Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5-20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.
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PMID:Interaction of apolipoprotein A-I with lecithin-cholesterol vesicles in the presence of phospholipase C. 1472 75

For an insight regarding the control of PtdEtn (phosphatidylethanolamine) synthesis via the CDPethanolamine pathway, rat liver cDNA encoding ECT (CTP:phosphoethanolamine cytidylyltransferase) was transiently or stably transfected in Chinese-hamster ovary cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26- and 4-fold increase in specific activity of ECT respectively. However, no effect of ECT overexpression on the rate of [3H]ethanolamine incorporation into PtdEtn was detected in both cell lines. This was explored further in cells overexpressing four times ECT activity (McA-ECT1). The rate of PtdEtn breakdown and PtdEtn mass were not changed in McA-ECT1 cells in comparison with control-transfected cells. Instead, an accumulation of CDPethanolamine (label and mass) was observed, suggesting that in McA-ECT1 cells the ethanolaminephosphotransferase-catalysed reaction became rate-limiting. However, overexpression of the human choline/ethanolaminephosphotransferase in McA-ECT1 and control-transfected cells had no effect on PtdEtn synthesis. To investigate whether the availability of DAG (diacylglycerol) limited PtdEtn synthesis in these cells, intracellular DAG levels were increased using PMA or phospholipase C. Exposure of cells to PMA or phospholipase C stimulated PtdEtn synthesis and this effect was much more pronounced in McA-ECT1 than in control-transfected cells. In line with this, the DAG produced after PMA exposure was consumed more rapidly in McA-ECT1 cells and the CDPethanolamine level decreased accordingly. In conclusion, our results suggest that the supply of CDPethanolamine, via the expression level of ECT, is an important factor governing the rate of PtdEtn biosynthesis in mammalian cells, under the condition that the amount of DAG is not limiting.
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PMID:Control of the CDPethanolamine pathway in mammalian cells: effect of CTP:phosphoethanolamine cytidylyltransferase overexpression and the amount of intracellular diacylglycerol. 1475 25

G-proteins transmit the signals from hormone receptors onto intracellular effector systems which take part in production of the second messengers such as cAMP, IP3, DAG and Ca2+. Molecular mechanisms of G-protein participation in the coupling of the seven-domain receptors to adenylate cyclase, phospholipase C and channels for Ca2+ and K+ ions are discussed in this paper. G-protein is a heterotrimers built of alpha-, beta- and gamma-subunits, which dissociate onto alpha- and beta gamma-subunits during interaction with hormone-receptor complex. alpha-subunit as well as beta gamma-dimmer may interact with effector system that leads to acceleration or slowing down of second messengers formation. Molecular mechanisms of such regulatory signal diversification are described. Seven-domain receptors possess very high recognition specificity of G-proteins. It is defined by combination of both alpha- and beta gamma-subunits in the G-protein structure. There is well-defined interaction specificity of G-protein alpha-subunit with effector systems. Combinations of different beta- and gamma-subunits involved in complex formation define interaction specificity of G-protein beta gamma-complex with effector systems. The highest interaction specificity of receptors with G-proteins and G-proteins with effector systems is found during triple complex formations: receptor--G-protein--effector. Such specificity is stronger in living cells than in membrane preparations. It can be an evidence of intracellular factors influence on the processes of interaction of the proteins involved in transmembrane regulatory signal transduction.
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PMID:[Molecular mechanisms of G-proteins coupling with membrane receptors and second messenger systems]. 1487 Apr 84

Actions of endocannabinoids in the cerebellum can be demonstrated following distinct stimulation protocols in Purkinje cells. First, depolarization-induced elevations of intracellular Ca2+ lead to the suppression of neurotransmitter release from both inhibitory and excitatory afferents. In another case, postsynaptic group I metabotropic glutamate receptors (mGluRs) trigger a strong inhibition of the glutamatergic inputs from parallel and climbing fibers. Both pathways involve endocannabinoids retrogradely acting on type 1 cannabinoid receptors (CB1Rs) at presynaptic terminals. Here, we show that group I mGluR activation also depresses GABAergic transmission at the synapses between molecular layer interneurons and Purkinje cells. Using paired recordings, we found that application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine reduced the evoked IPSCs in Purkinje cells. This effect was independent of postsynaptic Ca2+ increases and was completely blocked by a CB1R antagonist. Experiments performed with the GTP-analogues GDP-betaS and GTP-gammaS provided evidence that endocannabinoids released after G-protein activation can also inhibit GABAergic inputs onto nearby, unstimulated Purkinje cells. Block of the enzymes DAG lipase or phospholipase C reduced the group I mGluR-dependent inhibition, suggesting that 2-arachidonyl glycerol could act as retrograde messenger. Finally, group I mGluR activation by brief bursts of activity of the parallel fibers induced a short-lived depression of spontaneous IPSCs via presynaptic CB1Rs. Our results reveal a mechanism with potential physiological importance, by which glutamatergic synapses induce an endocannabinoid-mediated inhibition of the GABAergic inputs onto Purkinje cells.
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PMID:Group I metabotropic glutamate receptors inhibit GABA release at interneuron-Purkinje cell synapses through endocannabinoid production. 1515 47

Stimulation of G-protein coupled membrane receptors linked to phospholipase C results in production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum, which triggers increased Ca2+ influx across the plasma membrane, so-called capacitative calcium entry. DAG can also activate plasma membrane calcium-permeable channels but the mechanism is still not fully understood. In the pregnant human myometrial cell line PHM1 and in primary myometrial cells, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, induced variable oscillatory patterns of intracellular free Ca2+. Similar behavior was seen with Sr2+ entry. The Ca2+ oscillations were not blocked by a broad spectrum of protein kinase C inhibitors, including chelerytrine, bisindolylmaleimide I and calphostin C, and were enhanced and prolonged by RHC-80267, an inhibitor of diacylglycerol lipase. The OAG-induced oscillatory response was not dependent on Ca2+ release from the endoplasmic reticulum but required extracellular Ca2+. Our results indicate that diacylglycerol directly activates cation channels in PHM1 and primary myometrial cells and promotes intracellular Ca2+ oscillations by actions independent of intracellular Ca2+ -ATPase activity and protein kinase C involvement.
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PMID:Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells. 1519 63

In the present study we have investigated an inhibitory pathway regulating a constitutively active Ca(2+)-permeable non-selective cation conductance (I(cat)) in rabbit ear artery smooth muscle cells. Constitutive single channel activity of I(cat) was recorded in cell-attached and inside-out patches with similar unitary conductance values. In inside-out patches with relatively high constitutive activity the G-protein activator GTPgammaS inhibited channel activity which was reversed by the protein kinase C (PKC) inhibitor chelerythrine indicating a G-protein pathway inhibits channel activity via PKC. Spontaneous channel activity was also suppressed by the G-protein inhibitor GDPbetaS suggesting a G-protein is also involved in initiation of constitutive channel activity. Bath application of antibodies to G(alphaq)/G(alpha11) enhanced channel activity whereas anti-G(alpha1-3)/G(alphao) antibodies decreased basal channel activity which suggests that G(alphaq)/G(alpha11) and G(alphaiota)/G(alphao) proteins initiate, respectively, the inhibitory and excitatory cascades. The phospholipase C (PLC) inhibitor U73122 increased spontaneous activity which implies a role for PLC in the inhibitory pathway. Bath application of the diacylycerol (DAG) analogue 1-oeoyl-2-acetyl-sn-glycerol (OAG) decreased the probability of channel opening (NP(o)) and this was reversed by chelerythrine. Application of the PKC activator phorbol 12, 13-dibutyrate (PDBu) and chelerythrine, respectively, decreased and increased NP(o). These data indicate that spontaneously active cation channels are inhibited by a tonic inhibitory pathway involving G(alphaq)/G(alpha11)-mediated stimulation of PLC to generate DAG which activates PKC to inhibit channel opening. There were some patches with relatively low NP(o) and it was evident that the inhibitory pathway was particularly marked in these cases. Moreover in the latter patches GTPgammaS and OAG caused marked increases in NP(o). Together with inhibitory effects of GDPbetaS and anti-G(alpha1-3)/G(alphao) antibodies the results suggest that there is constitutive G(alphai)/G(alphao) protein activity leading to channel opening via a DAG-mediated but PKC-independent mechanism. Finally, with whole-cell recording it is shown that noradrenaline increases I(cat) and the noradrenaline-evoked response is markedly potentiated by PKC inhibition. This latter observation shows that PKC also limits agonist-evoked I(cat) in these arterial myocytes.
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PMID:Inhibitory regulation of constitutive transient receptor potential-like cation channels in rabbit ear artery myocytes. 1529 79

Prevention of diabetic gastrointestinal dysfunction is of utmost importance. The present study demonstrated that diacylglycerol kinase (DGK) activity in diabetic gastric smooth muscle in the resting state was approximately 3.5-fold greater than that in controls. However, oral administration of TJ-43 (1% of food intake) or subcutaneous insulin injection (12 units/kg/day) in streptozotocin-induced diabetic rats (DM) for 2 weeks prevented DGK abnormalities based on the control level. Increased DGK activity in the resting state of DM was inhibited significantly by R59022, neomycin or staurosporine; in contrast, these drugs did not affect DGK activity in controls, insulin-treated DM or TJ-43-treated DM. In controls, the endogenous phosphatidic acid (PA) level was inhibited significantly by R59022 or neomycin but not affected by staurosporine. On the other hand, these three drugs significantly inhibited endogenous PA levels in DM, and neomycin significantly inhibited endogenous PA levels in insulin-treated and TJ-43-treated DM. This suggests that TJ-43 could prevent alteration of DGK activity and PA formation without reduction of blood glucose levels. Moreover, these effects were greater than those of insulin treatment. Results suggested that TJ-43 treatment influenced the hyperreactivity of DGK and DAG formation via phospholipase C activity. In conclusion, TJ-43 can be recommended with respect to enhancement of the quality of life in patients displaying diabetic gastrointestinal complications.
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PMID:A traditional herbal medicine, rikkunshi-to (TJ-43), prevents intracellular signaling disorders in gastric smooth muscle of diabetic rats. 1531 62

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain.
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PMID:Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells. 1554 67

Nuclear envelope (NE) formation in a cell-free egg extract proceeds by precursor membrane vesicle binding to chromatin in an ATP-dependent manner, followed by a GTP-induced NE assembly step. The requirement for GTP in the latter step of this process can be mimicked by addition of bacterial PI-PLC [phosphoinositide (PtdIns)-specific phospholipase C]. The NE assembly process is here dissected in relation to the requirement for endogenous phosphoinositide metabolism, employing recombinant eukaryotic PI-PLC, inhibitors and direct phospholipid analysis using ESI-MS (electrospray ionization mass spectrometry). PtdIns (phosphatidylinositol) species analysis by ESI-MS indicates that the chromatin-bound NE precursor vesicles are enriched for specific PtdIns species. Moreover, during GTP-induced precursor vesicle fusion, the membrane vesicles become partially depleted of the PtdIns 18:0/20:4 species. These data indicate that eukaryotic PI-PLC can support NE formation, and the sensitivity to exogenous recombinant PtdIns-5-phosphatases shows that the endogenous PLC hydrolyses a 5-phosphorylated species. It is shown further that the downstream target of this DAG (diacylglycerol) pathway does not involve PKC (protein kinase C) catalytic function, but is mimicked by phorbol esters, indicating a possible engagement of one of the non-PKC phorbol ester receptors. The results show that ESI-MS can be used as a sensitive means to measure the lipid composition of biological membranes and their changes during, for example, membrane fusogenic events. We have exploited this and the intervention studies to illustrate a pivotal role for PI-PLC and its product DAG in the formation of NEs.
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PMID:Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes. 1555 72

The wake-promoting neuropeptides orexins (hypocretins) play a crucial role in controlling neuronal excitability and synaptic transmission in the CNS. In this study, using whole-cell patch-clamp recordings in an acute dorsal raphe nucleus (DRN) slice preparation, we report that orexin B (Orx-B) depresses the evoked glutamate-mediated synaptic currents in DRN 5-HT neurons. The Orx-B-induced depression is accompanied by an increase in the paired-pulse ratio and the coefficient of variance, suggesting a presynaptic site of action. Orx-B also reduces the frequency but not the amplitude of miniature EPSCs, indicating that depression of glutamatergic transmission is mediated by a decrease in glutamate release. Surprisingly, the Orx-B-induced inhibition of glutamatergic transmission is abolished by postsynaptic inhibition of G-protein signaling with GDPbetaS, suggesting that this effect is signaled by postsynaptic orexin receptors and expressed presynaptically, presumably through a retrograde messenger. Interestingly, the Orx-B-induced depression of glutamate release is mimicked and occluded by the cannabinoid receptor agonist WIN 55,212-2, and is abolished by the CB1 cannabinoid receptor antagonist AM 251. These results imply that the Orx-B-induced depression of glutamatergic transmission to DRN 5-HT neurons is mediated by retrograde endocannabinoid release. Examination of downstream signaling pathways involved in this response indicates that the effect of Orx-B requires the activation of phospholipase C and DAG lipase enzymatic pathways but not a rise in postsynaptic intracellular calcium. Therefore, our findings reveal a previously unsuspected mechanism by which postsynaptic orexin receptors can modulate glutamatergic synaptic transmission to DRN 5-HT neurons.
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PMID:The wake-promoting peptide orexin-B inhibits glutamatergic transmission to dorsal raphe nucleus serotonin neurons through retrograde endocannabinoid signaling. 1567 70


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