Phospholipase C inhibitors and prostaglandins differentially regulate phosphatidylcholine synthesis in rat renal papilla: Evidence of compartmental regulation of CTP:phosphocholine cytidylyltransferase and CDP-choline:1,2-diacylglycerol cholinephosphotransferase

Phosphatidylcholine (PC) is the most abundant phospholipid in mammalian cell membranes. Several lines of evidence support that PC homeostasis is preserved by the equilibrium between PC biosynthetic enzymes and phospholipases catabolic activities. We have previously shown that papillary synthesis of PC depends on prostaglandins (PGs) that modulate biosynthetic enzymes. In papillary tissue, under bradikynin stimulus, arachidonic acid (AA) mobilization (the substrate for PG synthesis) requires a previous phospholipase C (PLC) activation. Thus, in the present work, we study the possible involvement of PLC in PC biosynthesis and its relationship with PG biosynthetic pathway on the maintenance of phospholipid renewal in papillary membranes; we also evaluated the relevance of CDP-choline pathway enzymes compartmentalization. To this end, neomycin, U-73122 and dibutiryl cyclic AMP, reported as PLC inhibitors, were used to study PC synthesis in rat renal papilla. All the PLC inhibitors assayed impaired PC synthesis. PG synthesis was also blocked by PLC inhibitors without affecting cyclooxygenase activity, indicating a metabolic connection between both pathways. However, we found that PC biosynthesis decrease in the presence of PLC inhibitors was not a consequence of PG decreased synthesis, suggesting that basal PLC activity and PGs exert their effect on different targets of PC biosynthetic pathway. The study of PC biosynthetic enzymes showed that PLC inhibitors affect CTP:phosphocholine cytidylyltransferase (CCT) activity while PGD₂ operates on CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT), both activities associated to papillary enriched-nuclei fraction. The present results suggest that renal papillary PC synthesis is a highly regulated process under basal conditions. Such regulation might occur at least at two different levels of the CDP-choline pathway: on the one hand, PLC operates on CCT activity; on the other, while PGs regulate CPT activity.     

Phospholipase C inhibitors and prostaglandins differentially regulate phosphatidylcholine synthesis in rat renal papilla. Evidence of compartmental regulation of CTP:phosphocholine cytidylyltransferase and CDP-choline:1,2-diacylglycerol cholinephosphotransferase

Phosphatidylcholine (PC) is the most abundant phospholipid in mammalian cell membranes. Several lines of evidence support that PC homeostasis is preserved by the equilibrium between PC biosynthetic enzymes and phospholipases catabolic activities. We have previously shown that papillary synthesis of PC depends on prostaglandins (PGs) that modulate biosynthetic enzymes. In papillary tissue, under bradikynin stimulus, arachidonic acid (AA) mobilization (the substrate for PG synthesis) requires a previous phospholipase C (PLC) activation. Thus, in the present work, we study the possible involvement of PLC in PC biosynthesis and its relationship with PG biosynthetic pathway on the maintenance of phospholipid renewal in papillary membranes; we also evaluated the relevance of CDP-choline pathway enzymes compartmentalization. To this end, neomycin, U-73122 and dibutiryl cyclic AMP, reported as PLC inhibitors, were used to study PC synthesis in rat renal papilla. All the PLC inhibitors assayed impaired PC synthesis. PG synthesis was also blocked by PLC inhibitors without affecting cyclooxygenase activity, indicating a metabolic connection between both pathways. However, we found that PC biosynthesis decrease in the presence of PLC inhibitors was not a consequence of PG decreased synthesis, suggesting that basal PLC activity and PGs exert their effect on different targets of PC biosynthetic pathway. The study of PC biosynthetic enzymes showed that PLC inhibitors affect CTP:phosphocholine cytidylyltransferase (CCT) activity while PGD(2) operates on CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT), both activities associated to papillary enriched-nuclei fraction. The present results suggest that renal papillary PC synthesis is a highly regulated process under basal conditions. Such regulation might occur at least at two different levels of the CDP-choline pathway: on the one hand, PLC operates on CCT activity; on the other, while PGs regulate CPT activity.     

Solubilization and purification of rat liver microsomal 1,2-diacylglycerol: CDP-choline cholinephosphotransferase and 1,2-diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase.

1. The procedure, which involved 2-step sonication of microsomes at pH 7.4 and then at pH 8.5 in the presence of sodium deoxycholate and subsequent dialysis, resulted in 4-5-fold purification of choline-phosphotransferase and ethanolaminephosphotransferase with the yield of 40-50%. 2. Ethanolaminephosphotransferase was further purified 8.5-fold over microsomes by sucrose density gradient centrifugation of the partially purified preparation, while cholinephosphotransferase activity was considerably lost during this procedure. No separation of the two transferases from each other was achieved at this step. 3. Cholinephosphotransferase required Mg2+ as cofactor, and microsomal phospholipids for its maximal activity. On the other hand, Mn2+ was more effective than Mg2+ as cofactor for ethanol aminephosphotransferase, and this enzyme was inhibited by microsomal phospholipids. 4. Both transferases were stimulated several-fold by sodium deoxycholate and also showed similar optimal pH ranging from pH 8.0 to 8.5. 5. Km values for 1,2-diacylglycerol emulsion were 81.0 muM for cholinephosphotransferase and 63.0 muM for ethanolaminephosphotransferase, respectively. CDP-choline and CDP-ethanolamine competitively inhibited, with the same Ki value (both 350 muM), ethanolaminephosphotransferase and cholinephosphotransferase, respectively. The Ki values obtained were much greater than the corresponding Km values for the cytidine substrates (36.4 muM for CDP-choline and 22.0 muM for CDP-ethanolamine). 6. The partially purified enzymes were further treated with Triton X-100. When enzyme activities were assayed with Mg2+, cholinephosphotransferase, although considerably inactivated, was partially separated from ethanolaminephosphotransferase by sucrose density gradient centrifugation of Triton-treated preparations. Furthermore, cholinephosphotransferase (but not ethanol-aminephosphotransferase) itself was partially separated into Mg2+ -requiring and Mn2+ -requiring components. In contrast, ethanolaminephosphotransferase assayed with either Mg2+ or Mn2+ formed a single peak together with Mn2+ -requiring cholinephosphotransferase.     

A CDP-choline pathway for phosphatidylcholine biosynthesis in Treponema denticola

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP-choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35-40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline-dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylcholine. The rate of production of another phospholipid in T. denticola, phosphatidylethanolamine, was elevated considerably in the licCA mutant, suggesting that the elevated level of this lipid compensated for the loss of phosphatidylcholine in the membranes. Thus it appears that T. denticola does contain a licCA-dependent CDP-choline pathway for phosphatidylcholine biosynthesis.     

Effects of fatty acids on phosphatidylcholine biosynthesis in isolated hamster heart

The effects of stearic, oleic, and arachidonic acids on phosphatidylcholine biosynthesis in the hamster heart were investigated. When hamster hearts were perfused with labelled choline in the presence of fatty acids, biosynthesis of phosphatidylcholine was stimulated only by stearic acid. Stearic acid was found to accumulate in unesterified (free) form in the hamster heart after perfusion. The stimulation by stearic acid was mediated in vivo by an enhancement of CTP:phosphocholine cytidylyltransferase activity in the microsomal fraction of the hamster heart and the enzyme activity in the cytosolic fraction was not affected. In contrast with the observations in rat hepatocytes, cytidylyltransferase from the hamster heart was not stimulated directly by stearic acid. The selective activation of the microsomal enzyme when the heart was perfused with stearic acid suggests that activation of the enzyme was mediated via the modification of the membrane by stearic acid.     

Regulation of phosphatidylcholine biosynthesis in Chinese hamster ovary cells by reversible membrane association of CTP: phosphocholine cytidylyltransferase

Treatment of Chinese hamster ovary cells with phospholipase C was previously shown to stimulate the CDP-choline pathway for phosphatidylcholine biosynthesis, and to cause activation of the CTP:phosphocholine cytidylyltransferase with a concomitant change in subcellular location of the enzyme (Sleight, R., and Kent, C. (1983) J. Biol. Chem. 258, 831-835). This paper presents a detailed analysis of the early events in the phospholipase C treatment, and provides evidence that the increased cytidylyltransferase activity causes the increased flux through the pathway. The time courses for the increase in cytidylyltransferase activity, increase in amount of membrane-associated enzyme, decrease in phosphocholine levels, and increase in phosphatidylcholine synthesis were similar, with all changes occurring within 30 min after addition of phospholipase C. These events preceded a decrease in cellular choline levels which correlated with a decreased capacity for choline uptake. The rate at which radioactive label was lost from pulse-labeled phosphocholine was the same as the rate at which label was incorporated into phosphatidylcholine, and these rates were stimulated 2.2-fold by phospholipase C treatment. We have also shown that the association of cytidylyltransferase with membranes was rapidly reversible when phospholipase C was removed from the cultures, and that the rate of decrease in phosphatidylcholine synthesis paralleled the rate of decrease in cytidylyltransferase activity. Cytidylyltransferase became reassociated with membranes when phospholipase C was added back to cultures from which it was previously removed. These results represent the first detailed account of the time frame involved in regulating phosphatidylcholine synthesis by the reversible association of cytidylyltransferase with cellular membranes.     

CDP-choline: 1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. II. Photoaffinity labeling by radioactive CDP-choline analogs.

Photoaffinity labeling of cholinephosphotransferase from rat liver microsomes directly by its substrate, [32P]CDP-choline or by a synthetic photoreactive CDP-choline analog, 3'(2')-O-(4-benzoyl)benzoyl [32P]CDP-choline (BB-[32P]CDP-choline), was examined for the possible identification of its molecular form on subsequent SDS-PAGE followed by 32P-autoradiography. When the partially purified cholinephosphotransferase was photoirradiated in the presence of [32P]CDP-choline, a considerable amount of 32P-radioactivity was incorporated into the TCA-insoluble component. This incorporation was dependent on irradiation time, Mg2+ or Mn(2+)-requiring and inhibited strongly by the presence of Ca2+. Either CDP-choline or CDP-ethanolamine inhibited the ultraviolet irradiation-dependent incorporation of 32P-radioactivity into the TCA-insoluble component in a dose-dependent manner, whereas neither phosphocholine or 5'-CDP had any effect on this process. These results strongly suggested that the observed 32P-incorporation from [32P]CDP-choline into the protein component could be a consequence of the covalent interaction between cholinephosphotransferase and its substrate, [32P]CDP-choline. Two polypeptides, 25 kDa and 18 kDa, with high 32P-radioactivity were clearly identified on a SDS gel after the direct photoaffinity labeling with [32P]CDP-choline for more than 5 min of ultraviolet irradiation. On the other hand, when BB-[32P]CDP-choline was used as a photoaffinity ligand, a single polypeptide with apparent molecular size of 55 kDa could be rapidly photolabeled within 2.5 min, then this band gradually lost its 32P-radioactivity with increasing time of ultraviolet irradiation. Thus, the overall results strongly indicated that cholinephosphotransferase in rat liver microsomes exists most likely as a 55 kDa polypeptide (or subunit) and that 25 kDa and 18 kDa peptides identified after the direct photoaffinity labeling with [32P]CDP-choline were probably the photo-cleavage products of cholinephosphotransferase during the prolonged ultraviolet irradiation, both of which could contain the catalytic domain of the original enzyme protein(s).     

Alteration of 1,2-diacylglycerol content in ischemic and reperfused heart

Human term placenta contains an ATP diphosphohydrolase activity which hydrolyses ATP to ADP and inorganic phosphate and ADP to AMP and a second mole of inorganic phosphate. The activity has a pH optimum between 8.0 and 8.5. Magnesium or calcium ions are required for maximum activity. Other nucleoside phosphates, p-nitrophenyl phosphate or sodium pyrophosphate, are not hydrolysed. The activity is not due to ATPases, or to myokinase, as determined by the use of inhibitors. NaF and NaN₃ were found to inhibit strongly the activity thus identifying it as an ATP diphosphohydrolase.A sensitive enzymatic assay for measurement of AMP, one of the products of the reaction, was established, based on the strong inhibition of muscle fructose 1,6-biphosphatase by AMP. The range of the assay was 0.05–0.8 µM AMP. ATP diphosphohydrolase was found to have a rate of AMP production from ADP twice the rate from ATP. Under the same conditions, the assay for Pi release, on the other hand, gave velocities similar to each other for the two substrates.The activity appears to be identical to the ADP-hydrolysing activity in placenta reported by others.Abbreviations Ap₅A P¹ - P⁵-di(adenosine-5) Pentaphosphate - ATP-DPH ATP Diphosphohydrolase - DCCD N,N Dicyclohexycarbodiimide - Fru-P₂ase Fructose 1,6-biphosphatase - SDS Sodium Dodecyl Sulfate - TLC Thin Layer Chromatography     

Increased 1,2-diacylglycerol content in myopathic hamster hearts at a prenecrotic stage

1,2-Diacylglycerol (DAG) has been suggested to be a secondary messenger. In this study, we determined the amount of 1,2-DAG in heart tissue from Syrian hamsters with hereditary cardiomyopathy at 30 days (prenecrotic stage) and 90 days of age by thin-layer chromatography with flame ionization detection (TLC-FID). Myocardial triglyceride contents were higher at 30 days of age and lower at 90 days of age compared to the levels in age-matched normal hamsters. Decreases in major species of phospholipids in hearts were observed only at 90 days of age. However, elevated 1,2-DAG content in myopathic hearts was found at 30 days of age, whereas there was no difference between the two groups at 90 days of age. It is suggested that the increase in 1,2-DAG at the prenecrotic stage is involved in the pathogenesis of the cardiomyopathy.     

Effect of amino acids on choline uptake and phosphatidylcholine biosynthesis in the isolated hamster heart

Choline uptake by the hamster heart has been shown to be enhanced by exogenous glycine. In this study, the effect of neutral, basic, and acidic amino acids on choline uptake was assessed. Hamster hearts were perfused with labelled choline, and in the presence of L-alanine, L-serine, or L-phenylalanine (greater than or equal to 0.1 mM), choline uptake was enhanced 20-38%. L-Arginine, L-lysine, L-aspartate, and L-glutamate did not influence choline uptake. The rate of phosphatidylcholine biosynthesis was unaffected by all amino acids tested. Enhancement of choline uptake by neutral amino acids was not additive or dose dependent but required a concentration threshold. The enhancement of choline uptake by neutral amino acids was not influenced by preperfusion with the same amino acid. Exogenous choline had no effect on the uptake of amino acids. We postulate that choline and the neutral amino acids are not cotransported and modulation of choline uptake is facilitated by direct interaction of the neutral amino acids with the choline transport system.     

CDP-choline significantly restores phosphatidylcholine levels by differentially affecting phospholipase A2 and CTP: phosphocholine cytidylyltransferase after stroke

Phosphatidylcholine (PtdCho) is a major membrane phospholipid, and its loss is sufficient in itself to induce cell death. PtdCho homeostasis is regulated by the balance between hydrolysis and synthesis. PtdCho is hydrolyzed by phospholipase A2 (PLA2), PtdChospecific phospholipase C (PtdCho-PLC), and phospholipase D (PLD). PtdCho synthesis is rate-limited by CTP:phosphocholine cytidylyltransferase (CCT), which makes CDP-choline. The final step of PtdCho synthesis is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase. PtdCho synthesis in the brain is predominantly through the CDP-choline pathway. Transient middle cerebral artery occlusion (tMCAO) significantly increased PLA2 activity, secretory PLA2 (sPLA2)-IIA mRNA and protein levels, PtdCho-PLC activity, and PLD2 protein expression following reperfusion. CDP-choline treatment significantly attenuated PLA2 activity, sPLA2-IIA mRNA and protein levels, and PtdCho-PLC activity, but did not affect PLD2 protein expression. tMCAO also resulted in loss of CCT activity and CCTalpha protein, which were partially restored by CDP-choline. No changes were observed in cytosolic PLA2 or calcium-independent PLA2 tMCAO. protein levels after Up-regulation of PLA2, PtdCho-PLC, and PLD and regulation of CCT collectively down-resulted in loss of PtdCho, which was significantly restored by CDP-choline treatment. CDP-choline treatment significantly attenuated the infarction volume by 55 +/- 5% after 1 h of tMCAO and 1 day of reperfusion. Taken together, these results suggest that CDP-choline significantly restores Ptd-Cho levels by differentially affecting sPLA2-IIA, PtdCho-PLC, and CCTalpha after transient focal cerebral ischemia. A hypothetical scheme is proposed integrating results from this study and from other reports in the literature.     

Trifluoperazine and chlorpromazine inhibit phosphatidylcholine biosynthesis and CTP:phosphocholine cytidylyltransferase in HeLa cells

The influence of chlorpromazine and trifluoperazine on phosphatidylcholine biosynthesis in HeLa cells was investigated. HeLa cells were prelabeled with [Me-3H]choline for 1 h. The cells were subsequently incubated with various concentrations of drugs. Both compounds were potent inhibitors of phosphatidylcholine biosynthesis, with 50% inhibition by 5 micron of either drug. Analysis of the radioactivity in the soluble precursors indicated a block in the conversion of phosphocholine to CDPcholine catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15). Inhibition by these drugs was slowly reversed after incubation for more than 2 h, or was immediately abolished when 0.4 mM oleate was included in the cell medium or when the drug-containing medium was removed. The subcellular location of the cytidylyltransferase was unaffected by either drug, nor did the drugs alter the rate of release of cytidylyltransferase from HeLa cells by digitonin treatment. The drugs had a direct inhibitory effect on cytidylyltransferase activity in HeLa cell postmitochondrial supernatants. Half-maximal inhibition was achieved with 30 microM trifluoperazine and 50 microM chlorpromazine. These drugs did not change the apparent Km of the cytidylyltransferase for CTP or phosphocholine. Inhibition of cytidylyltransferase by these compounds was reversible with exogenous phospholipid or oleate in the enzyme assay. The data indicate that both drugs inhibit phosphatidylcholine synthesis by an effect on the cytidylyltransferase. The mechanism of action remains unknown at this time.     

sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism

A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation. Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods. Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km. Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition. A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor. Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 15062-15069). Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes. The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes.     

Involvement of 1,2-diacylglycerol in improvement of heart function by etomoxir in diabetic rats

Abnormal lipid metabolism has been proposed to be involved in the pathogenesis of diabetic cardiomyopathy. In this study, we measured myocardial lipid levels, including 1,2-diacylglycerol (1,2-DAG) and ceramide (CM), and myocardial function in diabetic rats. We also evaluated the effects of etomoxir (ETM), a carnitine palmitoyl transferase I inhibitor, on diabetic rat hearts from the viewpoints of alterations in lipid second messengers and myocardial function. Rats were injected with streptozotocin (60 mg/kg) to induce diabetes and were treated 5 weeks later with ETM (18 mg/kg) for 8 days. In diabetic rats, heart rate, systolic blood pressure, and fractional shortening were significantly reduced compared with those in controls. Treatment of diabetic rats with ETM ameliorated myocardial dysfunction other than heart rate. Myocardial 1,2-DAG levels in diabetic rats were significantly elevated compared with those in controls, while myocardial CM levels were not. ETM treatment caused an additional increase in myocardial 1,2-DAG levels in diabetic rats, but the CM levels did not change. There was a marked difference in fatty acid pattern of 1,2-DAG between diabetic and ETM-treated diabetic rat hearts. The fatty acids 18:1 and 18:2 were significantly increased and the fatty acids 16:0, 18:0, 20:4, and 22:6 were significantly reduced in ETM-treated diabetic rat hearts. These data suggest 1,2-DAG is involved in ameliorating myocardial dysfunction in diabetic rats and that its source is different between diabetic and ETM-treated diabetic rats. CM is unlikely to be involved in the pathogenesis of diabetic cardiomyopathy or the improvement of cardiac contractility in diabetic rats by ETM.     

Co-ordinate control of CDP-choline and phosphatidylethanolamine methyltransferase pathways for phosphatidylcholine biosynthesis occurs in response to change in diet fat

Brain microsomal and synaptic plasma membrane phosphatidylcholine composition and biosynthetic activity were examined in relation to the composition of diet fat fed. Phosphocholinetransferase and methyltransferase activities are shown to be modulated by the diet, and by changes in the membrane phospholipid content of long-chain polyunsaturated fatty acids. This physiological modulation is co-ordinated such that the rate of phosphatidylcholine synthesis via one route is inversely regulated with activity of the alternate pathway.     

Modulation of biosynthesis of phosphatidylcholine via CDP-choline in rat liver: Influence of ethanol on the microsomal cholinephosphotransferase activity

We have studied in vitro the effects of ethanol on the different enzymes involved in the biosynthesis of phosphatidylcholine (PC) via CDP-choline. Ethanol alters neither choline kinase (CK) nor CTP:phosphocholine cytidylyltransferase (CT) activities but, at levels higher than 50 mM, it does significantly inhibit microsomal cholinephosphotransferase (CPT) activity concomitantly with an increase in the ethanol concentration. A study of the kinetics of the reaction catalysed by CPT shows that ethanol decreases V_max without altering K_m, indicating a non-competitive inhibitory effect. An analysis of the thermodependence of CPT activity in the absence of ethanol reveals a break in the Arrhenius plot and thus a straight relationship between enzyme activity and the physico-chemical state of the microsomal membrane. Incubation of microsomes in the presence of ethanol increased the transition temperature from 25.8–28.2°C. Microsomes were also incubated with n-alkanols with chain-lengths of fewer than five carbon atoms at concentrations which, according to their partition coefficients, produce equimolar levels in the membrane. Under these conditions all the alkanols caused the same inhibitory effect. All these results demonstrate that ethanol modulate the PC biosynthesis at the level of CPT activity and does not affect the CT enzyme. The inhibition found on CPT is clearly dependent on the alteration produced by ethanol on the hepatic microsomal membrane.