 nmol/l) stimulated transphosphatidylation by phospholipase D resulting in the formation of phosphatidylethanol. This stimulation was abolished after down-regulation of protein kinase C by long-term pretreatment for 22 h with phorbol 12-myristate 13-acetate (PMA). The stimulation of phospholipase D by the simultaneous addition for 8 min of maximum concentrations of PMA and vasopressin (AVP), PMA and bradykinin, or AVP and bradykinin produced no additive phosphatidylethanol or choline response, suggesting that AVP, bradykinin and PMA stimulated phospholipase D-catalysed phosphatidylcholine hydrolysis by a similar protein kinase C-dependent mechanism. Furthermore, LH (10 ng/ml), insulin (500 nmol/l), GH (100 ng/ml), interleukin-1 beta (5 U/ml) and platelet-activating factor (200 nmol/l) were found not to activate phospholipase D, whereas the Ca2+ ionophore A23187 (10 mumol/l) stimulated phosphatidylethanol formation, suggesting that Ca2+ might be a regulator of phospholipase D in Leydig cells.

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.

Phospholipase D in heart: basal activity and stimulation by phorbol esters and aluminum fluoride.

Evidence for a general role of phospholipase D in signal transduction is accumulating. In the present study, the activity of the enzyme was investigated in heart tissue under basal conditions and after addition of phorbol esters or aluminum fluoride (AlF-4; 10 mM NaF plus 10 microM AlCl3). Atria of rats and chickens were incubated with [3H]-myristic acid in order to label preferentially phosphatidylcholine. Under basal conditions, the tissues generated choline and phosphatidic acid (PtdOH), the primary catalytic products of phospholipase D. When 0.5 or 2.0% ethanol was present, [3H]-phosphatidylethanol (PETH) was rapidly formed at the expense of [3H]-PtdOH. This transphosphatidylation reaction is specific for phospholipase D activity. The basal formation of PETH was not inhibited by a Ca(2+)-free, EGTA-containing medium. The phorbol ester 4 beta-phorbol-12 beta, 13 alpha-dibutyrate (PDB), which is known to activate protein kinase C, enhanced the net formation of choline, whereas the inactive 4 beta-phorbol-13 alpha-acetate (PAc) was ineffective. PDB (0.2 microM), in contrast to PAc, also increased the formation of [3H]-PtdOH and, in the presence of ethanol, of [3H]-PETH. The PDB-evoked formation of PETH occurred again at the expense of PtdOH. Treshold and maximum effective concentrations of PDB were 10 nM and 0.2-0.6 microM, respectively. The effects of PDB on either choline efflux and generation of PETH showed the same Ca(2+)-dependency, i.e., both effects were blocked by a Ca(2+)-free, EGTA-containing medium, but not by a Ca(2+)-free medium without EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid desensitization of vasopressin-stimulated phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine hydrolysis questions the role of these pathways in sustained diacylglycerol formation in A10 vascular-smooth-muscle cells.

The kinetics of vasopressin-stimulated PtdIns(4,5)P2 and phosphatidylcholine (PtdCho) hydrolysis in relation to sustained diacylglycerol (DAG) formation was investigated in A10 vascular-smooth-muscle cells in culture. Vasopressin stimulated a transient increase in Ins(1,4,5)P3 mass formation, which was mirrored by a decrease in PtdIns(4,5)P2 mass levels. Vasopressin stimulated sustained accumulation of total [3H]inositol phosphates ([3H]IP) in the presence of Li+; however, this was significantly decreased by adding a vasopressin-receptor antagonist at different times after initial stimulation. Vasopressin-stimulated phospholipase D (PLD) activity was found to be a transient phenomenon lasting approx. 2 min. Experiments involving agonist preincubation with subsequent addition of butanol confirmed that vasopressin-stimulated PLD activity was desensitized. Vasopressin stimulated an increase in formation of choline, but not of phosphocholine, suggesting that PLD was the major catalytic route of PtdCho hydrolysis in this cell line. The roles of choline and inositol phospholipid hydrolysis in the prolonged phase of DAG formation was examined by comparing vasopressin-stimulated changes in DAG levels in the presence of butanol, the protein kinase C inhibitor Ro-31-8220 or a V1a-receptor antagonist. Vasopressin-stimulated DAG formation was decreased by 40-50% in the presence of butanol between 1 and 10 min; however, during more prolonged stimulation butanol was without significant effect. In cells pretreated with Ro-31-8220, vasopressin-stimulated DAG formation was decreased by approx. 30% at 2 min, but was significantly potentiated at later times. This coincided with an enhancement of vasopressin-stimulated [3H]IP accumulation. In cells exposed to the V1a-receptor antagonist 5 min after addition of vasopressin, subsequent DAG formation was significantly decreased, indicating that sustained formation of DAG, like [3H]IP accumulation, was dependent on continual agonist receptor activation. The results are discussed in terms of different phospholipid-hydrolytic pathways providing DAG generation.

Stimulation of phosphatidic acid synthesis in bovine aortic endothelial cells in response to activation of P 2-purinergic receptors

In this study we used the bovine thoracic aorta endothelial cell line AG 4762 and primary bovine aortic endothelial cells to investigate the formation of phosphatidic acid (PA) in response to activation of P 2-purinergic receptors. 2-Methylthio ATP (2MeSATP) stimulated the formation of [ 32P]-PA in bovine aortic endothelial cells labelled with 32P i, for 2.5 hr. A comparison of the response to other ATP analogues suggests that this was mediated via a P 2y-purinergic receptor. Using various agonists at 30 μM there was a correlation between the formation of [ 32P]PA and of total inositol phosphates in the presence of lithium. The 2MeSATP-stimulated accumulation of [ 32P]PA showed an initial high rate, followed by a more sustained slower rate. The initial response was independent of extracellular calcium while the later response was dependent on calcium influx. The protein kinase C stimulator phorbol myristate acetate (PMA) produced only a very small enhancement of [ 32P]PA accumulation compared to 2MeSATP. The 2MeSATP stimulation of both inositol phosphates and [ 32P]PA was almost eliminated by the presence of PMA. Using cells prelabelled with [ 3H]methylcholine 2MeSATP produced only a small non-significant enhancement of [ 3H]choline formation; PMA by contrast formed a much larger amount of 3H]choline. There was no evidence of a change in [ 3H]phosphocholine. The dissociation between phospholipase D (PLD) activation and [ 32P]PA accumulation and the correlation between stimulation of [ 32P]PA accumulation and phospholipase C (PLC) activation all suggest that, using this protocol for labelling cells, the principle route of the stimulation of formation of [ 32P]PA is via the activation of PLC followed by metabolism of diacylglycerol (DAG) by DAG kinase. These results show that activation of P 2y-purinergic receptors on aortic endothelial cells leads to the formation of phosphatidic acid and that both PLD and PLC pathways are likely to contribute to this response.

Angiotensin II stimulation of phospholipase D in rat renal mesangial cells is mediated by the AT 1 receptor subtype

Angiotensin II stimulated a biphasic 1,2-diacylglycerol formation in [ 3H]arachidonic acid-labelled mesangial cells. In contrast, in cells labelled with [ 3H]myristic acid, a tracer that preferentially marks phosphatidylcholine, angiotensin II induced a delayed monophasic production of 1,2-diacylglycerol. This delayed peak of 1,2-diacylglycerol generation was associated with a concomitant increase in choline formation, suggesting that stimulation of mesangial cells with angiotensin II causes a phospholipase D-mediated phosphatidylcholine hydrolysis. This conclusion is supported by the observation that angiotensin II stimulated the accumulation of phosphatidylethanol, when ethanol was added to mesangial cells. The production of choline and phosphatidylethanol stimulated by angiotensin II was completely blocked by the angiotensin II AT 1 receptor-selective antagonist DuP 753 with an IC 50 value of 8 nM, but not by the angiotensin II AT 2 receptor selective ligand CGP 42112A. Furthermore, angiotensin(1–7) and angiotensin(1–6) had only weak effects on choline generation. These data clearly indicate that angiotensin II AT 1 receptors trigger phospholipase D-mediated phosphatidylcholine hydrolysis in rat mesangial cells.

Rapid and sensitive discriminating determination of acetylcholinesterase activity in amniotic fluid with a choline sensor

A simple method for the separate determination of acetylcholinesterase and butyrylcholinesterase activities in amniotic fluid is reported. This determination is performed with an enzyme electrode involving an immobilized choline oxidase membrane associated with the amperometric detection of hydrogen peroxide. Acetylcholine or butyrylcholine, in the presence of samples containing acetylcholinesterase or butyrylcholinesterase are specifically hydrolyzed, the formation of choline being detected vs time by the sensor with no need for a selective inhibitor. The dynamic linear ranges for acetylcholinesterase and butyrylcholinesterase are respectively 100 μU to 10 mU and 30 μU to 3 mU per ml sample.

Norepinephrine-induced phosphatidylcholine hydrolysis in intact rat aorta

The present study tests whether norepinephrine induces the hydrolysis of phosphatidylcholine (PC) in intact vascular smooth muscle. Norepinephrine and the phorbol ester, phorbol myristate acetate (PMA), increased the formation of choline and phosphorylcholine in rat aorta. The norepinephrine-induced PC hydrolysis was inhibited by the protein kinase C (PKC) antagonist, 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H7). These results suggest that the diacyclglycerol formed during the sustained phase of the contractile response to norepinephrine may be derived, at least in part, from PC hydrolysis. The hydrolysis may be mediated through PKC activation of phospholipase C and D.

Phorbol ester and vasopressin activate phospholipase D in leydig cells

In the present study evidence is provided for the existence of phospholipase D (PLD) activity in rat Leydig cells. Leydig cells were cultured and labelled with [ 3H]myristic acid. In the presence of ethanol, phorbol 12-myristate 13-acetate (PMA) stimulated the formation of [ 3H]phosphatidylethanol ([ 3H]PEt) in a dose-dependent manner at the expense of [ 3H]phosphatidic acid ([ 3H]PA). In cells prelabelled with [ 3H]choline, PMA caused a rapid increase in intracellular free [ 3H]choline. The time course of [ 3H]PEt formation was similar to the time course of intracellular [ 3H]choline formation. The data taken together support the notion that PMA stimulates phosphatidylcholine (PC) hydrolysis by a mechanism, which principally involves PLD. Activation of PLD by PMA was inhibited by long-term pretreatment of cells with PMA to downregulate protein kinase C (PKC) and by pretreatment with staurosporine. These data support the notion that activation of PLD by PMA is dependent on PKC. Arginine vasopressin (AVP) caused a rapid stimulation of PLD activity in the cells. This activation was inhibited after down-regulation of PKC, indicating that the agonist acts by a mechanism similar to that of PMA.

Antigen-induced biphasic diacylglycerol formation in RBL-2H3 cells: The late sustained phase due to phosphatidylcholine hydrolysis is dependent on protein kinase C

Exposure to antigen (Ag) caused a biphasic 1,2-diacylglycerol (DG) production in [ 3H]myristic acid-labeled RBL-2H3 cells; the early, small transient phase and the second large sustained phase. The accumulation of phosphatidic acid (PA) or phosphatidylethanol (PEt) in the presence of ethanol was paralleled by the second-phase DG generation. Ag-induced formation of phosphocholine and choline in [ 3H]choline-labeled cells suggested the hydrolysis of phosphatidylcholine (PC) by phospholipases C and D. Treatment with phorbol myristate (PMA) or A23187 caused increases in [ 3H]DG and water-soluble [ 3H]choline metabolites. In protein kinase C (PKC) down-regulated cells, PEt formation was markedly reduced. In these cells DG production induced by Ag and A23187 was largely suppressed, thus indicating that PKC would play an important regulatory role for PC hydrolysis. However, because the A23187 treatment showed significant accumulation of water-soluble choline metabolites in PKC down-regulated cells, an increase in intracellular Ca 2+ is another factor regulating PC hydrolysis. Taken together, these results may indicate that PC hydrolysis in response to Ag is dependent of PKC and Ca 2+.

Formation of Free Choline in Brain Tissue during in vitro Energy Deprivation

Free choline and ATP contents were measured in Mongolian gerbil hippocampal slices (tissue) and incubation media (media) during exposure to 30 min of aglycemia, high potassium, anoxia, or ischemia. Changes in choline levels reflected the degree of energy reduction, lower ATP levels being associated with high choline (4-fold increase during exposure to high potassium and anoxia, and 11-fold increase during ischemia). Media (extracellular) choline was particularly affected and increased about twofold during relatively mild energy depletion (e.g., aglycemia), but tissue choline content was less sensitive to energy reduction. A plot of choline vs. ATP levels indicated a nonlinear correlation, and the sharp increase in choline occurred when ATP values fell to about 2.5 nmol/mg of protein. Inhibition of acetylcholine sterase by 10 microM physostigmine during ischemia did not prevent an increase in choline contents but rather enhanced them, indicating that acetylcholine hydrolysis was not the source of free choline. Formation of free choline was Ca2+ independent. These findings suggest the involvement of phospholipase D and phosphatidylcholine hydrolysis in free choline formation during energy stress. The extent of choline formation may be an indicator of the degree of membranal damage, which in turn reflects damage to the metabolic machinery of the cell.

Sphingosine inhibits phosphatidate phosphohydrolase in human neutrophils by a protein kinase C-independent mechanism

Human neutrophils have been labeled in 1-O-alkyl-phosphatidylcholine with 3H in both the alkyl chain and the choline moiety. Upon stimulation of these labeled cells with formyl-Met-Leu-Phe, C5a, or phorbol 12-myristate 13-acetate, phospholipase D is activated to produce 1-O-[3H]alkylphosphatidic acid ([3H]alkyl-PA) and [3H]choline. The [3H]alkyl-PA is then dephosphorylated by phosphatidate phosphohydrolase (PPH) to produce 1-O-[3H]alkyldiglyceride ([3H]alkyl-DG). Sphingosine, a sphingoid base known to inhibit protein kinase C (PKC), causes a dose-dependent inhibition of [3H]alkyl-DG formation. This inhibition is accompanied by increased accumulation of [3H]alkyl-PA without alterations in [3H]choline formation. Studies using various other sphingoid bases demonstrate that a long hydrocarbon chain and an amino group are required for the inhibition of DG formation. These results suggest that sphingoid bases inhibit PPH activity without altering phospholipase D activation and that they exhibit a similar structure-activity relationship for both PPH and PKC. K252a, a PKC inhibitor which acts by competing for ATP binding sites, does not inhibit the formation of [3H]alkyl-DG, [3H]alkyl-PA, or [3H]choline at a concentration (3 microM) that completely blocks phorbol 12-myristate 13-acetate-induced protein phosphorylation. Moreover, in neutrophil homogenates, sphingosine but not octylamine, inhibits PPH activity in a dose-dependent manner. Thus sphingosine inhibits PPH activity by a PKC-independent mechanism, raising the possibility that sphingoid bases may play a role in regulating PPH-mediated lipid metabolism in stimulated cells.

Effects of homo- and heterodimeric isoforms of PDGF on signalling events in rat renal mesangial cells

Plaetlet-derived growth factor (PDGF) exists in three dimeric isoforms, AA, BB and AB. Mesangial cells exclusively bound to BB homodimer and responded only to the BB isoform in terms of DNA synthesis and phosphoinositide hydrolysis. PDGF-BB stimulated a dose-dependent formation of inositol trisphosphate (InsP 3). Neither pertussis toxin nor short-term (10 min) treatment with 12- O-tetradecanoylphorbol 13-acetate (TPA) inhibited the PDGF-BB-evoked production of InsP 3. In contrast, the response to PDGF-BB was attenuated in cells in which protein kinase C has been down-regulated by long-term (24 h) treatment with TPA. In parallel to the generation of InsP 3, there was a biphasic increase in 1,2-diacylglycerol (DAG). The second peak of DAG generation was associated with a concomitant 2-fold increase in choline formation. In addition, PDGF-BB stimulated the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity, when 1-propanol was was added to mesangial cells. Stimulation of mesangial cells with PDGF-BB caused a dose-dependent formation of prostaglandin E 2. Furthermore, mesanigal cells secreted PDGF-AA into the cultrue supernatant.

Phosphatidylcholine hydrolysis stimulated by phorbol myristate acetate is mediated principally by phospholipase D in endothelial cells

The mechanism of phosphatidylcholine (PC) degradation stimulated by phorbol myristate acetate (PMA) was investigated in bovine pulmonary artery endothelial cells prelabeled with [ methyl- 3H]choline ([ 3H]choline) or [9,10- 3H]myristic acid ([ 3H]myristic acid). Both labels were selectively incorporated into PC, and addition of PMA stimulated comparable losses of 3H from PC in cells prelabeled with [ 3H]choline or [ 3H]myristate. In cells prelabeled with [ 3H]choline, the loss of 3H from PC correlated with a rapid increase in intracellular free [ 3H]choline. The increase in intracellular [ 3H]choline stimulated by PMA was not preceded by an increase in any other 3H-labeled PC degradation product. PMA did not stimulate the formation of PC deacylation products in cells prelabeled with [ 3H]choline. In permeabilized cells prelabeled with [ 3H]choline, PMA stimulated the formation of [ 3H]choline but not [ 3H]phosphocholine. In intact cells prelabeled with [ 3H]myristate, the loss of 3H from PC induced by PMA correlated with the formation of [ 3H]phosphatidic acid ([ 3H]PA) and [ 3H]diacylglycerol. In the presence of ethanol, PMA stimulated the formation of [ 3H]phosphatidylethanol ([ 3H]PEt) at the expense of [ 3H]PA. The time-course of [ 3H]PEt formation was similar to the time-course of intracellular [ 3H]choline formation in cells stimulated with PMA. These data taken together support the notion that PC degradation in endothelial cells stimulated with PMA is mediated principally by phospholipase D. PC breakdown via phospholipase D was not observed in cells treated with phorbol esters incapable of interacting with protein kinase C. Activation of phospholipase D by phorbol esters was inhibited by long-term pretreatment of cells with PMA to down-regulate protein kinase C and by pretreatment of the cells with staurosporine. These data support the notion that activation of phospholipase D by phorbol esters is dependent upon protein kinase C.

P2-Purinergic Agonists Stimulate Phosphodiesteratic Cleavage of Phosphatidylcholine in Endothelial Cells: Evidence for Activation of Phospholipase D

The effects of purinergic agonists on phosphatidylcholine (PC) breakdown and prostacyclin synthesis were investigated in cultured bovine pulmonary artery endothelial cells (BPAEC) prelabeled with [3H]choline and [14C]myristic acid. Both labels were selectively incorporated into PC. In BPAEC prelabeled with [3H]choline, ATP stimulated a rapid 5-fold increase in intracellular free [3H]choline. [3H]Choline formation was associated with a concomitant loss of 3H from PC, and it was not preceded by an increase in the 3H content of other PC degradation products. In BPAEC prelabeled with [14C]myristic acid, ATP stimulated a rapid increase in [14C]phosphatidic acid and [14C]diacylglycerol. These changes were associated with a loss of 14C from PC but not from phosphatidylinositol. In permeabilized BPAEC prelabeled with [3H]choline, ATP and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) stimulated an increase in [3H]choline but not [3H]phosphocholine. The effects of ATP and GTP gamma S were synergistic at low GTP gamma S concentrations. Permeabilized BPAEC did not convert exogenous [3H]phosphocholine to [3H]choline. These data are consistent with the notion that purinergic agonists stimulate PC breakdown by a phospholipase D mechanism. This suggestion was supported by the observation that in the presence of ethanol, ATP stimulated the formation of [14C]phosphatidylethanol in BPAEC prelabeled with [14C]myristic acid. Several adenine nucleotides and related purine derivatives were compared for their effectiveness in stimulating PC breakdown and prostacyclin synthesis. The chemical specificity observed for the two responses indicated that both were mediated by the P2Y purinoceptor subtype. PC breakdown stimulated by ATP occurred prior to the maximum rate of release of prostacyclin into the medium. Taken together these data indicate that PC breakdown in endothelial cells stimulated with purinergic agonists is due, at least in part, to the activation of a phospholipase D that is coupled to purinoceptors by a guanine nucleotide-binding protein. In conjunction with previous data on the effects of bradykinin, these findings suggest an important role for PC breakdown in the mechanism of signal transduction in endothelial cells stimulated with Ca2+-mobilizing agonists.

On the mechanism of muscarinic hydrolysis of choline phospholipids in the heart

In the heart, choline phospholipids were by far the largest fraction (about 50%) of phospho-lipids, much larger than that of inositol phospholipids (less than 6%) and phosphatidic acid (0.3%). The choline phospholipids (11 μmol/g) maintained a constant efflux of choline of about 1.5 nmol g −1 min −1 into the perfusate. Carbachol (10 μm) rapidly enhanced the choline efflux by a muscarinic mechanism, that was independent of mepacrine, an inhibitor of phospholipase A 2, as well as of extracellular Ca 2+ the maximum acceleration was reached within 2 min. In contrast, the accumulation of inositol phosphates by carbachol was blocked in the presence of a Ca 2+-free perfusion medium. Similar to the carbachol-evoked choline efflux, the increase in tissue content of phosphatidic acid by carbachol was unaffected by infusion of a Ca 2+-free, EGTA-containing solution. Sodium oleate (20 μM), an activator of phospholipase D, imitated the effects of carbachol on choline and phosphatidic acid, whereas NaF (5 mM), which has been reported to inhibit phospholipase D, blocked carbachol-evoked efflux of choline. In conclusion, muscarinic receptor stimulation enhanced the hydrolysis of choline phospholipids presumably via activation of phospholipase D. The immediate formation of choline, phosphatidic acid and presumably diacylglycerol is discussed including its possible physiological importance.

Characterization of choline efflux from the perfused heart at rest and after muscarine receptor activation.

The resting efflux of choline from perfused chicken hearts varied from 0.4 to 2.6 nmol/g min, but was constant for at least 80 min in the individual experiments. The rate of choline efflux was found to be equal to the rate of choline formation in the heart, which, from the following reasons, was essentially due to hydrolysis of choline phospholipids. Cardiac content of choline phospholipids (7,200 nmol/g) was much higher than that of acetylcholine (5.5 nmol/g). Resting release of acetylcholine was 0.016 nmol/g min and, after inhibition of cholinesterase, only about 0.1 nmol/g min. Resting efflux of choline was reduced by mepacrine, a phospholipase A2 inhibitor, by perfusion with a Ca2+-free Tyrode's solution containing EGTA and by the combination mepacrine plus Ca2+-free/EGTA solution. In all experiments the reduced choline efflux levelled off within 10 min at about 50%. Omission or elevation of Mg2+ from 1.05 to 10.5 mmol/l had no effect. Resting efflux was increased to 150% by oleic acid (as sodium salt; 2 X 10(-5) mol/l) which is known to activate phospholipase D. Likewise muscarinic agonists (carbachol and acetylcholine) caused facilitation of the efflux of endogenous choline that was blocked by 3 X 10(-7) mol/l atropine. This effect was not reduced, but even slightly enhanced, by mepacrine and by infusion of EGTA in a modified Tyrode's solution (Ca2+-free, 10.5 mmol/l Mg2+). It is concluded that the resting efflux of choline from the heart is essentially due to hydrolysis of choline phospholipids, that half of the efflux is insensitive to mepacrine and is Ca2+-independent (excluding an involvement of phospholipase A2).(ABSTRACT TRUNCATED AT 250 WORDS)