Production Of Choline Research Articles

Separation of dipalmitoyl phosphatidyl choline, cholesterol and their degradation products by high-performance liquid chromatography on a perfluorinated stationary bonded phase

The behaviour of a perfluorinated stationary phase, Fluofix, has been investigated for HPLC of lipid compounds. The lipid compounds investigated were those usually composed of liposomes, including dipalmitoyl phosphatidyl choline (DPPC), cholesterol, their impurities and degradation products. Some other phospholipids and lipids of interest like aliphatic amines and detergents were included. Because of the specific behaviour of the perfluorinated bonded phase towards hydrogenated compounds, a great variety of lipids can be separated using simple ethanol–water mobile phases. This method separates DPPC and cholesterol from their degradation products and also separates phospholipids differing by their polar head group or their fatty acid residues. It could a very useful tool for chromatographic analysis, stability studies and formulation characterisation of liposome suspensions.

Activation of cPLA2 in vascular smooth muscle.

Force regulation in vascular smooth muscle is under the control of a variety of lipid-dependent second messengers, such as inositol 1,4,5 trisphosphate and diacylglycerol (Hashimoto et al.,1986; Gu et al., 1991). Our laboratory has demonstrated that norepinephrine can induce force in rat tail artery via the stimulation of IP3 production and the resulting release of calcium ions from intracellular stores (Gu et al.,1991; Somlyo et al,1985). It appears that IP3 production in this tissue is under the control of a G-protein-dependent isoform of phospholipase C known as phospholipase Cb2 (LaBelle and Murray, 1990; LaBelle and Polyak,1996). We have also demonstrated that norepinephrine can activate phospholipase D in rat tail artery, with the resulting production of choline and phosphatidic acid from the lipid phosphatidylcholine (Gu et al, 1992).KeywordsArachidonic AcidArachidonic Acid ReleaseMyosin Light Chain PhosphataseDiacylglycerol LipaseContractile AgonistThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Analysis of Choline and Phosphorylcholine Content in Human Neutrophils Stimulated by f-Met-Leu-Phe and Phorbol Myristate Acetate: Contribution of Phospholipase D and C

We analysed changes in choline (CHO) and phosphorylcholine (PCHO) content of stimulated human polymorphonuclear leukocytes (PMNs) by a chemiluminescence assay to further examine the relative contributions of phospholipase D (PLD) and PLC to phosphatidylcholine (PC) breakdown. PLD activation was also analysed by measuring tritiated phosphatidic acid (PA) and diglycerides (GDs) in PMNs labelled with tritiated alkyl-lyso PC. Stimulation of PMNs with formyl-methionyl-leucyl-phenylalanine fMLP; 0.1 μM induced a weak elevation of mass choline (+25% of basal level) that was strongly potentiated in PMNs primed with cytochalasin B (+350% relative to the control value of 657±53 pmol/10 7 cells). CHO production was rapid and transient, peaking within 1 min, and ran parallel to that of tritiated PA. Thereafter, the amount of tritiated PA declined strongly (40% of maximum by 3 min), whereas the elevated choline content induced by fMLP plateaued for at least 5 min. Phorbol myristate acetate (PMA) sustained the formation of CHO for as long as 20 min, which correlated with that of [ 3H]PA in a time- and concentration-dependent manner. PCHO content of resting PMN leukocytes (1560 ± 56 pmol/10 7 cells) was not modified after stimulation of PMNs with fMLP or PMA for at least 10 min, which argues against breakdown of phosphatidylcholine by PLC. For longer treatment (10–20 min), fMLP stimulated a significant enhancement of PCHO level, which occurred concomitantly with a decrease in CHO level, suggesting that choline kinase rather than PLC may be activated. Unlike fMLP, PMA stimulated a fall in PCHO between 10 and 15 min after PMN stimulation, pointing to different regulatory mechanisms of PCHO level. These data indicate that DG formation from PC in PMNs is mediated by PLD but not by PLC and show that chemiluminescence measurement of choline is a reliable index of PLD activation.

Cross-talk Between Receptor-mediated Phospholipase C-βand D via Protein Kinase C as Intracellular Signal Possibly Leading to Hypertrophy in Serum-free Cultured Cardiomyocytes

Phospholipase C-β(PLC-β) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such asα1-adrenergic agonists, endothelin-1 (ET-1) and angiotensin II (Ang II) induce hypertrophy of myocytes. The goal of this study was to evaluate the role of phospholipase D (PLD) in contributing to the formation of the PKC activator 1,2-diacylglycerol (1,2-DAG) and to study the mechanism(s) of PLD activation by agonists. Stimulation of serum-free cultured neonatal rat cardiomyocytes with ET-1 (10−8m), phenylephrine (PHE, 10−5m) or Ang II (10−7m) resulted in a rapid (0–10 min) activation of PLC-βto an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([3H]leucine incorporation into protein) measured after 24 h. Phorbol 12-myristate 13-acetate (PMA, 10−6m) and ET-1 were equipotent in stimulating protein synthesis. ET-1 and PMA, but not PHE and Ang II stimulated [3H]choline formation from labelled PtdCho after a lag-phase of about 10 min. That this [3H]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [14C]palmitoyl-phosphatidylcholine to exogenous ethanol. ET-1 and PHE, to much lesser extent, produced a rapid (0–5 min) translocation of PKC-εimmunoreactivity from the cytosol to the membrane fraction, whereas no intracellular redistribution of PKC-α, -δand -ξimmunoreactivities was observed. PMA caused translocation of PKC-α, PKC-εas well as PKC-δ. Cellular redistribution of PKC activity measured by [32P]-incorporation into histone III-S was not observed with ET-1 and PHE, but only with PMA stimulation. Down-regulation of PKC isozymes by 24 h pretreatment of cells with PMA or blockade of PKC by chelerythrine (10−4m) inhibited ET-1 and PMA stimulated [3H]choline production. Staurosporine (10−6m) had, however, no effect. In conclusion, the results indicate that in serum-free cultured cardiomyocytes, ET-1 initially activates PLC-βand after a lag-phase PLD, whereas PHE and Ang II activate only PLC-β. PLC-βstimulated by ET-1, may cross-talk with PLD via translocation of PKC-ε. These signals are possibly linked to the hypertrophic response.

Role of the Yeast Phosphatidylinositol/Phosphatidylcholine Transfer Protein (Sec14p) in Phosphatidylcholine Turnover andINO1 Regulation

In yeast, mutations in the CDP-choline pathway for phosphatidylcholine biosynthesis permit the cell to grow even when the SEC14 gene is completely deleted (Cleves, A., McGee, T., Whitters, E., Champion, K., Aitken, J., Dowhan, W., Goebl, M., and Bankaitis, V. (1991) Cell 64, 789-800). We report that strains carrying mutations in the CDP-choline pathway, such as cki1, exhibit a choline excretion phenotype due to production of choline during normal turnover of phosphatidylcholine. Cells carrying cki1 in combination with sec14(ts), a temperature-sensitive allele in the gene encoding the phosphatidylinositol/phosphatidylcholine transporter, have a dramatically increased choline excretion phenotype when grown at the sec14(ts)-restrictive temperature. We show that the increased choline excretion in sec14(ts) cki1 cells is due to increased turnover of phosphatidylcholine via a mechanism consistent with phospholipase D-mediated turnover. We propose that the elevated rate of phosphatidylcholine turnover in sec14(ts) cki1 cells provides the metabolic condition that permits the secretory pathway to function when Sec14p is inactivated. As phosphatidylcholine turnover increases in sec14(ts) cki1 cells shifted to the restrictive temperature, the INO1 gene (encoding inositol-1-phosphate synthase) is also derepressed, leading to an inositol excretion phenotype (Opi-). Misregulation of the INO1 gene has been observed in many strains with altered phospholipid metabolism, and the relationship between phosphatidylcholine turnover and regulation of INO1 and other co-regulated genes of phospholipid biosynthesis is discussed.

Phosphatase activity regulates superoxide anion generation and intracellular signaling in human neutrophils

Phosphorylation of components of the neutrophil NADPH oxidase plays a critical role in activation and maintenance of superoxide anion (O 2 −) generation. To investigate the role of dephosphorylation by phosphatases in regulating O 2 − production, human neutrophils were treated with calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, prior to stimulation. Calyculin A alone did not stimulate O 2 − production. However, neutrophils exposed to 50 nM calyculin A and the chemotactic peptide formyl-met-leu-phe (FMLP, 100 nM) displayed markedly enhanced O 2 − production in comparison to cells stimulated with FMLP alone (28.63±7.00 versus 8.69±3.69 nmol O 2 −/1.5×10 6 neutrophils/5 min, respectively, n=18, p<0.001), with an increased duration of O 2 − production. In contrast, phosphatase-inhibition decreased oxidative responsiveness to phorbol myristate acetate (PMA, ≥16 nM). We next examined the effect of calyculin A on products of the phosphatidylcholine-specific phospholipase D (PLD) pathway by assaying the mass levels of phosphatidic acid (PA), choline and diacylglycerol (DAG). Calyculin A increased both PA and choline production to 224±28% and 315±61% of FMLP-stimulated controls, respectively ( p<0.01, n=7) without significantly increasing DAG. Also, membrane protein kinase C activity increased more than 10-fold in FMLP-stimulated cells exposed to calyculin A but decreased in cells stimulated with PMA following calyculin A pre-treatment. These results suggest that phosphatases exert variable and stimulus-dependent effects on pathways leading to O 2 − production. Further, it appears that phospholipase D activity and PA generation represent important steps in the pathway for NADPH activation triggered by FMLP.

Product-retardation and -activation of catalytic hydrolysis by phospholipase D in small unilamellar vesicles of egg yolk phosphatidylcholine

We evaluated the hydrolysis of egg yolk phosphatidylcholine (PC) by phospholipase D from Streptomyces chromofuscus (PLD) in small unilamellar vesicles (SUV) in presence of 50 µM Ca2+. After initial choline production (hydrolysis of 1.5% of the PC at the outer leaflets of the vesicle bilayers), the hydrolysis was reduced to 5% of the initial velocity. The kinetic behavior in SUV of premixed PC and a low percentage of the hydrolysis product, phosphatidic acid (PA), was similar to that of PC SUV. The reduced velocity disappeared when the membrane structure was disintegrated by means of a nonionic surfactant. In the retardation phase, the partially hydrolyzed vesicles (postsubstrates) had much higher affinity for PLD than fresh PC SUV. These results indicated that small clusters of the product, PA, at the vesicle surface were responsible for the reduced velocity of hydrolysis. The initial velocity increased in a biphasic manner with the substrate concentration. At a PC concentration range up to 4 mM, the experimental data fit Michaelis-Menten kinetics. At concentrations above 6 mM, the velocity again markedly increased. Negatively charged mixed vesicles of PC and PA did not have such kinetics. Furthermore, adding PC SUV to the postsubstrates, where the fraction of free PLD was less than 0.05, induced steep choline production. These results showed that PLD bound to vesicles had higher activity than free PLD. We speculated that PLD bound to vesicles collided with and was directly transferred to PC SUV when the fraction of free PLD in aqueous medium was very small.

Insulin and insulin-like growth factor-I responsiveness and signalling mechanisms in C 2C 12 satellite cells: effect of differentiation and fusion

In proliferating C 2C 12 myoblasts, serum and physiological concentrations of insulin and IGF-I stimulated protein synthesis and RNA accretion. After fusion, the multinucleated myotubes remained responsive to serum but not to insulin or IGF-I, even though both insulin and type-1 IGF receptor mRNAs increased in abundance. Protein synthetic responses to insulin and IGF-I in myoblasts were not inhibited by dexamethasone, ibuprofen or Ro-31-8220, thus phospholipase A 2, cyclo-oxygenase and protein kinase C did not appear to be involved in the signalling mechanisms. Neither apparently were polyphosphoinositide-specific phospholipase C or phospholipase D since neither hormone increased inositol phosphate, phosphatidic acid, choline or phosphatidylbutanol production. Only the phosphatidylinositol-3-kinase inhibitor, wortmannin, and the 70 kDa S6-kinase inhibitor, rapamycin, wholly or partially blocked the effects of insulin and IGF-I on protein synthesis. 2-deoxyglucose uptake remained responsive to insulin and IGF-I after fusion and was also inhibited by wortmannin. The results suggest that the loss of responsiveness after fusion is not due to loss of receptors, but to the uncoupling of a post-receptor pathway, occurring after the divergence of the glucose transport and protein synthesis signalling systems, and that, if wortmannin acts at a single site, this is prior to that point of divergence.

Construction of a plasmid carrying both CTP synthetase and a fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes and its application to industrial CDP-choline production: enzymatic production of CDP-choline from orotic acid (Part II).

A new method for enzymatic production of cytidine diphosphate choline (CDP-choline) from orotic acid and choline chloride was developed. To establish an industrial manufacturing process, we constructed a plasmid, pCKG55, which simultaneously expressed in Escherichia coli the three following enzymes; CTP synthetase (encoded by the pyrG gene from E. coli), cholinephosphate cytidylyltransferase (encoded by the CCT gene from Saccharomyces cerevisiae), and choline kinase (encoded by the CKI gene from S. cerevisiae). CCT and CKI genes on pCKG55 were designed to be expressed as a single CCT/CKI fused protein. This CCT/CKI fused protein retained both activities and the thermal stability of its cholinephosphate cytidylyltransferase activity was nearly the same as the native CCT enzyme. Corynebacterium ammoniagenes KY13505 and E. coli MM294/pCKG55 were cultured in 5-liter jar fermentor independently. Equal volumes of each broth were mixed in a 2-liter jar fermentor, and then the enzymatic reaction was done using 47 mM orotic acid and 60 mM choline chloride as substrates. After 23 h of the reaction at 32 degrees C, 21.5 mM (11 g/liter) of CDP-choline was accumulated.

Enzymatic production of pyrimidine nucleotides using Corynebacterium ammoniagenes cells and recombinant Escherichia coli cells: enzymatic production of CDP-choline from orotic acid and choline chloride (Part I).

Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Cornybacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter (77.6 mM) from orotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY12505 for the enzymatic reaction using orotic acid as a substrate. CTP was produced at 8.95 g/liter (15.1 mM) after 23 h of this reaction. To produce CDP-choline, two additional enzyme activities were needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CKIase; encoded by the CKI gene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system. After 23 h of the reaction using orotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mM) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.

Regulatory mechanisms of choline production

This paper gives a short account of the mechanisms regulating choline production. Although choline is absorbed from the GI tract and biosynthesized in the liver, its subsequent metabolism to choline esters and phospholipids seems to dominate in importance as to its regulatory role in maintaining a constant source of free choline extracellularly.

13C NMR Analysis of the Use of Alternative Donors to the Tetrahydrofolate-Dependent One-Carbon Pools inSaccharomyces cerevisiae

Serine is generally accepted as the major one-carbon donor in folate-mediated one-carbon metabolism in most cells. Previous work from our laboratory with the yeastSaccharomyces cerevisiaehas demonstrated that glycine and formate can also provide one-carbon units. Under normal growth conditions, it is likely that cells utilize serine, glycine, formate, and perhaps other one-carbon donors simultaneously, but to differing degrees. In the present work, we have used13C NMR to monitor how yeast cells distribute alternative, competing one-carbon sources into various pools. Cells were grown with [2-13C]glycine and unlabeled formate or folinic acid (leucovorin, 5-formyl-tetrahydrofolate) as competing one-carbon sources. The relative contribution of each one-carbon donor to the three oxidation states of the tetrahydrofolate-bound one-carbon pool [5-methyl-tetrahydrofolate (CH3-THF), 5,10-methylene-THF (CH2-THF), and 10-formyl-THF (10-CHO-THF)] was determined by analysis of two metabolic end products of one-carbon metabolism, choline and adenine. Glycine-derived13C-labeled one-carbon units are incorporated into these two metabolites; dilution of the13C indicates competition by the unlabeled one-carbon source. The results reveal that the contribution from formate, folinic acid, and glycine is different for each of the one-carbon pools. Formate competed most dramatically at the 10-CHO-THF pool, with decreasing competition into the CH2-THF and CH3-THF pools. In a mutant strain lacking cytosolic CH2-THF dehydrogenase activity, a distinct shift toward the use of glycine instead of formate as the source of one-carbon units for the more reduced pools (CH2-THF and CH3-THF) was observed, while 10-CHO-THF pools were not affected. In contrast, the formyl group of folinic acid competed almost exclusively at the 10-CHO-THF level, with barely detectable dilution of the CH2-THF and CH3-THF pools in wild-type cells. The mutant strain exhibited essentially identical results, confirming that 5-formyl-THF enters the active one-carbon pool at the level of 10-CHO-THF, presumably via 5,10-methenyl-THF. Furthermore, donation of one-carbon units by folinic acid was observed only when cells were depleted of THF by treatment with the dihydrofolate reductase inhibitor methotrexate. These results reveal that the state of equilibrium between one-carbon pools in a growing cell depends on the source of the one-carbon units. This work illustrates the power of13C NMR for examining thein vivoutilization of alternative one-carbon donors under a variety of conditions.

Efficient product clearance through exit channels in substrate hydrolysis by acetylcholinesterase

The channels connecting the active site of acetylcholinesterase (AChE) to the protein exterior were mapped by computational techniques in order to find potential exit routes for charged reaction products. 3.9% of the total volume of the AChE monomer is hollow space and over 50% of the void is located in the center; it is partitioned into three chambers, a deep entry channel, below it a wide channel located in a slightly positive region of AChE and ideally suitable for the exit of negatively charged fragments and a small chamber above Trp 84 and Met 83. The latter serve as gates for the departure of the positively charged choline product of the hydrolysis of acetylcholine into the small cavity. An efficient product clearance is a prerequisite to a very low energy pathway for the irreversible hydrolysis of acetylcholine.

Effects of ?-tocopherol and its acetate on the hydrolytic activity of phospholipase D in egg yolk phosphatidylcholine bilayers

Effects of α-tocopherol (Toc) and α-tocopherol acetate (TocA) on the hydrolysis activity of phospholipase D (fromStreptomyces chromofuscus) were studied in small unilamellar vesicles (SUV) of egg yolk phosphatidylcholine (PC). Choline produced in the reaction was monitored by use of a choline oxidase — oxygen electrode. Addition of 18 mol% Toc into SUV (2 mM PC) brought about a twofold rate of choline production. On the other hand, the effect of 18 mol% TocA in SUV was very small. The apparent maximum velocity,Vmax(app), increased by addition of Toc in SUV. The apparent Michaelis constant,Km(app), was unchanged by addition of Toc and TocA in SUV. The Toc and TocA molecules did not have significant effects when PC was solubilized in the micelles of heptaethylene glycol dodecyl ether. The effects of Toc and TocA are, therefore, not due to specific ones on the enzyme itself, but rather upon the bilayer-organization of the substrate. Measurements of spreading pressure showed complete miscibility of PC and Toc, and limited mutual solubility of PC and TocA, suggesting stronger attractive interactions between Toc and PC than those between TocA and PC in the bilayers.

ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity

The hydrolysis of phosphatidylcholine by phospholipase D (PLD) results in the production of phosphatidic acid and choline. An assay that uses an exogenous substrate was developed to measure this activity in membranes and solubilized preparations from HL60 cells. A cytosolic factor markedly enhanced PLD activity in membranes and was essential for GTPγS-dependent stimulation of an enriched preparation of PLD. The factor was purified to homogeneity from bovine brain cytosol and identified as a member of the ADP-Ribosylation Factor (ARF) subfamily of small G proteins. Subsequently, recombinant myristoylated ARF1 was found to be a better activator of PLD activity than was the nonmyristoylated form. ARF proteins have been implicated recently as factors for regulation of intracellular vesicle traffic. The current finding suggests that PLD activity plays a prominent role in the action of ARF and that ARF may be a key component in the generation of second messengers via phospholipase D.

Effects of 1,2-diacylglycerol and cholesterol on the hydrolysis activity of phospholipase D in egg-yolk phosphatidylcholine bilayers

Effects of cholesterol (Chol) and 1,2-diacylglycerol (DAG) on the hydrolysis activity of phospholipase D (from Streptomyces chromofuscus) were studied in small unilamellar vesicles (SUV) of egg-yolk phosphatidylcholine (PC). 1,2-Diacylglycerol used here is derived from PC. Choline produced in the reaction was monitored by using a choline oxidase-oxygen electrode. Addition of 18.3 mol% Chol into SUV (2 mM PC) led to a small increase in the reaction rate. On the other hand, 18.3 mol% DAG in SUV brought about at 5–6-fold rate of choline production. The apparent maximum velocity, V max(app), increased by addition of DAG and Chol in SUV. In PC/Chol-SUV, the effect of increase in V max(app) was largely compensated by the increase in the apparent Michaelis constant, K m(app). The Chol and DAG molecules did not have significant effects on the kinetic parameters, when PC was solubilized in the micelles of heptaethylene glycol dodecyl ether. The effects of Chol and DAG are, therefore, not due to specific ones on the enzyme itself, but rather upon the bilayer-organization of the substrate. We discuss the activation of phospholipase D in terms of the influences of DAG and Chol on the structure of hydrophilic region and fluidity of the bilayers.

Anticholinesterase activity measurement by a choline biosensor: application in water analysis

A choline amperometric biosensor was assembled and used to measure the anticholinesterase activity due to compounds (which have the property to inhibit cholinesterase enzymes) present in water samples. This parameter can be used as a ‘toxicological index’, defined as the amount of compound which causes a certain percentage of cholinesterase inhibition equivalent to a known amount of a reference compound causing the same percentage inhibition. The organophosphorus insecticide Paraoxon, which has proved to be a strong inhibitor of cholinesterase enzymes, was chosen as the reference compound. The analysis was carried out by monitoring the decrease of cholinesterase activity in the presence of a pesticide and a substrate specific for the enzyme whose reaction produces choline. The decrease in choline production was measured by the choline sensor and correlated to the concentration of anticholinesterase compound present in the solution. Parameters such as buffer, pH, temperature and incubation time were optimized. The rate constant K i was calculated experimentally for Paraoxon and used in the anticholinesterase activity measurements at different fixed incubation times. The probe was calibrated with different standard solutions of Paraoxon. The effect of Paraoxon and heavy metals on the choline probe was evaluated. This probe was then used for the determination of anticholinesterase activity of some organophosphorus pesticides, and heavy metals in spiked waters. Samples were also analysed by liquid/liquid extraction and GC determination. Results seem to correlate with acute toxicity expressed as LD 50 (oral, rat). Analysis of water samples from different sources in central Italy were analysed for total anticholinesterase activity (TAA) and compared with a reference procedure.

Differential pathways (phospholipase C and phospholipase D) of bradykinin-induced biphasic 1,2-diacylglycerol formation in non-transformed and K-ras-transformed NIH-3T3 fibroblasts. Involvement of intracellular Ca2+ oscillations in phosphatidylcholine breakdown.

Bradykinin (BK) induced a biphasic increase in 1,2-diacylglycerol (DAG) in both K-ras-transformed fibroblasts (DT) and the parent NIH-3T3 cells. The first phase was coincident with the increase in Ins(1,4,5)P3 resulting from PtdIns(4,5)P2 hydrolysis, and the second, sustained, phase was derived from phosphatidylcholine (PtdCho) hydrolysis. In NIH-3T3 cells, stimulation by BK induced greater production of choline than phosphocholine in [3H]choline-labelled cells and appreciable phosphatidylethanol (PtdEtOH) formation in [3H]myristic acid-labelled cells, suggesting that PtdCho was hydrolysed mainly by a phospholipase D (PLD) activity. Pretreatment with propranolol, an inhibitor of phosphatidate phosphohydrolase, markedly diminished the second DAG accumulation, supporting the above notion. In DT cells, BK induced predominantly phosphocholine generation and little PtdEtOH formation, indicating that the PtdCho hydrolysis was due to a phospholipase C (PLC) activity. The BK-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) observed in single DT cells [Fu, Sugimoto, Oki, Murakami, Okano & Nozawa (1991) FEBS Lett. 281, 263-266] were detected as a sustained [Ca2+]i elevation when assayed in a cell suspension. A receptor-operated Ca2+ channel blocker, SK&F 96365, suppressed both the BK-induced phosphocholine generation and the sustained [Ca2+]i elevation in a similar dose-dependent manner. These results thus suggested that oscillations in [Ca2+]i are involved in the activation of PtdCho-specific PLC in DT cells.

Activation of phospholipase D by protein kinase C. Evidence for a phosphorylation-independent mechanism.

The role of protein kinase C (PKC) in the regulation of phosphatidylcholine-hydrolyzing phospholipase D (PLD) was investigated. In membranes from Chinese hamster lung fibroblasts that had been incubated with [14C]choline to label endogenous phosphatidylcholine, phorbol 12-myristate 13-acetate (PMA) failed to stimulate production of [14C]choline. However, stimulation was observed if fibroblast cytosolic fraction or PKC partially purified from this fraction was added. When incubated with membranes in the presence of PMA, pure PKC from rat brain stimulated [14C]choline production in a concentration-dependent manner, with a maximal 2-3-fold effect. PMA similarly stimulated [14C]phosphatidylpropanol formation from propanol using membranes from [14C]myristic acid-prelabeled cells, confirming the activation of PLD. None of the effects described required exogenous ATP. To probe the role of phosphorylation in the PKC effect, we included high concentrations of apyrase in the assay. This ATPase had no effect on the ability of PKC to activate PLD, but under exactly the same conditions, it eliminated autophosphorylation of PKC. The results provide conclusive evidence for the involvement of PKC in the activation of PLD and suggest that ATP-dependent phosphorylation is not required.

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.