Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices

Noradrenaline- and ionomycin-stimulated as well as basal phospholipase D activity from rat hippocampus produced, in the presence of ethanol, two different classes of [32P]phosphatidylethanol (designated I and II), which were separated by thin layer chromatography. Endogenous labeling experiments using 3H-fatty acids showed that two different classes of phosphatidylcholine, separated by two-dimensional TLC, one enriched with high incorporation of [3H]arachidonic acid (B) and the other with [3H]myristic acid (A), were the most likely sources for the two classes of phosphatidylethanol. Experiments where individual 32P-phospholipids extracted from [32P]Pi-labeled hippocampal slices were incubated with cabbage phospholipase D, in the presence of ethanol, showed that each class of [32P]phosphatidylcholine, i.e. A and B, produced a different band of [32P]phosphatidylethanol, with the same mobility in TLC as phosphatidylethanol II and I, respectively.     

Contribution of phosphoinositides and phosphatidylcholines to the production of phosphatidic acid upon concanavalin A stimulation of rat thymocytes

Stimulation of rat thymocytes by concanavalin A (Con A) results in a very early increase of the cellular level of phosphatidic acid (PA), while that of diacylglycerol (DAG) was not affected. As the biological activity of PA is very likely to be determined by its molecular species composition, the present study aims to investigate the pathways leading to the production of PA in Con A-stimulated rat thymocytes. Prelabeling the cells with [3H]arachidonic acid, [3H]myristic acid, [3H]choline, or [14C]lysophosphatidylcholine allowed us to determine that PA is formed by both phosphoinositide (PIs) and phosphatidylcholine (PC) hydrolysis. We then investigated whether PA derived from PC was formed by phospholipase C (PLC) or phospholipase D (PLD) hydrolysis. In the presence of 1-butanol, the production of phosphatidylbutanol was only observed in tetradecanoyl phorbol acetate (TPA)-stimulated cells. The use of a specific PC phospholipase C inhibitor resulted in a decrease of Con A-stimulated PA production in cells labeled with [3H]myristate. When cells were labeled with [3H]choline, only TPA stimulation induced a release of labeled choline. All together, these experiments suggest that PA is originated from two phospholipid sources, predominantly PI via PLC hydrolysis and to a lesser extent PC, by PLC hydrolysis also. Molecular species analyses by reverse phase HPLC are in agreement with this hypothesis, as diacyl-GP molecular species composition is similar to that of diacyl-GPC and DAG in resting cells, but resembles that of diacyl-GPI in Con A-treated cells. Thus, in stimulated cells, the amount of 18:0/20:4 species doubled while those of saturated and monounsaturated species decreased.     

Evidence that ceramide selectively inhibits protein kinase C-alpha translocation and modulates bradykinin activation of phospholipase D

Sphingomyelinase (SMase) treatment (0.1 unit/ml for up to 30 min) of mouse epidermal (HEL-37) or human skin fibroblast (SF 3155) cells preincubated with [3H]serine to label the sphingomyelin pool caused the accumulation of labeled ceramide but not sphingosine or ceramide 1-phosphate. Incubation of HEL-37 cells with dioctanoylglycerol (diC8) or SF 3155 cells with bradykinin caused translocation of calcium/phosphatidylserine-dependent protein kinase C (PKC) activity to particulate material. In both cell lines the translocation was blocked by SMase treatment of the cells or by incubation with the cell-permeable ceramide analogue N-acetylsphingosine (C2-Cer). Western blot analysis indicated that treatment of HEL-37 cells with diC8 or SF 3155 cells with bradykinin resulted in the translocation of both PKC-alpha and PKC-espilon to particulate material. Treatment with SMase or C2-Cer specifically blocked the translocation of PKC-alpha but not that of PKC-epsilon. Pretreatment of cells with SMase or C2-Cer also inhibited the activation of phospholipase D activity induced by either diC8 (HEL-37 cells) or bradykinin (SF 3155 cells). The data provide strong evidence that ceramide can negatively regulate the translocation of PKC-alpha but not PKC-epsilon and further suggest that PKC-alpha may be involved in regulating phospholipase D activity.     

Regulation of phospholipase D activity in a human oligodendroglioma cell line (HOG)

Oligodendroglial cells express many specific proteins, such as myelin basic protein (MBP), which are physiologically phosphorylated by protein kinase C (PKC). Diacylglycerols are physiological activators of PKC and can be liberated from phospholipids by the direct receptor-mediated activation of phospholipase C (PL-C) or indirectly via the activation of phospholipase D (PL-D). In a well-characterized human oligodendroglioma (HOG) cell line, PL-C (measured by release of [3H]inositol phosphates) and PL-D (formation of [3H]myristoylated or palmitoylated phosphatidylethanol) were activated by both carbachol (blocked by pirenzepine, suggesting an M1 receptor) and histamine (H1 receptor) but not glutamate, bradykinin, or phenylephrine. PL-C stimulation by carbachol or histamine was completely inhibited by short-term treatment (< 30 min) with phorbol ester (TPA), a PKC activator. In contrast, PL-D activation by either carbachol or histamine was stimulated in additive fashion by TPA, suggesting at least two distinct mechanisms for PL-D activation. Down regulation of PKC by prolonged (24 hr) treatment with TPA reversed the inhibitory effects of TPA on PL-C and the stimulatory effects on PL-D. However, the PKC inhibitors H-7 and galactosylsphingosine did not inhibit the TPA-mediated stimulation of PLD while the less-specific PKC inhibitor, staurosporine, was only partially inhibitory. Preexposure of cells to carbachol, greatly reduced both PL-C and PL-D activation by carbachol, suggesting homologous desensitization. Time-course studies indicated that PL-D activation (10 sec or less) was at least as fast as PL-C activation, and the affinity of carbachol and histamine for the receptor coupled to either phospholipase (EC50 = 5-10 microM) was about the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

8-OH-DPAT influences extracellular levels of serotonin and dopamine in the medial preoptic area of male rats

The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C. In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids. Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from [1,3-3H]glycerol indicating the involvement of 1,2-diacyl-sn glycerol. The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5'triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacylsn-glycerol levels. Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from [methyl-3H]choline was markedly stimulated. These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells.     

Sustained activation of phospholipase D via adenosine A3 receptors is associated with enhancement of antigen- and Ca(2+)-ionophore-induced secretion in a rat mast cell line

The adenosine analog, N-ethylcarboxamidoadenosine (NECA), causes transient activation of phospholipase C and an enhancement of antigen-induced secretion in a rat mast cell (RBL-2H3) line via adenosine A3-receptors (Ramkumar et al., J. Biol. Chem. 268:16887, 1993) by a mechanism that is inhibited by bacterial toxins and potentiated by dexamethasone (Ali et al., J. Biol. Chem. 265:745-753, 1990). Here we show that NECA synergizes the secretory response to Ca(2+)-ionophore as well as to antigen. The ability of NECA to synergize the secretory responses persisted for 10 to 20 min, long after the early phospholipase C-mediated reactions to NECA had subsided. NECA caused, however, a dose-dependent sustained activation of phospholipase D, as indicated by the formation of [3H]phosphatidic acid, or in the presence of 0.3% ethanol, [3H]phosphatidylethanol. This activation was associated with a sustained increase in diglycerides, in protein kinase C activity and in the phosphorylation of myosin light chains by protein kinase C. The generation of diglycerides was enhanced in dexamethasone-treated cells and suppressed in cells that had been treated with cholera toxin or pertussis toxin. Collectively, the studies suggested that the generation of diglycerides via phospholipase D and the associated activation of protein kinase C were, by themselves, insufficient signals for secretion in RBL-2H3 cells, but that these reactions synergized responses to stimulants such as antigen or A23187 that caused substantial increases in [Ca2+]i.     

Phospholipase D- and protein kinase C isoenzyme-dependent signal transduction pathways activated by the calcitonin receptor

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [3H] choline with an EC50 = 2.5 +/-0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 +/-1.0 x 10(-8)M). The hormone failed to induce release of [3H]phosphocholine and [3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-C alpha, but not the delta isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-C zeta, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.     

Lysosomal phospholipase activity is decreased in mucolipidosis II and III fibroblasts

Mucolipidosis (ML) II and III are rare autosomal recessively inherited diseases characterized by deficiency of multiple lysosomal enzymes and, as a result, a generalized storage of macromolecules in lysosomes of cells of mesenchymal origin. In ML II and ML III fibroblasts, most, but not all, newly synthesized lysosomal enzymes are secreted into the medium instead of being targeted correctly to lysosomes. Defects in the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase underlie this effect. It is unknown how lysosomal phospholipases are targeted to the lysosomes of fibroblasts. In the present study lysosomal phospholipase activity was determined in delipidated fibroblast homogenates and plasma from ML II and ML III patients and controls using a [3H]choline-labeled phosphatidylcholine. After incubation, residual phosphatidylcholine and its labeled degradation products (lysophosphatidylcholine, glycerophosphorylcholine and choline phosphate) were quantified. We found that ML II and ML III fibroblasts are deficient in lysosomal phospholipase A and C activity. These enzymes were present in elevated amounts in plasma of ML II and ML III patients. These data indicate that phospholipases, like most other lysosomal enzymes in these diseases, are secreted into the blood instead of being targeted specifically to lysosomes. Thus, the mannose-6-phosphate receptor pathway is needed for proper delivery of lysosomal phospholipases to lysosomes. We also found that production of labeled choline phosphate was mainly due to the activity of acid sphingomyelinase instead of phospholipase C under the assay conditions used. Other active lipolytic enzymes were phospholipase A and lysophospholipase. No evidence for phospholipase D activity was found.     

Acetylated low density lipoprotein inhibits the incorporation of arachidonic acid in phospholipids with a concomitant increase of cholesterol arachidonate in rat peritoneal macrophages

The aim of our work was to evaluate the influence of native low density lipoproteins (LDL) and LDL chemically modified by acetylation (acLDL) on incorporation and release of arachidonic acid (AA) in rat peritoneal macrophages. Compared to a control group without treatment, 100 micrograms/ml of acLDL for 15 h considerably increased the incorporation of [3H]AA in cholesterol-ester (CE) of rat peritoneal macrophages and induced a decrease of 3H-labeled membrane phospholipids (PL). No effect was shown with LDL treatment. In the presence of acLDL, LS3251 (100 nM), an acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, inhibited the [3H]AA incorporation into CE in macrophages. [3H]AA-prelabeled macrophages cultured for 15 h with acLDL (compared to macrophages untreated or treated with LDL) showed an increase of labeled CE and a decrease of labeled PL and of cyclooxygenase and lipoxygenase eicosanoid production. After zymosan stimulation of macrophages prelabeled with [3H]AA and treated with or without LDL or acLDL, AA release and eicosanoid production increased in all groups of macrophages. The inhibition of eicosanoid production in foam cells does not seem to be linked to an inhibition of phospholipase but rather paralleled to an increase of the cholesterol [3H]arachidonate. A significant portion of cellular arachidonate released from phospholipids, in particular from phosphatidylcholine, could serve as a substrate to ACAT in this foam cell.     

Health indices as predictors of cognition among older African Americans: MacArthur studies of successful aging

The purpose of this study was to visualize muscarinic receptors and their distribution on cardiomyocytes and to examine the effects of muscarinic cholinergic receptor (mACh-R) stimulation with carbachol on phosphatidylcholine hydrolysis. Cardiomyocytes were prepared as primary culture from 7-day-old chick embryo hearts. Cardiomyocytes, grown on cover slips, were labelled with BODIPY PZ, a fluorescent analog of the muscarinic receptor antagonist pirenzepine, and examined with a laser scanning confocal microscope, mACh-R clusters were visualized and were fairly homogeneous in size with diameters ranging from 0.5 to 1.0 micron. The number of receptor clusters per cell was 83.5 +/- 6.8 (mean +/- SEM) and clusters were found at the periphery of the cell. Cardiomyocytes, grown as a monolayer in dishes, were treated with the 10(-4) M carbachol, a mACh-R agonist, and the effects on phosphatidylcholine hydrolysis were ascertained in cells preincubated with [methyl-3H]choline for 18 h. Cells were washed, lysed, and subjected to thin-layer chromatography to separate [3H]choline in various metabolites of phosphatidylcholine. Carbachol significantly (p < 0.05) increased intracellular free choline and decreased cellular phospholipid consistent with phosphatidylcholine hydrolysis. Carbachol increased the amount of [3H]choline that effluxed out of the cardiomyocyte into the medium. Carbachol-induced choline efflux was not prevented by pretreatment with n-butanol, a phospholipase D inhibitor, suggesting that other lipases such as phospholipase C are the major enzyme involved in phosphatidylcholine hydrolysis. Pertussis toxin prevented carbachol-induced choline efflux and the changes in intracellular free choline and phospholipid. An action of carbachol through G proteins was supported by the ability of pertussis toxin to antagonize the carbachol-induced reduction in cAMP generation from isoproterenol. In summary, mACh-Rs, visualized in living cardiomyocytes, were peripheral to the nucleus. Phosphatidylcholine hydrolysis induced by mACh-R stimulation may be a signal transduction pathway for mACh-R in the cardiomyocyte, operating through inhibitory G proteins sensitive to pertussis toxin.     

Sphingosine induces phospholipase D and mitogen activated protein kinase in vascular smooth muscle cells

The enzymes phospholipase D and diacylglycerol kinase generate phosphatidic acid which is considered to be a mitogen. Here we report that sphingosine produced a significant amount of phosphatidic acid in vascular smooth muscle cells from the rat aorta. The diacylglycerol kinase inhibitor R59 949 partially depressed sphingosine induced phosphatidic acid formation, suggesting that activation of phospholipase C and diacylglycerol kinase can not account for the bulk of phosphatidic acid produced and that additional pathways such as phospholipase D may contribute to this. Further, we have shown that phosphatidylethanol was produced by sphingosine when vascular smooth muscle cells were stimulated in the presence of ethanol. Finally, as previously shown for other cell types, sphingosine stimulated mitogen-activated protein kinase in vascular smooth muscle cells.     

The deletion of six ORFs of unknown function from Saccharomyces cerevisiae chromosome VII reveals two essential genes: YGR195w and YGR198w

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2(3)H]glycerol-3-phosphocholine (PC), or [14C]ethanol. Using [3H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [3H]phosphatidic acid and phosphatidylethanol [3H]PtdEt; whereas with [14C]ethanol or [3H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [14C]PtdEt or [3H]phosphatidylglycerol, respectively). The use of varying concentrations of [3H]PC and 400 mM of ethanol gave an apparent Km value for PC of 0.51 mM and a Vmax value of 111 nmol x h(-1) x (mg protein)(-1). The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent Km of 202 mM and a Vmax value for ethanol of 125 nmol x h(-1) x (mg protein)(-1). A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

Characterization of phospholipase D activation by muscarinic receptors in human neuroblastoma SH-SY5Y cells

The cholinergic regulation of phospholipase D activity was studied in SH-SY5Y human neuroblastoma cells with phosphatidylethanol formation as a specific marker for the enzyme activity. The muscarinic antagonists, hexahydrosiladifenidol and pirenzepine, inhibited carbachol-induced phosphatidylethanol formation in a concentration-dependent manner and the inhibitory constants indicated that muscarinic M1 receptors are responsible for the major part of the phospholipase D activation. The mechanism of receptor-mediated phospholipase D activation varies between different cell types and receptors. In SH-SY5Y cells, the carbachol-induced phospholipase D activity was inhibited by protein kinase C inhibitors. Since both phospholipases D and C are activated by muscarinic stimulation in SH-SY5Y cells, most of the phospholipase D activation is probably secondary to the protein kinase C activation that follows phospholipase C-mediated increase in diacylglycerols. Other kinases may be involved in the regulation since also a tyrosine kinase inhibitor decreased the phosphatidylethanol formation. Stimulation of G-protein(s) and increase in the intracellular Ca2+ concentration activated phospholipase D and may be additional mechanisms for the muscarinic regulation of phospholipase D in SH-SY5Y cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, increased the carbachol-induced formation of phosphatidic acid at the expense of 1,2-diacylglycerol. This indicates that phospholipase D contributes to the formation of 1,2-diacylglycerol after carbachol stimulation in SH-SY5Y cells.     

Cholesterol efflux effect of high density lipoprotein is impaired by whole cigarette smoke extracts through lipid peroxidation

OBJECTIVE: The aim was to investigate the consequences of simultaneous stimulation of phospholipase C and D by agonists for the molecular species composition of 1,2-diacylglycerol and phospholipids in cardiomyocytes. METHODS: Serum-free cultured neonatal rat cardiomyocytes were stimulated by endothelin-1, phenylephrine or phorbolester. The molecular species of 1,2-diacylglycerol (in mol%) and those derived from phosphatidylcholine and phosphatidylinositol were analyzed by high-performance liquid chromatography and their absolute total concentration (nmol per dish) by gas-liquid chromatography. Phospholipids were labelled with [14C]glycerol or double-labelled with [14C]16:0 and [3H]20:4n6 for measurements of respectively, the amount of or relative rate of label incorporation into 1,2-diacylglycerol. RESULTS: The major molecular species of 1,2-diacylglycerol in unstimulated cells was found to be 18:0/20:4 (57 mol%). The same species was observed predominantly in phosphatidylinositol (73 mol% compared to 11 mol% in phosphatidylcholine). A significant decrease (about 10 mol%) was found for the 18:0/20:4 species of 1,2-diacylglycerol during stimulation (10-40 min) with endothelin-1 or phorbolester, but not phenylephrine. The results of the double-labelling experiments were consistent with the latter finding: the ratio [3H]20:4 over [14C]16:0 in 1,2-diacylglycerol decreased from 1.70 in the control to 1.40 during 10-min endothelin-1 or phorbolester stimulation, but not during phenylephrine stimulation. The [14C]glycerol incorporation into 1,2-diacylglycerol remained relatively constant under agonist-stimulated conditions as did the total concentration of 1,2-diacylglycerol. CONCLUSIONS: 1,2-Diacylglycerol present in unstimulated cardiomyocytes is likely derived from phosphatidylinositol. During stimulation with endothelin-1 and phorbolester, but not phenylephrine, phosphatidylcholine becomes an increasingly important source for 1,2-diacylglycerol due to sustained activation of phospholipase D. The 1,2-diacylglycerol level remains relatively constant during agonist stimulation which strongly indicates that particular molecular species of 1,2-diacylglycerol more than its total concentration determine the activation of protein kinase C isoenzymes.     

A proposed new mammalian cell protein kinase, associated with microtubules]

The effect of phospholipase A2 treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with Naja mocambique mocambique phospholipase A2 reduced the pool sizes of phosphatidylcholine and phosphatidylethanolamine compared with controls. The pool sizes of lysophosphatidylcholine and lysophosphatidylethanolamine were elevated, whereas the pool sizes of cardiolipin and other phospholipids were unaffected by phospholipase A2 treatment. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or preincubated in the absence or presence of phospholipase A2. In all experiments, radioactivity incorporated into cardiolipin was reduced in phospholipase A2-treated cells with time compared with controls, indicating attenuated de novo biosynthesis of cardiolipin. The mechanism for the reduction in cardiolipin biosynthesis in phospholipase A2-treated cells was a decrease in the activity of phosphatidic acid:cytidine-5'-triphosphate cytidylyltransferase, the rate-limiting enzyme of cardiolipin biosynthesis, mediated by elevated cellular lysophosphatidylcholine levels. The results suggest that de novo cardiolipin biosynthesis in H9c2 cells may be regulated by the cellular level of lysophosphatidylcholine.     

Mechanical assistance in cardiogenic shock after cardiac surgery

With [14C]oleate-labeled phosphatidylcholine as a substrate for phospholipase D the hydrolytic activity was measured by phosphatidic acid formation and the transphosphatidylation activity was measured by the phosphatidylethanol formed in the presence of ethanol. The pH optimum was 6.5 with dimethylglutarate as the buffer. EGTA inhibited the transphosphatidylation activity to a greater extent than the hydrolytic activity. In contrast CaCl2, BaCl2, MgCl2 and SrCl2 stimulated the hydrolytic activity without effecting the transphosphatidylation activity. BeCl2 another member of the group IIa transition metals was a very potent inhibitor of both the hydrolytic and transphophatidylation activity. GTP gamma S, an activator of G protein-mediated events, was an inhibitor of both activities.     

Dietary choline supplementation in pregnant rats increases hippocampal phospholipase D activity of the offspring

Supplementation with choline during pregnancy in rats causes a long-lasting improvement of visuospatial memory of the offspring. The biochemical mechanism of this effect may be related to the function of choline as a precursor of phosphatidylcholine (PC), the substrate of a receptor-stimulated enzyme, phospholipase D (PLD). PLD activation initiates the sequential formation of two intracellular messengers, phosphatidic acid and 1,2-sn-diacylglycerol. We hypothesized that prenatal choline status may cause long-term modulation of PLD-catalyzed PC hydrolysis in the hippocampus, a brain region implicated in visuospatial memory functions. PLD activity was determined in hippocampal slices prelabeled with [3H]glycerol or [3H]oleic acid by measuring the PLD-catalyzed formation of [3H]phosphatidylpropanol in the presence of 1-propanol. Slices were obtained from male pups born to mothers consuming a control diet, a choline-supplemented diet, or a choline-free diet from days 11 to 17 of pregnancy. The radiolabeling of phospholipid classes was unaffected by the treatments. Prenatal choline supplementation significantly increased basal PLD activity in [3H]glycerol-labeled slices [by 46% of controls on postnatal day (P) 7 and by 36% on P21], and [3H]oleate-labeled slices (by 91% on P7), as well as glutamate-stimulated PLD activity in [3H]oleate-labeled slices (by 60% on P7). Prenatal choline deficiency failed to alter PLD activity. The actions of choline apparently required intact cells because in vitro assays of PLD activity in hippocampal homogenates, using fluorescent NBD-PC as substrate, revealed no differences between groups. The results show that prenatal choline supplementation up-regulates basal and receptor-stimulated PLD activity in the hippocampus during postnatal development.     

Intestinal absorption of polyunsaturated phosphatidylcholine in the rat

The mechanism of intestinal absorption of polyunsaturated phosphatidylcholine in an oil medium was studied with 1,2-di-[9,10,12,13-3H4]linoleoyl-sn-glycero-3-phospho-[N-14CH3]-choline, 1-[1-14C]linoleoyl-2-[9,10,12,13-3H4]-linoleoyl- and 1-[9,10,12,13-3H4]linoleoyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine, especially with regard to the stability of the ester bonds in position 1 and 2 of the phospholipid molecule. The absorption rate, as measured by the disappearance from the gastro-intestinal tract, was comparatively rapid in the first 6 - 8 h, but then became considerably slower. After 24 h more than 90% of the applied radioactivity was absorbed from the intestinal tract. Respiratory 14CO2 from the degradation of the unsaturated acyl moiety in position 2 is produced much more rapidly than that from the acyl group attached to the 1-position of the glycerophosphocholine backbone. Analyses of the liver phosphatidylcholine by specific enzymatic hydrolysis with phospholipase A2, 6 h after the application, showed that in the isolated PC 9 times more labelled fatty acids from the original 1-position were present than from the corresponding 2-position. In rats with lymph fistula it was shown that more than 90% of the acyl moieties of the administered 1,2-di-[9,10,12,-13-3H4]linoleoyl-[N-14CH3] glycerophosphocholine was transported by the chylomicrons. About one half of the 14C choline radioactivity of the glycerophosphocholine backbone was found in the chylomicrons and the other half in the liver. The 3H radioactivity distribution in the chylomicrons amounted to 25% in the phosphatidylcholine fraction and 75% in the neutral lipids. Positional specific analyses of the phosphatidylcholine present in chylomicrons confirmed the fact that the 1-position remained practically intact while the 2-position underwent considerable exchange with unlabelled fatty acids. Analysis of the liver of the animals with lymph fistula indicated that it was practically free of the 3H radioactivity derived from the acyl moieties but contained a high percentage of the 14C radioactivity of the choline group. The methyl groups of choline were oxidized only to a very small extent. These results demonstrate that during the absorption process, about one half of the absorbed polyunsaturated phosphatidylcholine is hydrolyzed to 1-acyl-lysophosphatidylcholine and reacylated again to phosphatidylcholine upon entering the mucosa cell, while the other half is completely hydrolyzed to free fatty acids and glycerophosphocholine or its hydrolysis products. The fatty acids released are utilized for the reassembly of triacylglycerides and phosphatidylcholine found in the chylomicrons.     

Expression of members of a novel membrane linked metalloproteinase family (ADAM) in human articular chondrocytes

The I1-imidazoline receptor is expressed in the rostral ventrolateral medulla (RVLM) where it mediates vasodepression, and in PC12 pheochromocytoma cells where it elicits generation of diacylglycerol independent of phosphatidylinositol turnover or activation of phospholipase D. We hypothesized that the I1-imidazoline receptor couples to a phosphatidylcholine-selective phospholipase C (PC-PLC). The I1-agonist moxonidine elicited diacyglyceride accumulation and release of [3H]phosphocholine from PC12 cells prelabeled with [3H]choline. The PC-PLC inhibitor D609 abolished both responses. Microinjection of D609 into the RVLM of hypertensive rats blocked the vasodepressor response to intravenous moxonidine. These data implicate PC-PLC in cellular and organismic responses to I1-receptor stimulation.