PC-PLC Activity Research Articles

Human sphingomyelin synthase 1 generates diacylglycerol in the presence and absence of ceramide via multiple enzymatic activities.

Sphingomyelin (SM) synthase 1 (SMS1), which is involved in lipodystrophy, deafness, and thrombasthenia, generates diacylglycerol (DG) and SM using phosphatidylcholine (PC) and ceramide as substrates. Here, we found that SMS1 possesses DG-generating activities via hydrolysis of PC and phosphatidylethanolamine (PE) in the absence of ceramide and ceramide phosphoethanolamine synthase (CPES) activity. In the presence of the same concentration (4.7 mol%) of PC and ceramide, the amounts of DG produced by SMS and PC-phospholipase C (PLC) activities of SMS1 were approximately 65% and 35% of total DG production, respectively. PC-PLC activity showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PC species. A PC-PLC/SMS inhibitor, D609, inhibited only SMS activity. Mn2+ inhibited only PC-PLC activity. Intriguingly, DG attenuated SMS/CPES activities. Our study indicates that SMS1 is a unique enzyme with PC-PLC/PE-PLC/SMS/CPES activities.

Sphingomyelin synthases 1 and 2 exhibit phosphatidylcholine phospholipase C activity

Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo.

A Phospholipase C-Like Protein From Ricinus communis Increases Hydroxy Fatty Acids Accumulation in Transgenic Seeds of Camelina sativa.

There have been strong interests in producing unusual fatty acids in oilseed crops to provide renewable industrial feedstock. Results are so far largely disappointing since much lower amounts of such fatty acids accumulate in genetically engineered seeds than in their original natural sources. It has been suggested that the flux of unusual fatty acids through phosphatidylcholine (PC) represents a major bottleneck for high accumulation of such fatty acids in triacylglycerol (TAG). We show here that a phospholipase C-like protein (RcPLCL1) from castor bean, which accumulates nearly 90% of the hydroxylated ricinoleic acid in its seed TAG, increases the amount of hydroxy fatty acids (HFAs) when co-expresses with the fatty acid hydroxylase (RcFAH12) in transgenic seed of Camelina sativa. RcPLCL1 shows hydrolyzing activities on both PC and phosphatidylinositol substrates in our in vitro assay conditions. The PC-PLC activity of the RcPLCL1 may have increased the efficiency of HFA-PC to diacylglycerol conversion, which explains our observation of increased HFA contents in TAG concomitant with decreased HFA in the membrane lipid PC during seed development. Consequently, this may also alleviate the potential detrimental effect of HFA on germination of the engineered camelina seeds. Our results provide new knowledge that will help design effective strategies to engineer high levels of HFAs in transgenic oilseeds.

Activation of gill Ca2+-sensing receptor as a protective pathway to reduce Ca2+-induced cytotoxicity.

The expression of the Ca(2) (+)-sensing receptor (Casr) in the endocrine gland known as the corpuscle of Stannius (CS) regulates the secretion of the hypocalcemic hormone stanniocalcin-1 (STC1) to inhibit gill Ca(2) (+) uptake. Although numerous studies have reported the branchial expression of Casr and Stc1, the functions of these proteins in gills have not been elucidated yet. On the basis of recent findings regarding the autocrine/paracrine functions of STC1 in mammalian models, we proposed the hypothesis that branchial CaSR has an in situ 'sensing' function to regulate STC1 that maintains local Ca(2) (+) homeostasis. In this study, we investigated Casr-mediated signaling and its regulation of Stc1 and cyclooxygenase-2 (Cox2) expression/function using a primary gill-cell culture model. The biochemical responses of gill cells isolated from Japanese eels to an increasing concentration of extracellular Ca(2) (+) (0.1-1 mM) were tested. This stimulation led to a transient increase in phosphatidylcholine-phospholipase C (PC-PLC) activity, followed by activation of ERK and inositol 1,4,5-trisphosphate-Ca(2) (+)/calmodulin-dependent protein kinase 2 (CaMK2) signaling pathways. Cotreatment with the calcimimetic R467 caused synergistic effects on Ca(2) (+)-stimulated PC-PLC activity, ERK signaling, and CaMK2 signaling. The activation of the CaSR-PLC-ERK pathway was associated with increased expression levels of Stc1 and Cox2 as confirmed by the inhibition of Erk using a chemical inhibitor, PD98059. Functionally, Ca(2) (+)/R-467 pretreatment was found to protect cells from thapsigargin-induced cell death. Inhibition of COX2 activity using NS398 abolished this protection, while transduction of STC1 lentiviral particles in the gill cells increased the protective effects. Collectively, our data revealed the expression of functional CaSR in gill tissues. The identification of the CaSR-STC1/COX2-mediated protective pathway in gill cells sheds light on a possible cellular protective mechanism against an increase in intracellular Ca(2) (+) levels associated with transepithelial Ca(2) (+) transport.

Innate Immunity Signaling in Neonatal Rat Cardiac Myocytes

Neonates have a heightened inflammatory response to pathogens & are uniquely susceptible to sepsis‐related cardiac dysfunction. MAP‐kinase signaling plays a critical role in innate immunity/inflammation wherein MAPK phosphatase‐1 (MKP‐1) is a key negative regulator, but its roles in cardiac myocyte inflammatory signaling are not defined. Our hypothesis was that MKP‐1 deficiency amplifies inflammatory responses in neonatal rat cardiomyocytes, and is related to MAPK, Gαs & Gαq signaling pathways. H9c2 cells were transfected with siMKP‐1 or scrambled control (H9WT) and treated with 5μg/ml LPS. IL6 & TNFα mRNA and protein expression was detected by qRTPCR & ELISA. cAMP, PC‐PLC & activated PKC were detected by EIA. siMKP‐1 cells had a greater increase in TNFα (p<0.001) & IL6 (p<0.001) mRNA & protein expression at 1h & 4h after LPS vs. H9WT. MKP‐1 deficient cells also had greater increases in cAMP, PC‐PLC & PKC activity (P<0.001) after LPS. Inhibition of P38 completely blocked the increases in TNFα, IL6, cAMP & PC‐PLC, but significantly increased activated PKC (P<0.001) in siMKP‐1 cells. Neonatal rat cardiac myocytes generate inflammatory cytokines in direct response to LPS, which is enhanced by MKP‐1 deficiency, mediated by the MAP kinase P38 & associated with increased cAMP, PC‐PLC & PKC. Defining these cardiac myocyte signaling processes may lead to novel therapeutic targets for sepsis‐related cardiac dysfunction.

Activation of Phosphatidylcholine Cycle Enzymes in Human Epithelial Ovarian Cancer Cells

Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) could provide choline-based imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. The increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and nontumoral immortalized counterparts (EONT) may derive from (a) enhanced choline transport and choline kinase (ChoK)-mediated phosphorylation, (b) increased PC-specific phospholipase C (PC-plc) activity, and (c) increased intracellular choline production by PC deacylation plus glycerophosphocholine-phosphodiesterase (GPC-pd) or by phospholipase D (pld)-mediated PC catabolism followed by choline phosphorylation. Biochemical, protein, and mRNA expression analyses showed that the most relevant changes in EOC cells were (a) 12-fold to 25-fold ChoK activation, consistent with higher protein content and increased ChoKalpha (but not ChoKbeta) mRNA expression levels; and (b) 5-fold to 17-fold PC-plc activation, consistent with higher, previously reported, protein expression. PC-plc inhibition by tricyclodecan-9-yl-potassium xanthate (D609) in OVCAR3 and SKOV3 cancer cells induced a 30% to 40% reduction of PCho content and blocked cell proliferation. More limited and variable sources of PCho could derive, in some EOC cells, from 2-fold to 4-fold activation of pld or GPC-pd. Phospholipase A2 activity and isoform expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKalpha mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from patients with EOC. Overall, we showed that the elevated PCho pool detected in EOC cells primarily resulted from upregulation/activation of ChoK and PC-plc involved in PC biosynthesis and degradation, respectively.

Promotion of autophagy and inhibition of apoptosis by low concentrations of cadmium in vascular endothelial cells

Recent reports, highlighting the relationships of cadmium exposure and vascular diseases, indicated that vascular endothelial cell was the target of cadmium (Cd) toxicity. However, the underlying mechanisms have not been fully elucidated. In this study, we evaluated the internalization of Cd2+ into human umbilical vein endothelial cells (HUVECs) by a novel Cd2+-selective sensor suitable for living cells. Then, we detected apoptosis in the treated cells. Our results showed that Cd2+ at low concentrations (< 10 micromol/l) inhibited apoptosis induced by deprivation of serum and basic fibroblast growth factor (bFGF). To investigate the corresponding molecular mechanisms, we employed acridine orange staining and Western blotting of MAP1 LC3 to detect autophagy, and analyzed the levels of integrin beta4, caveolin-1 and activity of PC-PLC. Our results showed that low concentrations of Cd2+ promoted autophagy and depressed the levels of integrin beta4, caveolin-1 and PC-PLC activity. The data suggested that autophagy played a key role in Cd2+ induced endothelial dysfunction; integrin beta4, caveolin-1 and PC-PLC might be the targets of Cd2+ in vascular endothelial cells.

Monitoring growth phase-related changes in phosphatidylcholine-specific phospholipase C production, adhesion properties and physiology of Bacillus cereus vegetative cells

The physiological status and metabolic heterogeneity of Bacillus cereus cells within a culture during an 8-h batch fermentation process was measured using flow cytometry (FCM). Concurrently, production of the toxin, PC-PLC, and the extent of cell adhesion of live and dead cells were monitored using novel fluorescent assays. Flow cytometry analysis detected growth phase-related changes in the physiological profiles of cells over the course of the fermentation, with variation in the percentage of cells displaying membrane damage and intracellular esterase and redox activities. As the exponential phase proceeded, populations became more uniform in terms of protein content as measured using FCM in tandem with a cell tracking dye, with the majority of cells becoming membrane intact, esterase positive and redox active. PC-PLC activity appeared strongly related to cell density. Permeabilisation of cells was accompanied by a loss in adherent properties, while 25-100% of cells with intracellular esterase activity possessed adhesion properties. Cells in late exponential phase appeared to have reduced adherence properties compared to cells in early exponential or lag phase. As well as demonstrating the utility of FCM for measuring heterogeneity in terms of cell physiological status throughout the course of batch cultures, the methods utilised in this study could be used to relate processes such as toxin production or cell adhesion to cell physiological state.

Conversion of Low Density Lipoprotein-associated Phosphatidylcholine to Triacylglycerol by Primary Hepatocytes

We have studied the uptake and metabolism of phosphatidylcholine (PC), the major phospholipid of low density lipoproteins (LDL), by cultures of primary hepatocytes. Strikingly, in the absence of the LDL receptor, PC incorporation into hepatocytes was inhibited by only 30%, whereas cholesteryl ether uptake was inhibited by 60-70%. On the other hand, scavenger receptor class B, type I, the other important receptor for LDL in the liver, was found to be responsible for the uptake of the remaining 30-40% of LDL-cholesteryl ether. PC uptake was, however, only partially inhibited (30%) in scavenger receptor class B, type I, knock-out hepatocytes. Once LDL-PC was taken up by hepatocytes, approximately 50% of LDL-[(3)H]oleate-PC was converted to triacylglycerol rather than degraded in lysosomes as occurs for LDL-derived cholesteryl esters. The remainder of the LDL-derived PC was not significantly metabolized to other products. Triacylglycerol synthesis from LDL-PC requires a PC-phospholipase C activity as demonstrated by inhibition with the phospholipase C inhibitor D609 or activation with rattlesnake venom. Small interfering RNA-mediated suppression of acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), but not DGAT1, decreased the acylation of the LDL-derived diacylglycerol. These findings show that PC in LDL particles is taken up not only by the classical receptors but also by additional mechanism(s) followed by metabolism that is completely different from the cholesteryl esters or apoB100, the other main components of LDL.

Compartmentalization of the Broad-Range Phospholipase C Activity to the Spreading Vacuole Is Critical forListeria monocytogenesVirulence

Listeria monocytogenes is a bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell by using an actin-based mechanism of motility. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from vacuoles formed upon cell-to-cell spread. PC-PLC is made as an inactive proenzyme whose activation requires cleavage of an N-terminal propeptide. During infection, PC-PLC is activated specifically in acidified vacuoles. To assess the importance of compartmentalizing PC-PLC activity during infection, we created a mutant that makes constitutively active PC-PLC (the plcBDelta pro mutant). Results from intracellular growth and cell-to-cell spread assays showed that the plcBDelta pro mutant was sensitive to gentamicin, suggesting that unregulated PC-PLC activity causes damage to host cell membranes. This was confirmed by the observation of a twofold increase in staining of live infected cells by a non-membrane-permeant DNA fluorescent dye. However, membrane damage was not sufficient to cause cell lysis and was dependent on bacterial cell-to-cell spread, suggesting that damage was localized to bacterium-containing filopodia. Using an in vivo competitive infection assay, we observed that the plcBDelta pro mutant was outcompeted up to 200-fold by the wild-type strain in BALB/c mice. Virulence attenuation was greater when mice were infected orally than when they were infected intravenously, presumably because the plcBDelta pro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance for L. monocytogenes virulence of compartmentalizing the activity of PC-PLC during infection.

Upregulating of Fas, integrin β4 and P53 and depressing of PC-PLC activity and ROS level in VEC apoptosis by safrole oxide

Previously, we found that safrole oxide could trigger vascular endothelial cell (VEC) apoptosis. In this study, to investigate its mechanism to induce apoptosis in VECs, the activities of nitric oxide synthetase and phosphatidylcholine specific phospholipase C, the level of reactive oxygen species and the expressions of Fas, integrin β4 and P53 were analyzed. The data showed that safrole oxide induced apoptosis by increasing the expressions of Fas, integrin β4 and P53, and depressing the activity of Ca 2+-independent phosphatidylcholine-specific phospholipase C and intracellular reactive oxygen species levels in VECs.

Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells.

In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.

Involvement of phospholipids in the mechanism of insulin action in HEPG2 cells

The mechanism of action by which insulin increases phosphatidic acid (PA) and diacylglycerol (DAG) levels was investigated in cultured hepatoma cells (HEPG2). Insulin stimulated phosphatidylcholine (PC) and phosphatidyl-inositol (PI) degradation through the activation of specific phospholipases C (PLC). The DAG increase appears to be biphasic. The early DAG production seems to be due to PI breakdown, probably through phosphatidyl-inositol-3-kinase (PI3K) involvement, whereas the delayed DAG increase is derived directly from the PC-PLC activity. The absence of phospholipase D (PLD) involvement was confirmed by the lack of PC-derived phosphatidylethanol production. Experiments performed in the presence of R59022, an inhibitor of DAG-kinase, indicated that PA release is the result of the DAG-kinase activity on the DAG produced in the early phase of insulin action.

Proteolytic pathways of activation and degradation of a bacterial phospholipase C during intracellular infection by Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that spreads cell to cell without exposure to the extracellular environment. Bacterial cell-to-cell spread is mediated in part by two secreted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositolspecific PLC (PI-PLC). PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme (proPC-PLC) whose activation is mediated in vitro by an L. monocytogenes metalloprotease (Mpl). Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread of an infection, but suggested that proPC-PLC has an Mpl-independent activation pathway. Using biochemical and microscopic approaches, we describe three intracellular proteolytic pathways regulating PCPLC activity. Initially, proPC-PLC secreted in the cytosol of infected cells was rapidly degraded in a proteasome-dependent manner. Later during infection, PCPLC colocalized with bacteria in lysosome-associated membrane protein 1-positive vacuoles. Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sensitive to inhibitors of cysteine proteases. Lastly, proPC-PLC activation by either pathway was sensitive to bafilomycin A1, a specific inhibitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment. These results are consistent with a model in which proPC-PLC activation is compartment specific and controlled by a combination of bacterial and host factors.

A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START.

In an effort to study further the mechanism of Cdc28 function and cell cycle commitment, we describe here a genetic approach to identify components of pathways downstream of the Cdc28 kinase at START by screening for mutations that decrease the effectiveness of signaling by Cdc28. The first locus to be characterized in detail using this approach was PKC1 which encodes a homolog of the Ca(2+)-dependent isozymes of the mammalian protein kinase C (PKC) superfamily (Levin et al., 1990). By several genetic criteria, we show a functional interaction between CDC28 and PKC1 with PKC1 apparently functioning with respect to bud emergence downstream of START. Consistent with this, activity of the MAP kinase homolog Mpk1 (a putative Pkc1 effector) is stimulated by activation of Cdc28. Furthermore, we demonstrate a cell cycle-dependent hydrolysis of phosphatidylcholine to diacylglycerol (a PKC activator) and choline phosphate at START. Diacylglycerol production is stimulated by Cdc28 in cycling cells and is closely associated with Cdc28 activation at START. These results imply that the activation of Pkc1, which is known to be necessary during bud morphogenesis, is mediated via the CDC28-dependent stimulation of PC-PLC activity in a novel cell cycle-regulated signaling pathway.

Lipid signal transduction pathways in angiotensin II type 1 receptor-transfected fibroblasts.

A stable Chinese hamster ovary fibroblast line expressing the rat vascular type 1a angiotensin II (ANG II) receptor was used to study the lipid-derived signal transduction pathways elicited by type 1a ANG II receptor activation. ANG II caused a biphasic and dose-dependent increase in diacylglycerol (DAG) accumulation with an initial peak at 15 s (181 +/- 11% of control, P < 0.02) and a second sustained peak at 5-10 min (214 +/- 10% of control, P < 0.02). The late DAG peak was derived from phosphatidylcholine (PC), and the formation was blocked by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANG II also increased phosphatidic acid (PA) production nearly fourfold by 7.5 min. In the presence of ethanol, ANG II markedly increased phosphatidylethanol (PEt) formation, indicating activation of phospholipase D (PLD). ANG II was shown to increase the mass of three separate PA species, one of which apparently originated from DAG kinase action on PC-phospholipase C (PLC)-produced DAG, providing evidence for PC-PLC activity. ANG II also formed a third PA species, which originated neither from PLD nor from DAG kinase. These results demonstrate that multiple lipid signals propagated via collateral stimulation of PLC and PLD are generated by specific activation of the vascular type 1a ANG II receptor.

Insulin-stimulated hydrolysis of phosphatidylcholine by phospholipase C and phospholipase D in cultured rat hepatocytes

We have investigated the mechanism of action by which insulin increases phosphatidate (PA) and diacylglycerol (DAG) levels in cultured rat hepatocytes. Insulin initially stimulated phosphatidylcholine-dependent phospholipase D (PC-PLD) with a significant increase in both PA and intracellular as well as extracellular choline. The involvement of phospholipase D was confirmed by the formation of PC-derived phosphatidylethanol in the presence of ethanol. The DAG increase appeared to be biphasic. Only the early phase of DAG production was inhibited by propranolol, an inhibitor of the phosphatidate phosphatase (PAP) responsible for the conversion of PA into DAG, suggesting that initially the DAG increase is due to the PLD-PAP pathway. The delayed DAG increase was in parallel with increased intracellular and extracellular phosphocholine and probably derived directly from PC-PLC activity. Experiments performed in the presence of 1 μM phorbol 12-myristate 13-acetate (PMA) indicated that protein kinase C (PKC) mediated the insulin effect on PC-PLC, but not on PC-PLD. These findings were confirmed using the PKC inhibitors calphostin, H7 and staurosporine. The dual activation of these phospholipases with a biphasic elevation of DAG levels and activation of specific PKC isoenzymes could be necessary to elicit both early and delayed effects of insulin.

Phosphatidylcholine metabolism in hypoxic and phospholipase C exposed rat ventricular myocytes.

A phospholipase C specific for choline and ethanolamine acyl and plasmalogen glycerophospholipids (PC-PLC) has been described in myocardial tissue. In the present study we investigated whether an endogenous PC-PLC is activated in hypoxic, substrate-free incubations of rat ventricular myocytes. The phosphatidylcholine pool of the myocytes was prelabelled with [14C]choline during a 4-h preincubation (pulse) period. The myocytes were subsequently washed and incubated for another 2 h (chase period) in normoxic, hypoxic, or hypoxic buffer supplemented with PC-PLC from Bacillus cereus. We hypothesized that an increase in the total (intracellular plus extracellular) content of [14C]phosphocholine (one of the products resulting from PC-PLC action on phosphatidylcholine) throughout the chase period would indicate PC-PLC activity. Instead, an apparent decrease was observed for this parameter in all myocyte groups (17-29%), even in the one exposed to exogenous PC-PLC. However, 60 min after the start of the chase period, the level of total [14C]phosphocholine was higher in hypoxic (p = 0.022) and hypoxic + PC-PLC exposed (p = 0.013) myocytes compared with normoxic controls. The total content of [14C]choline increased significantly (p < 0.017) in all myocyte groups during the incubation period (98-153%) as a result of an increment of this metabolite in the buffer. Furthermore, the values measured in hypoxic and hypoxic + PC-PLC exposed myocytes during the first hour of the chase period were significantly (p < 0.017) higher than the corresponding values in normoxic myocytes. The present results do not allow firm conclusions regarding endogenous PC-PLC activation in energy-depleted rat cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for phosphatidylcholine hydrolysis by phospholipase C in rat platelets

Production of [ 3H]1,2-dipalmitoylglycerol ([ 3H]DAG) from 1-palmitoyl-2-[9,10- 3H]palmitoyl- sn-glycero-3- phosphocholine and [ 3H]phosphorylcholine from 1,2-dipalmitoyl- sn-glycero-3-[ Me- 3H]phosphocholine was studied using sonicated rat platelets. The formation of [ 3H]DAG and [ 3H]phosphorylcholine occurred at a comparable rate. [ 3H]Phosphorylcholine formation was dependent on the concentration of the substrate, platelet sonicates and calcium in the incubation medium. The [ 3H]phosphorylcholine formation increased in presence of 0.01% deoxycholate and 0.01% Triton X-100. The phosphatidylcholine-phospholipase C (PC-PLC) in the platelet sonicates was recovered in both the supernatant and particulate fractions obtained after ultracentrifugation at 105000 × g for 1 h. The PC-PLC activity in both fractions was inhibited by 2 mM EDTA. In the presence of 0.01% deoxycholate and 0.01% Triton X-100 the activity in the particulate fraction increased compared to the activity in the supernatant, which was inhibited by 0.01% Triton X-100. The pH optima for PC-PLC in both fractions was between pH 7.2 and 7.6. PC-PLC activity was also found in rabbit and human platelet sonicates, but the activity was significantly lower than in rat platelet sonicates. There was no evidence to suggest presence of phosphatidylcholine-specific phospholipase D activity in rat sonicated platelets. This data, therefore, provides direct evidence for the presence of PC-PLC activity in rat platelets.