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)

Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established a stable Chinese hamster ovary cell line expressing both Y1-NPY and alpha 1b-adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 microM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium concentration ([Ca2+]i) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca2+, but abolished by prior depletion of intracellular Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[3H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at > 15 s post-stimulation) was too slow to play a causative role in Ca2+ mobilization. Combination of PE and PYY resulted in increases in [Ca2+]i which were at best additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P3 at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [3H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca2+ in an Ins(1,4,5)P3-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P3 generation. Instead the major consequences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.
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PMID:Synergistic interaction of Y1-neuropeptide Y and alpha 1b-adrenergic receptors in the regulation of phospholipase C, protein kinase C, and arachidonic acid production. 774 27

In the present study, we investigated the effect of hypoxia on the chronotropic response to norepinephrine (NE) of cultured neonatal rat ventricular myocytes. We measured beating of myocytes with the Fotonic sensor, using a newly developed method for a noncontact displacement measurement. The beating rate counted with the sensor had a high correlation coefficient with that counted visually under a microscope (r = 0.997, P < 0.01). NE concentrations of 10(-8) - 10(-4) M caused negative chronotropy dose dependently in the presence of 5 x 10(-7) M propranolol. NE-induced chronotropy was completely antagonized by 10(-6) M prazosin. Three hours hypoxia decreased the spontaneous beating rate 40% (P < 0.01). Negative chronotropy induced by 10(-4) M NE in normoxia was inverted to positive and was antagonized by prazosin. Hypoxia increased the basal level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to 190% (P < 0.01), while NE-stimulated Ins(1,4,5)P3 production was significantly suppressed. Immunoblotting analysis of G protein subunits demonstrated no quantitative changes in Gi alpha, Gq alpha, Go alpha and G beta common subunits in hypoxia. In a saturation binding assay with [3H]prazosin, Kd values were increased to 152% by hypoxia (P < 0.05) without significant change in Bmax. Basal activity of low Km-GTPase was increased to 122% by hypoxia (P < 0.05). These results suggest that the hypoxia-induced increase in low-Km GTPase activity, which could stimulate phospholipase C by an activated alpha GTP subunit of G protein and consequently induce receptor-independent increase in Ins(1,4,5)P3, may be responsible for the inversion of the NE-induced negative chronotropic response in normoxia.
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PMID:Hypoxia inverts the negative chronotropic response to norepinephrine in normoxia in cultured neonatal rat cardiac myocytes: role of the alpha 1 adrenergic signal transduction system. 774 96

Inhibitory effects of the anti-manic agent lithium on carbachol-stimulated phosphoinositide signaling have been investigated in Chinese hamster ovary (CHO) cells transfected with human m1 muscarinic receptor cDNA (Bmax, 816 fmol/mg of protein). In the presence of Li+, a time-dependent inhibition of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] mass accumulation was observed within 10 min of agonist addition (IC50 for lithium inhibition at 20 min after carbachol addition, 0.5 mM). The Li(+)-induced decrease in agonist-stimulated Ins(1,4,5)P3 levels was preceded by a dramatic increase in CMP-phosphatidate accumulation. The idea that Li+ blockade of inositol monophosphatase caused a rapid depletion of the cellular myo-inositol pool in CHO-m1 cells was supported by the reversal of Li+ effects by exogenous myo-inositol. Carbachol (1 mM) alone caused a rapid and dramatic decrease in phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)-P2]in CHO-m1 cells labeled to equilibrium with [3H]-inositol. Carbachol-evoked decreases in PtdIns(4,5)P2 were time-dependently accentuated by Li+ (IC50 for Li+ inhibition at 20 min after carbachol addition, 1.2 mM). Measurements of changes in PtdIns(4,5)P2 mass demonstrated that the effect of Li+ was completely and concentration-dependently reversed by addition of myo-inositol. Sequential 30-min periods of carbachol stimulation resulted in similar time courses of Ins(1,4,5)P3 accumulation when an intervening 20-min recovery period was included in the protocol. Inclusion of Li+ throughout resulted in a more rapid and dramatic attenuation of Ins(1,4,5)P3 during the agonist rechallenge period, which could be correlated with accentuated changes in PtdIns(4,5)P2. These data demonstrate that, although mechanisms operate to efficiently resynthesize PtdIns(4,5)P2, the temporal correlation of carbachol-evoked decreases in PtdIns(4,5)P2 levels in the presence of Li+ strongly suggests that phosphoinositide-specific phospholipase C substrate depletion may be causal in the subsequent decrease in Ins(1,4,5)P3 levels.
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PMID:Disruption by lithium of phosphatidylinositol-4,5-bisphosphate supply and inositol-1,4,5-trisphosphate generation in Chinese hamster ovary cells expressing human recombinant m1 muscarinic receptors. 780 34

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding domain of phospholipase C-delta 1 (PLC-delta 1) was determined by examining binding activity of the synthetic peptide corresponding to residues 30-43 of PLC-delta 1. The peptide coupled with carrier proteins such as keyhole limpet hemocyanin or bovine serum albumin bound Ins(1,4,5)P3, whereas a scrambled peptide with the same amino acids did not do so. Polyclonal antibody against the peptide was examined to determine whether it would cause inhibition of the Ins(1,4,5)P3 binding to PLC-delta 1. Fab fragment of antibody to the peptide did inhibit binding to PLC-delta 1, in a dose-dependent manner. Thus it seems likely that the region of residues 30-43 of PLC-delta 1 is responsible for the binding of Ins(1,4,5)P3.
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PMID:D-myo-inositol 1,4,5-trisphosphate binding domain of phospholipase C-delta 1. 781 Dec 37

Depending on the vascular bed considered, the actions of ATP on the endothelium are mediated by either P2Y or P2U receptors. The two types of receptors seem to coexist on bovine aortic endothelial cells, where they are both coupled to phospholipase C. In this study, we have investigated whether they are truly coexpressed on the same cells and whether their signaling pathways diverge beyond phospholipase C activation. Measurements of [Ca2+]i in single cells showed that almost all bovine aortic endothelial cells are responsive to both 2-methylthio-ATP (2MeSATP), an agonist of P2Y receptors, and UTP, an agonist of P2U receptors. UTP stimulated the release of prostacyclin from freshly isolated bovine aortic endothelial cells, even when they were exposed to cycloheximide at the time of their collection: this indicates that P2U receptors must already be expressed on endothelial cells in situ and do not appear during cell culture. The time course of inositol phosphate (InsP) accumulation and the relative proportion of Ins(1,4,5)P3, Ins(1,3,4,5)P4, and Ins(1,3,4)P3 were similar in cells stimulated by 2MeSATP or UTP. UTP and 2MeSATP both stimulated the hydrolysis of phosphatidylcholine by phospholipase D, as reflected by the release of [3H]choline from prelabeled cells. The responses to both agents were blocked after downregulation of protein kinase C, resulting from a prolonged exposure to phorbol 12-myristate 13-acetate: this blockade occurred at a step distal to phospholipase C activation. A single difference between the two pathways has been identified: the effect of 2MeSATP on InsP3 was significantly more inhibited after a short exposure to phorbol 12-myristate 13-acetate than that of UTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Coexpression of P2Y and P2U receptors on aortic endothelial cells. Comparison of cell localization and signaling pathways. 783 29

A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [3H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6), as well as inositol mono- to tetrakisphosphate, fractions was observed. Addition of carbachol (1 mM) caused an immediate increase in level of Ins(1,4,5)P3 (peak increase two-fold over basal by 60 s), which was well-maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP5 and InsP6 was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P3 metabolism occurs predominantly via the inositol polyphosphate 5-phosphatase route, with metabolism via the Ins(1,4,5)P3 3-kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide-specific phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, although some hydrolysis of phosphatidyl-inositol 4-phosphate cannot be excluded.
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PMID:Muscarinic cholinoceptor-stimulated synthesis and degradation of inositol 1,4,5-trisphosphate in the rat cerebellar granule cell. 786 Nov 45

Rabbit aortic muscles were stretched from a holding length of 0.6 maximum length (Lmax) to lengths as great as 1.0 Lmax and the new length maintained. When muscles were stretched to 1.0 Lmax, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] contents were increased at 375 ms (uncorrected for freezing time) poststretch to 209 +/- 27 and 139.8 +/- 12% (SE), respectively, of control values. Increases in Ins(1,4,5)P3 and Ins(1,4)P2 contents reached an apparent maximum at approximately 500 ms, i.e., to 243.7 +/- 15.8 and 180.9 +/- 16.2% of control, and were decreased to near control levels at 1,700 ms poststretch. The stretch threshold for phospholipase C (PLC) activation was 0.85 Lmax. The latency to onset of PLC activation, correcting for the time for freezing, was 275 to 375 ms. Maximal PLC activity was 91 pmol.s-1.100 nmol total lipid P(i)-1, which corresponded to 10% of total phosphatidylinositol bisphosphate being hydrolyzed per second. The mechanism of stretch-activated PLC activity involved influx of Ca2+ via gadolinium-sensitive ion channels, but not via nifedipine-sensitive ion channels.
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PMID:Smooth muscle stretch-activated phospholipase C activity. 786 85

1. Stimulation of P2U-purinoceptors with UTP or histamine H1-receptors with histamine gave rise to the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) in DDT1 MF-2 smooth muscle cells. 2. Stimulation of P2U-purinoceptors or histamine H1-receptors caused an increase in cytoplasmic Ca2+, consisting of an initial peak, representing the release of Ca2+ from internal stores and a sustained phase representing Ca2+ influx. 3. The P2U-purinoceptor-mediated Ca(2+)-entry mechanism was more sensitive to UTP than Ca(2+)-mobilization (EC50: 3.3 microM +/- 0.4 microM vs 55.1 microM +/- 9.2 microM), in contrast to these processes activated by histamine H1-receptors (EC50: 5.8 microM +/- 0.6 microM vs 3.1 microM +/- 0.5 microM). 4. Pre-stimulation of cells with several adenosine 3':5'-cyclic monophosphate (cyclic AMP) elevating agents, reduced the histamine H1-receptor-mediated formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin completely inhibited Ins(1,4,5)P3 formation (IC50: 158 +/- 24 nM) whereas Ins(1,3,4,5)P4 formation was inhibited by only 45% (IC50: 173 +/- 16 nM). The P2U-purinoceptor-mediated production of these inositol phosphates was not affected by cyclic AMP. 5. Forskolin and isoprenaline reduced the histamine-induced increase in cytoplasmic Ca2+, as measured in Ca2+ containing medium and in nominally Ca(2+)-free medium but did not change the UTP-induced increase in cytoplasmic Ca2+. 6. These results clearly demonstrate that cyclic AMP differentially regulates components of the histamine induced phospholipase C signal transduction pathway. Furthermore, cyclic AMP does not affect the phospholipase C pathway activated by stimulation of P2U-purinoceptors in DDT1 MF-2 cells.
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PMID:Regulation of histamine- and UTP-induced increases in Ins(1,4,5)P3, Ins (1,3,4,5)P4 and Ca2+ by cyclic AMP in DDT1 MF-2 cells. 788 38

The coupling of two endothelin receptor subtypes (ET(A) and ETB) to several types of guanine-nucleotide-binding regulatory protein (G protein) was examined. Two subtypes of receptor cDNAs were transfected alone or together with four different G protein alpha subunit cDNAs in COS-7 cells. In ET(A) receptor-transfected cells, endothelin-1 (ET-1) activated phosphatidylinositol-specific phospholipase C as measured by the production of phosphatidylinositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. ETB-receptor-transfected cells also produced Ins(1,4,5)P3 on stimulation by ET-1. The ET-1-induced production of Ins(1,4,5)P3 was markedly higher in G alpha q-cotransfected or G alpha 11-cotransfected cells than in cells transfected with each receptor alone. ET-1 also stimulated production of cAMP in ET(A) or ETB receptor-transfected cells. The production of cAMP was synergistically amplified by G alpha s co-transfection with each receptor. In contrast, when G alpha i2 was co-transfected with the ET(A) or ETB receptor, ET-1 displayed an inhibitory action on forskolin-stimulated cAMP accumulation. Pertussis-toxin treatment of the G alpha i2-transfected cells resulted in abolition of the endothelin-induced inhibition of cAMP accumulation. These observations indicate that both ET(A) and ETB receptors are able to couple to Gq, G11, Gs and Gi2, and suggest that endothelin receptors stimulate multiple effectors via several types of G protein simultaneously. The overall effects induced by endothelin may differ in cell types depending on the level of expression of each G-protein subtype in the cell.
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PMID:Molecular identification of guanine-nucleotide-binding regulatory proteins which couple to endothelin receptors. 788 89

The presence of specific receptors for gastrointestinal hormones on T cells and their involvement in the immune response are still matters of debate. We reported the effects of gastrin-cholecystokinin (CCK)-related peptides on J.RT3-T3.5 Jurkat cells. A single class of high-affinity binding sites (dissociation constant approximately 0.1 nM) for gastrin and CCK-8 was evident on these cells. These peptides dose-dependently induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i), which was independent of extracellular Ca2-. L-365,260 was 150- to 300-fold more potent than L-364,718 to inhibit radiolabeled ligand binding or peptide-stimulated [Ca2+]i increase, confirming the gastrin-CCK-B nature of the receptor. Gastrin caused a rise in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] level within 5 s of stimulation. Finally, gastrin increased interleukin (IL)-2 secretion in J.RT3-T3.5 cells. We conclude that 1) J.RT3-T3.5 cells possess "gastrin-CCK-B type" receptors coupled to phospholipase C activation, Ins(1,4,5)P3 formation, and Ca2+ release from intracellular Ca2+ pools, and 2) these receptors could be involved in the regulation of IL-2 production.
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PMID:Gastrin-CCK-B type receptors on human T lymphoblastoid Jurkat cells. 790 Aug 13


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