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PubMed1986

Selective inhibition of human platelet phospholipase A2 by buffering cytoplasmic calcium with the fluorescent indicator quin 2. Evidence for different calcium sensitivities of phospholipases A2 and C.

Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076).

Aminoquinolines↗

PubMed1989

The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils.

The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5'-[gamma-thio]triphosphate (ATP[S]), adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.

Arachidonic Acid↗

PubMed1990

Phospholipase C from Clostridium perfringens stimulates phospholipase A2-mediated arachidonic acid release in cultured intestinal epithelial cells (INT 407).

The mechanisms by which phospholipase C from Clostridium perfringens stimulates release of arachidonic acid (AA) in cultured intestinal epithelial cells (INT-407) were investigated. INT-407 cells were first allowed to incorporate 14C-labeled AA into their phospholipids; the labeled cells were then exposed to phospholipase C, and the release of free 14C-AA was determined. Phospholipase C caused a rapid (3 min) intracellular rise of free 14C-AA, followed by a considerable, dose- and time-dependent release of 14C-AA into the extracellular medium. For comparison, the calcium ionophore A23187 also caused a rapid mobilization of free 14C-AA, but a much lower extracellular 14C-AA release than phospholipase C during longer (1 h) incubation. The 14C-AA release was accompanied by a degradation of 14C-myo-inositol-labeled phosphatidylinositols and was reduced by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). Both phospholipase C- and A23187-stimulated 14C-AA release was associated with degradation of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol and was reduced by nordihydroguaiaretic acid and 4-bromophenacyl bromide, two known phospholipase A2 inhibitors. In addition, the 14C-AA release was reduced by the calmodulin inhibitors trifluoperazine, compound 48/80, and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). These findings indicate that phospholipase C from C. perfringens stimulates phospholipase A2-mediated AA release from human intestinal epithelial cells and suggest that this stimulation is brought about via processes involving phosphatidylinositol breakdown and activation of calmodulin and protein kinase C. It is possible that this phospholipase C-evoked AA release may contribute to the mucosal pathologic condition in diseases with altered intestinal microbial flora.

Arachidonic Acids↗

PubMed1980

Phospholipase A2 and phospholipase C activities of platelets. Differential substrate specificity, Ca2+ requirement, pH dependence, and cellular localization.

Deoxycholate treatment of horse platelets previously labeled in their phospholipids with [14C]arachidonate produces selective conversion of [14C]phosphatidylinositol (PI) to [14C]1,2-diacylglycerol. This phospholipase C activity, which has a pH optimum of 7.5, is specific for phosphatidylinositol since other phospholipids or neutral lipids are not affected. Although exogenous Ca2+ is not required for activity, ethylene glycol bis(beta-aminoethyl ether)N,N,N',N-tetraacetic acid or EDTA abolishes phosphatidylinositol degradation. However, in the presence of added Ca2+, other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) are also degraded but by a phospholipase A2 activity. This activity generates the respective lyso-derivatives as well as various [14C]arachidonate metabolites. The phospholipase A2 activity is further enhanced by increasing the pH (7.5 to 9.5), a condition which severely suppresses the phospholipase C activity. Most of the platelet phospholipase A2 activity is associated with the particulate fractions of the cell, while the phospholipase C activity appears to be almost completely restricted to the soluble fraction. Deoxycholate treatment of the particulate fractions results in cleavage by phospholipase A2 of phosphatidylcholine and phosphatidylethanolamine but not of phosphatidylinositol. The preferred substrates for platelet phospholipase A2 appear to be phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine, while phosphatidylinositol seems to be degraded nearly exclusively by phospholipase C.

Animals↗

PubMed1998

Ammodytin L, an inactive phospholipase A2 homologue with myotoxicity in mice, binds to the presynaptic acceptor of the beta-neurotoxic ammodytoxin C in Torpedo: an indication for a phospholipase A2 activity-independent mechanism of action of beta-neurotoxins in fish?

A Ser48 phospholipase A2-homologue, ammodytin L, which is myotoxic in mammals and devoid of any phospholipase A2 activity, completely inhibits the specific binding of the neurotoxic phospholipase A2, ammodytoxin C, to fish presynaptic membranes from Torpedo marmorata electric organ. In cross-linking experiments, 125I-ammodytin L labels the same membrane proteins as 125I-ammodytoxin C (70, 38.5-57.4 and 19.7 kDa). The formation of these adducts is completely prevented by the presence of ammodytoxin C but not of a non-toxic phospholipase A2, ammodytin I2. A chimeric phospholipase A2, constructed by associating the N-terminal half of ammodytoxin to the C-terminal half of ammodytin L, possesses a low, but significant phospholipase A2 activity, however it is not toxic to mice, probably due to abolition of the specific neuronal acceptor binding in mammals. Nevertheless, the chimeric phospholipase A2 is able to interact with the ammodytoxin acceptor in Torpedo marmorata electric organ. The existence of neuronal acceptors for ammodytin L and for the chimeric phospholipase A2 suggests that they may act as neurotoxins in fish. As ammodytin L does not possess any enzymatic activity it, therefore, appears to be an excellent tool to investigate the mechanism of action of beta-neurotoxins independently of their phospholipase A2 activity.

Amino Acid Sequence↗

PubMed1987

Monoclonal antibodies against an intracellular phospholipase A2 from rat liver and their cross-reactivity with other phospholipases A2.

The membrane-associated phospholipase A2 from rat liver mitochondria was solubilized and partially purified by AcA 54 gel filtration and Matrex gel blue A chromatography. The approximately 2500-fold purified preparation was injected into mice to prepare monoclonal antibodies against phospholipase A2 after fusion of spleen cells and mouse SP2/0 myeloma cells. Hybridoma supernatants were assayed for antibody production in enzyme-linked immunosorbent assay with partially purified phospholipase A2 as antigen. Positive clones were tested for their ability to bind phospholipase A2 in a specific immunoprecipitation assay involving protein-A--Sepharose to which rabbit anti-(mouse immunoglobulins) and monoclonal antibodies from hybridoma supernatants were complexed. Twelve clones producing antibodies that bound mitochondrial phospholipase A2 were identified. The binding of all of these antibodies to protein fractions eluted from AcA 54 and Matrex gel blue A columns coincided with the phospholipase A2 activity in these fractions. All monoclonal antibodies showed cross-reactivity with rat liver cytosolic and solubilized rat platelet phospholipase A2. Extracellular phospholipase A2 from rat and pig pancreas or Crotalus atrox were not recognized by the anti-(mitochondrial phospholipase A2) antibodies.

Animals↗

PubMed2004

Presence of secretory group IIa and V phospholipase A2 and cytosolic group IValpha phospholipase A2 in chondrocytes from patients with rheumatoid arthritis.

Both secretory and cytosolic phospholipase A2 enzymes have been implicated in the pathogenesis of arthritis in animal models, but the exact expression patterns of the enzymes in diseased human joint tissue are uncertain. We investigated the messenger RNA expression of group IIa, IValpha and V phospholipase A2 and localized the presence of group IIa and IValpha phospholipase A2 at protein levels in articular cartilage from patients with rheumatoid arthritis, osteoarthritis and patients with non-arthritic joints. Both group IIa phospholipase A2 messenger RNA and protein were detected in all samples independent of diagnosis, but were far more prominent in cartilage from rheumatoid arthritis samples. In cartilage with rheumatoid arthritis, the enzyme was detected both within the chondrocytes and in the extracellular matrix, whereas only few osteoarthritic cartilage samples showed positive staining in the matrix. In the cartilage matrix of non-arthritic controls, group IIa phospholipase A2 was totally absent. Messenger RNA for the group IValpha and V phospholipase A2 was, except for one osteoarthritic cartilage sample, exclusively detected in rheumatoid arthritic cartilage. For group IValpha phospholipase A2 this was also confirmed at the protein level. These results suggest that each phospholipase A2 enzyme has distinct roles in both healthy and diseased joint tissue, and that the diversity and amount of enzyme correlate with the grade of inflammation and disease severity.

Adult↗

PubMed1992

Stimulation of phosphatidate synthesis in endothelial cells in response to P2-receptor activation. Evidence for phospholipase C and phospholipase D involvement, phosphatidate and diacylglycerol interconversion and the role of protein kinase C.

To investigate the stimulation of phosphatidic acid formation in bovine aortic endothelial cells by P2-purinergic agonists, we labelled AG4762 cells with [32P]P1 and stimulated in the presence of butanol. Under these conditions phospholipase D generated [32P]phosphatidylbutanol, whereas the [32P]phosphatidic acid from phospholipase C and diacylglycerol kinase was unchanged. The action of various purinergic agonists on both [32P]phosphatidic acid and [32P]phosphatidylbutanol was consistent with the presence of a P2Y receptor. The stimulation of phospholipase D was dependent on extracellular Ca2+ and was mostly transient (completed within 3 min), whereas the initial stimulation of phospholipase C was independent of extracellular Ca2+, followed by a Ca(2+)-dependent phase. The agonist stimulation of phospholipase D was dependent on protein kinase C, as judged by its sensitivity to the relatively selective protein kinase C inhibitor Ro 31-8220. These results show that purinergic-receptor-mediated stimulation of phosphatidic acid has three phases: an initial Ca(2+)-independent stimulation of phospholipase C, an early but transient Ca(2+)- and protein kinase C-dependent stimulation of phospholipase D, and a sustained Ca(2+)-dependent stimulation of phospholipase C. Using propranolol to inhibit phosphatidate phosphohydrolase, we provide evidence that phosphatidic acid derived from purinergic-receptor-mediated stimulation of the phospholipase C/diacylglycerol kinase route can itself be converted back into diacylglycerol.

Adenosine Triphosphate↗

PubMed1992

Phospholipase D activation in human natural killer cells through the Kp43 and CD16 surface antigens takes place by different mechanisms. Involvement of the phospholipase D pathway in tumor necrosis factor alpha synthesis.

We have recently described a novel glycoprotein, Kp43, expressed on the surface of human natural killer (NK) cells that appears to regulate their functional activity. In this report, signaling mechanisms through the Kp43 surface antigen have been studied. Incubation of interleukin 2 (IL-2)-treated NK cells with anti-Kp43 monoclonal antibody F(ab')2 fragments resulted in the time- and dose-dependent stimulation of NK cell phospholipase D. Phospholipase D activation through the Kp43 surface antigen was found to take place in the absence of polyphosphoinositide turnover and appeared not to depend on the presence of Ca2+ in the extracellular medium. On the other hand, signaling mechanisms through the CD16 receptor (FcR-III) on NK cells were comparatively studied. Stimulation of IL-2-treated NK cells with anti-CD16 monoclonal antibody F(ab')2 fragment also resulted in time- and dose-dependent activation of phospholipase D. However, CD16-triggered phospholipase D activation took place concomitant to phospholipase C-mediated polyphosphoinositide breakdown and showed a strong dependence on extracellular Ca2+. These results provide, to our knowledge, the first evidence for the presence of activatable phospholipase D in NK cells, as well as the first indication that distinct receptor-modulated pathways exist for activation of phospholipase D within the same cell type. On the other hand, phosphatidic acid, the physiologic product of phospholipase D action on phospholipids, was found to mimic the effect of anti-Kp43 monoclonal antibody regarding tumor necrosis factor alpha (TNF-alpha) biosynthesis and secretion by NK cells. Addition of phosphatidic acid vesicles to IL-2-treated NK cell cultures stimulated a TNF-alpha production that was abolished when the cells were previously treated with actinomycin D. Other phospholipids, including lysophosphatidic acid, were ineffective. However, phosphatidic acid-induced TNF-alpha production was strongly inhibited by the presence of propranolol, an inhibitor of phosphatidic acid phosphohydrolase. Moreover, in cells responding to phorbol myristate acetate, a compound that triggers activation of phospholipase D, TNF-alpha synthesis was also inhibited by propranolol. Thus, these data suggest a second messenger role for phosphatidic acid-derived diradylglycerol in the induction of TNF-alpha gene expression.

Antigens, Differentiation↗

PubMed2000

Parallel secretion of pancreatic phospholipase A(2), phospholipase A(1), lipase, and colipase in children with exocrine pancreatic dysfunction.

The cosecretion of pancreatic lipase and colipase are important in normal fat digestion. As adsorption of phosphatidylcholine to the lipid substrate interferes with lipase activity, hydrolysis to lysophosphatidylcholine with subsequent desorption is also essential for fat digestion. There are some data regarding the secretion of pancreatic phospholipases in normal adults but none in children or patients with pancreatic disease. In the present study, we aimed a) to develop an accurate fast assay method to measure phospholipase A(2) and b) to determine the secretion rate of pancreatic phospholipase A(2) and whether it is cosecreted with lipase and colipase in children with exocrine pancreatic dysfunction. Nine male patients aged 0.5 to 16 y (seven with cystic fibrosis, two with malabsorption) underwent pancreatic stimulation tests. Their colipase and lipase secretion rates were measured by titrimetric methods and phospholipase A(2) and A(1) by phosphorus magnetic resonance spectroscopy ((31)P NMR). It was found that the phospholipases, colipase, and lipase were absent in the two patients with pancreatic insufficiency. In patients with normal absorption, there were marked inter-and intrasubject variations of lipase, colipase, and phospholipase secretion rates that were consistent with the degree of exocrine pancreatic dysfunction. However, in the three 20-min stimulation periods of the pancreatic function test, pancreatic phospholipase is cosecreted with lipase and colipase, and average colipase and phospholipase A(2) secretion rates follow a similar or parallel pattern. These findings are consistent with the important role of pancreatic phospholipases in intestinal phospholipid hydrolysis leading to the desorption of phospholipids from the lipid substrate and enhancing lipid hydrolysis and phospholipid absorption.

Adolescent↗

PubMed1989

Association of the intestinal brush-border membrane phospholipase A2 and lysophospholipase activities (phospholipase B) with a stalked membrane protein.

We have attempted to determine the size and membrane orientation of a recently described rat jejunal brush-border protein possessing phospholipase A2 and lysophospholipase activities (phospholipase B) (Pind, S. and Kuksis, A. [1988] Biochim, Biophys. Acta 938, 211-221). The phospholipase A2 and lysophospholipase activities were renatured following nonreducing sodium dodecyl sulphate polyacrylamide gel electrophoresis of the total membrane proteins and were shown to migrate as a component of a protein band having a relative molecular mass of 170 kDa. This band accounted for approximately 1% of the total Coomassie Blue staining proteins. Phospholipase B was also shown to be solubilized from the membranes, in an active form, by a proteolytic digestion with papain. Papain solubilization resulted in a loss of the hydrophobic properties observed for the intact phospholipase. These results suggest that the active site of the phospholipase projects from the luminal surface of the membrane vesicles. In support of this, phospholipase activity towards exogenous, detergent-solubilized phosphatidylcholine was demonstrated under conditions in which the membranes remained intact. We conclude that the phospholipase B has the characteristics of a stalked, brush-border membrane protein and may be considered as another digestive enzyme anchored in this membrane.

Animals↗

PubMed1986

Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex.

Phospholipase A has been isolated from a crude lysosomal fraction from rat kidney cortex and purified 7600-fold with a recovery of 9.8% of the starting activity. The purified enzyme is a glycoprotein having an isoelectric point of pH 5.4 and an apparent molecular weight of 30,000 by high-pressure liquid chromatography gel permeation. Naturally occurring inhibitors of lysosomal phospholipase A are present in two of the lysosomal-soluble protein fractions obtained in the purification. They inhibit hydrolysis of 1,2-di[1-14C]oleoylphosphatidylcholine by purified phospholipase A1 with IC50 values of 7-11 micrograms. The inhibition is abolished by preincubation with trypsin at 37 degrees C, but preincubation with trypsin at 4 degrees C has no effect, providing evidence that the inhibitors are proteins. The results suggest that the activity of lysosomal phospholipase A may be regulated in part by inhibitory proteins. Lysosomal phospholipase A from rat kidney hydrolyzes the sn-1 acyl group of phosphatidylcholine, does not require divalent cations for full activity, and is not inhibited by ethylenediaminetetraacetic acid. It has an acid pH optimum of 3.6-3.8. Neither p-bromophenacyl bromide, diisopropyl fluorophosphate, nor mercuric ion inhibits phospholipase A1. In contrast to rat liver, which has two major isoenzymes of acid phospholipase A1, kidney cortex has only one isoenzyme of lysosomal phospholipase A1.

Animals↗

PubMed1991

Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts.

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.

Cells, Cultured↗

PubMed1995

Evidence for receptor and G-protein regulation of a phosphatidylethanolamine-hydrolysing phospholipase A1 in guinea-pig heart microsomes: stimulation of phospholipase A1 activity by DL-isoprenaline and guanine nucleotides.

While evidence has been presented for the receptor-mediated activation of phospholipases A2, C and D, the activation of phospholipase A1 subsequent to receptor activation has not been established. Phospholipase A1-catalysed hydrolysis of 1-palmitoyl-2-linoleoyl-glycerophosphoethanolamine (GPE) by guinea-pig heart microsomes was stimulated 40-60% by isoprenaline. This isoprenaline-mediated increase in activity was blocked by propranolol and butoxamine, a specific beta 2-adrenergic antagonist, but not by atenolol, a specific beta 1-adrenergic antagonist. Neither clonidine nor phenylephrine, alpha 1- and alpha 2-adrenergic agonists respectively, had a stimulatory effect on the hydrolysis of the PE substrate. Guanosine 5'(-)[gamma-thio]triphosphate (GTP[S]) and guanosine 5'(-)[beta,gamma-imido]triphosphate, but not guanosine 5'(-)[beta-thio]diphosphate (GDP[S]) or adenosine 5'(-)[gamma-thio]triphosphate, stimulated the hydrolysis of 1-palmitoyl-2-linoleoyl-GPE by phospholipase A1. GDP[S] inhibited the isoprenaline-mediated stimulation of phospholipase A1 activity. Phospholipase A1 hydrolysis of 1-palmitoyl-2-linoleoyl-GPE was not dependent on cations; however, the stimulatory effects of isoprenaline and GTP[S] on the hydrolytic activity were abolished by cation chelators. The above data suggest that phospholipase A1 activity in guinea-pig heart microsomes is activated by the binding of isoprenaline to beta 2-adrenergic receptors. Furthermore the stimulation of phospholipase A1 activity by the agonist may be mediated via activation of G-proteins.

Adrenergic beta-Agonists↗

PubMed1991

Cross-talk between receptor-regulated phospholipase D and phospholipase C in brain.

Because receptors, G proteins, and phospholipases all exist within a membrane lipid environment, it is not unreasonable to assume that an enzyme capable of changing the lipid environment can affect the coupling relationship among these signal transducing components. Our previous study showed that a muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D via a G protein in brain. We demonstrate here that phosphatidylinositol phospholipase C and phosphatidylcholine phospholipase D are simultaneously activated within 15 s by muscarine in the presence of 1 microM GTP gamma S. More important, inhibition of phospholipase D by zinc attenuated carbamylcholine-induced activation of phospholipase C by 30%. Our additional evidence strongly indicates that the receptor-regulated phospholipase D plays an important modulatory role in agonist-stimulated phosphatidylinositol breakdown. This modulatory effect may be achieved by changing the membrane microenvironment in which phospholipase C and phosphoinositol lipids reside, consequently amplifying the inositol phospholipid signaling process. Our results lead us to postulate that the potential interaction between two different signaling pathways may provide a cell with intracellular coordination and enable the cell to achieve functional responses.

Animals↗

PubMed1992

Neuronal "nucleotide" receptor linked to phospholipase C and phospholipase D? Stimulation of PC12 cells by ATP analogues and UTP.

We have investigated the characteristics of the receptor for ATP on neuronal cells and the involvement of phospholipase C and phospholipase D in the effector mechanisms, using PC12 rat phaeochromocytoma cells in culture. We show that the cells respond, with generation of total inositol phosphates, to ATP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) but not to 2-methylthioadenosine5'-triphosphate (2MeSATP), beta,gamma-methylene ATP, or adenosine 5'-O-(2-thiodiphosphate) (ADP beta S). The largest response to ATP gamma S was mainly independent of extracellular calcium, had an EC50 of 7.93 +/- 0.76 microM, and was competitively inhibited by the nonspecific antagonist suramin. The pyrimidine nucleotide UTP also elicited a response in these cells. Measurement of [3H]inositol triphosphate showed a rapid rise to maximum (10-15 sec) in response to both ATP gamma S and UTP but no response to 2MeSATP. Cells prelabeled with 32Pi and stimulated in the presence of 50 mM butanol responded to ATP gamma S, ATP, and UTP with enhanced formation of [32P]phosphatidylbutanol as well as [32P]phosphatidic acid, indicating that agonist-stimulated phosphatidic acid occurs by both phospholipase D and phospholipase C activity. The stimulation of phospholipase D was inhibited by the presence of a protein kinase C inhibitor, Ro 31-8220. The dose-response curve for the stimulation by ATP gamma S of phospholipase C was shifted to the right by the presence of UTP, indicating that both compounds act on the same receptors. The data provide the first evidence for the existence of a nucleotide receptor on neuronal cells (insensitive to both purines and pyrimidines) and show that this receptor is linked to both phospholipase C and phospholipase D.

Adenosine Triphosphate↗

PubMed1989

Modification of phospholipase C and phospholipase A2 activities during poliovirus infection.

The infection of HeLa cells by poliovirus leads to profound alterations in the activities of both phospholipase C and the A23187-stimulated phospholipase A2. As early as the third hour after poliovirus infection, the activity of phospholipase C is enhanced, as measured by the increase in inositol triphosphate (IP3) in the cells. By the fifth hour post-infection there is a 5-fold increase in IP3 in the infected cells. Therefore, the synthesis of the bulk of poliovirus proteins and poliovirus genomes takes place in cells containing a high and sustained increase in IP3. This augmentation in IP3 is dependent on the multiplicity of infection used. Poliovirus gene expression is required to induce the increase in phospholipase C activity, since the presence of cycloheximide or guanidine blocked it. In contrast to the activation of phospholipase C induced by poliovirus, there is a drastic blockade of the A23187-induced phospholipase A2 activity, measured as the release of [3H]arachidonic acid to the medium. This action on phospholipase A2 is dependent on poliovirus gene expression because it was prevented by cycloheximide or 3-methylquercetin. To our knowledge this is the first report analyzing these two activities in animal virus-infected cells. The findings described may help to explain the profound modifications of both membrane permeability and lipid metabolism undergone by poliovirus-infected cells.

Antiviral Agents↗

PubMed1985

Identification of neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipase A2 in canine myocardium.

Two novel phospholipase activities have been identified in the cytosolic fraction of canine myocardium. Neutral active phospholipase C activity was partially purified by anion exchange, hydroxylapatite, chromatofocusing, and gel filtration chromatographies. The partially purified enzyme had similar maximum velocities (237 versus 241 nmol/mg X h) and apparent Michaelis constants (20 versus 14 microM) utilizing either plasmenylcholine or phosphatidylcholine as substrate. Myocardial phospholipase C had a pH optimum between 7 and 8, required divalent cations for maximal activity, and did not hydrolyze phosphatidylinositol or sphingomyelin. Myocardial cytosol contained a potent inhibitor of phospholipase C which masked enzymic activity until it was removed during the purification procedure. A plasmalogen selective phospholipase A2 activity was also identified in the cytosolic fraction of canine myocardium. The protein catalyzing this activity was partially purified by DEAE-Sephacel-hydroxylapatite tandem chromatography and exhibited a maximum velocity of 5 nmol/mg X h for plasmenylcholine but only 1 nmol/mg X h for phosphatidylcholine, had a pH optimum between 6 and 7 for both substrates, and did not require calcium ion for activity. These results constitute the first demonstration of a neutral active phospholipase C specific for choline and ethanolamine glycerophospholipids and a plasmalogen selective phospholipase A2 in mammalian tissue.

Animals↗