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)

We have investigated the effects of hypotonic stress on intracellular calcium concentration ([Ca(2+)](i)) in bovine aortic endothelial cells. Reducing extracellular osmolarity by 5% to 40% elicited a steep Ca(2+) transient both in normal Krebs and Ca(2+)-free solutions. The hypotonic stress-induced Ca(2+) transient was inhibited by phospholipase C inhibitors (neomycin and U-73122), a P(2)-receptor antagonist (suramin), and an ATP-hydrolyzing enzyme (apyrase), suggesting that the hypotonic stress-induced Ca(2+) transient is mediated by ATP. A luciferin-luciferase assay confirmed that 40% hypotonic stress released 91.0 amol/cell of ATP in 10 min. When the hypotonic stress-induced fast Ca(2+) transient was inhibited by neomycin, suramin, or apyrase, a gradual [Ca(2+)](i) increase was observed instead. This hypotonic stress-induced gradual [Ca(2+)](i) increase was inhibited by a phospholipase A(2) inhibitor, 4-bromophenacyl bromide. Furthermore, exogenously applied arachidonic acid induced a gradual [Ca(2+)](i) increase with an ED(50) of 13.3 microM. These observations indicate that hypotonic stress induces a dual Ca(2+) response in bovine aortic endothelial cells, i.e., an ATP-mediated fast Ca(2+) transient and an arachidonic acid-mediated gradual Ca(2+) increase, the former being the predominant response in normal conditions.
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PMID:Hypotonic stress-induced dual Ca(2+) responses in bovine aortic endothelial cells. 1092 62

We developed a protocol for quantification of relative gene expression using reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA(A) receptor beta(1) subunit (GABA(A)Rbeta(1)) and phospholipase C beta(4) subtype (PLCbeta(4)) in rat cerebrum and cerebellum were determined by comparing the ratio of PCR products generated by linear amplification of the target cDNA segments and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a reference. The density of PCR products was measured from digitized images of photographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a single band) of the detection system. The accuracy of the present method was <2-fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLCbeta(4) was significantly higher in cerebellum than cerebrum, and that of GABA(A)Rbeta(1) was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter receptor and intracellular signaling enzyme gene families.
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PMID:Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining. 1093 41

(1) The vasorelaxation produced by the phosphodiesterase 3 (PDE3) inhibitor, amrinone was investigated in isolated rat aorta denuded of endothelium. In the presence of extracellular Ca(2+), amrinone, milrinone and 3-isobutyl-1-methylxanthine (IBMX), relaxed endothelium-denuded rat aortic rings constricted with phenylephrine. While the actions of milrinone and IBMX were inhibited by the protein kinase G (PKG) inhibitor, Rp-8-Bromo guanosine-3',5' monophosphothioate (Rp-8-Br-cGMPS; 0.5 mM), that of amrinone was only slightly affected; whereas the protein kinase A (PKA) inhibitor, Rp-adenosine-3',5' cyclic monophosphothioate (Rp-cAMPS; 0.5 mM) had no effect on any agent. (2) Amrinone (100 microM) inhibited (45)Ca(2+) influx through receptor- or store-operated Ca(2+) channels following stimulation with phenylephrine (1 microM) or thapsigargin (1 microM). In contrast, amrinone had no effect on KCl (120 mM)-stimulated Ca(2+) influx. (3) In the absence of extracellular Ca(2+), amrinone (30 microM) inhibited the constriction produced by phenylephrine, 5-hydroxytryptamine (5HT) and U46619, and this effect was not affected by Rp-cAMPS or Rp-8-Br-cGMPS. (4) The intracellular mechanism of action of amrinone may involve the phospholipase C (PLC)-inositol 1,4,5 trisphosphate (IP(3))-intracellular Ca(2+) signal transduction pathway. However, amrinone (100 microM) had no effect on either basal- or noradrenaline (100 microM)-stimulated PLC activity. Similarly, IP(3) stimulated a concentration-dependent release of Ca(2+) from rat brain microsomes that was not affected by amrinone (30 and 100 microM). (5) In conclusion, the vasorelaxant action of amrinone does not involve adenosine 3',5' cyclic monophosphate (cAMP) or involve guanosine 3',5' cyclic monophosphate (cGMP) but may include an inhibition of Ca(2+) influx through receptor- or store-operated Ca(2+) channels, although it does not directly affect intracellular Ca(2+) release.
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PMID:The role of cyclic nucleotides and calcium in the relaxation produced by amrinone in rat aorta. 1128 18

The mode of action of venom from the ectoparasitic wasp Nasonia vitripennis in eliciting cell death was examined using an in vitro approach with BTI-TN-5B1-4 cells, and the cell responses were compared to those evoked by the extensively studied wasp toxin mastoparan. Wasp venom increased plasma membrane permeability to Na+, resulting in cellular swelling and death due to oncosis. When ouabain was used to disable Na+, K+-ATPases, the effects of venom were enhanced. Measurements of intracellular calcium using fluo-4 AM revealed a rearrangement and an increase in cytosolic [Ca+2]i within 30 min after exposure of BTI-TN-5B1-4 cells to venom. This venom-mediated increase in Ca+2 was apparently due to mobilization of intracellular stores since the changes occurred in the absence of extracellular Ca+2. Phospholipase C (PLC) inhibitors, neomycin and U-73122, blocked the venom-induced death temporarily (<3h), but by 24h, all venom-treated cells swelled and lysed. Pre-treatment of cells with caffeine or theophylline but not ryanodine attenuated the induction of oncosis by wasp venom. Anti-inflammatory peptide 1 (antiflammin 1) but not bromophenacyl bromide, agents that block phospholipase A2 (PLA2) activity, abolished the responsiveness of BTI-TN-5B1-4 cells to venom. These results suggest that venom initiates cell death by inducing Ca+2 release from intracellular stores probably via phospholipase C and IP3. A possible mode of action for venom from N. vitripennis requiring dual activation of PLC and PLA2 is discussed and compared to the pathways known to be activated by mastoparan.
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PMID:Venom from the ectoparasitic wasp Nasonia vitripennis increases Na+ influx and activates phospholipase C and phospholipase A2 dependent signal transduction pathways in cultured insect cells. 1160 74

The effect of trichloroethanol (TCEt), the active metabolite of chloral hydrate, on the intracellular concentration of calcium ([Ca(2+)](i)) was investigated in rat submandibular glands (RSMG) acini loaded with fura-2. TCEt (1 - 10 mM) increased the [Ca(2+)](i) independently of the presence of calcium in the extracellular medium. Dichloroethanol (DCEt) and monochloroethanol (MCEt) reproduced the stimulatory effect of TCEt but at much higher concentrations (about 6 fold higher for DCEt and 20 fold higher for MCEt). TCEt mobilized an intracellular pool of calcium, which was depleted by a pretreatment with thapsigargin, an inhibitor of the sarcoplasmic and endoplasmic reticulum calcium-dependent ATPases, but not with FCCP, an uncoupler of mitochondria. TCEt 10 mM inhibited by 50% the thapsigargin-sensitive microsomal Ca(2+)-ATPase. DCEt 10 mM and MCEt 10 mM inhibited the ATPase by 20 and 10%, respectively. TCEt inhibited the increase of the [Ca(2+)](i) and the production of inositol phosphates in response to carbachol, epinephrine and substance P. TCEt inhibited the uptake of calcium mediated by the store-operated calcium channel (SOCC). ATP and Bz-ATP increased the [Ca(2+)](i) in RSMG acini and this effect was blocked by extracellular magnesium, by Coomassie blue and by oxydized ATP (oATP). TCEt potentiated the increase of the [Ca(2+)](i) and of the uptake of extracellular calcium in response to ATP and Bz-ATP. TCEt had no effect on the uptake of barium and of ethidium bromide in response to purinergic agonists. These results suggest that TCEt, at sedative concentrations, exerts various effects on the calcium regulation: (1) it mobilizes a thapsigargin-sensitive intracellular pool of calcium in RSMG acini; (2) it inhibits the uptake of calcium via the SOCC; (3) it inhibits the activation by G protein-coupled receptors of a polyphosphoinositide-specific phospholipase C. It does not interfere with the activation of the ionotropic P2X receptors. The use of chloral hydrate should be avoided in studies exploring the in vivo responses to sialagogues.
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PMID:Multiple effects of trichloroethanol on calcium handling in rat submandibular acinar cells. 1205 35

Parathyroid hormone-related protein (PTHrP) promotes or suppresses apoptosis in various settings depending on cell type and context. PTHrP 1-34 and PTHrP 67-86 are type II cell growth factors with effects on pneumocyte growth and surfactant secretion. This study investigated the effects of 24 h pretreatment with these two peptides on rat type II cell apoptosis after 0.3 J/cm2 ultraviolet-B irradiation. Adherent cells decreased in number by 15 +/- 5% and nonadherent cells increased > 5-fold 24 h after ultraviolet irradiation. Cell loss was due predominantly to apoptosis, based on ethidium bromide exclusion, nuclear condensation, and caspase 3 activity. Nuclear condensation increased from 15.6 +/- 2.2% of irradiated cells with no treatment to 25.6 +/- 4.9 and 22.9 +/- 1.8% of cells in ultraviolet/PTHrP 1-34 and ultraviolet/PTHrP 67-86 groups, respectively (P < 0.01), along with a 60% increase in caspase 3 activity. Effects on apoptosis were unaffected by the presence or absence of serum, but were ameliorated by growth to confluence or adherence to fibronectin. PTHrP 1-34 and PTHrP 67-86 augmented inositol phosphate levels, but had minimal effects on cAMP. Thus, PTHrP 1-34 and PTHrP 67-86 sensitize type II cells to apoptosis, possibly by a phospholipase C-dependent mechanism. The effects appear to be regulated by cell-matrix and cell-cell interactions.
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PMID:Proapoptotic effects of parathyroid hormone-related protein in type II pneumocytes. 1279 77

The aim of this study was to determine the role of intracellular proteins in phagocytosis of opsonized Porphyromonas gingivalis by RAW264.7 cells, a murine macrophage-like cell line. This periodontopathogen was grown anaerobically and opsonized with an IgG2a murine monoclonal anti-P. gingivalis lipopolysaccharide antibody. RAW264.7 cells were preincubated with protein tyrosine kinase inhibitors (staurosporine and genistein), protein kinase C inhibitors (phorbol myristic acetate and bisindolylmaleimide), a serine/threonine phosphatase inhibitor (okadaic acid), a phosphatidylinositol 3-kinase inhibitor (worthmannin), phospholipase A2 inhibitors (bromophenacyl bromide and nordihydroguaiaretic acid), phospholipase C inhibitors (p-chloromercuriphenyl sulfonate and neomycin sulfate), an actin-filament depolymerizer (cytochalasin D), and a microtubule disrupting agent (colchicine). Inhibitor-treated macrophages were then incubated with the opsonized P. gingivalis and the phagocytosed cells determined microscopically. The results showed the percentage of the phagocytosed organisms decreased when the cells were preincubated with protein tyrosine kinase, protein kinase C, protein phosphatase and phosphatidylinositol 3-kinase inhibitors. Of interest, preincubation with phorbol myristic acetate for 30 min increased the ability of RAW264.7 cells to phagocytose the opsonized organisms. Phospholipase A2 and phospholipase C inhibitors only slightly reduced the number of phagocytosed organisms. The results indicated that opsonophagocytosis of P. gingivalis by RAW264.7 cells might be determined by the activation of protein tyrosine kinase, protein kinase C, protein phosphatases, and phosphatidylinositol 3-kinase inhibitor. Both phospholipase A2 and phospholipase C would appear to be involved to a lesser extent. The opsonophagocytosis of this periodontopathogen would also appear to be dependent upon actin and microtubule polymerization.
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PMID:Intracellular proteins involved in Porphyromonas gingivalis-induced opsonophagocytic activities of a murine macrophage cell line (RAW264.7 cells). 1472 50

Dopamine D(1)-mediated inhibition of Na(+),K(+)-ATPase activity in opossum kidney (OK) cells involves the sequential activation of the adenylyl cyclase-protein kinase A (PKA) and the phospholipase C-protein kinase C (PKC) pathways. The present study evaluated the signalling cascades involved in dopamine-mediated inhibition of Na(+)/H(+) exchanger isoform 3 (NHE3) in OK cells. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na(+) of 25+/-6 mM. Dopamine and the dopamine D(1)-like receptor agonist SKF 38393 ((+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol) inhibited NHE3 activity in a concentration-dependent manner; the dopamine D(2)-like receptor agonist quinerolane was devoid of effect. The SKF 38393-mediated inhibition of NHE3 was prevented either by the dopamine D(1)-like receptor antagonist SKF 83566 ((+/-)-7-Bromo-8-8-hydroxy-3 methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; 1 microM), overnight treatment with cholera toxin (500 ng/ml), the PKA antagonist H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5 isoquinolinesulfonamide hydrochloride; 10 microM), the PKC antagonist chelerythrine (1 microM), or the phospholipase C inhibitor U-73,122 (1-(6-[(17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl) amino] hexyl)-1H-pyrrole-2,5-dione; 3 microM). In addition, dibutyril cAMP (dB-cAMP; 500 microM) was found to increase phospholipase C activity, both in membranes and in cytosol from OK cells; in contrast, phorbol-12,13-dibutyrate (PDB) (1 microM) did not have a significant effect on phospholipase C activity. Pre-treatment of OK cells with the anti-G(s)alpha antibody, but not the anti-G(q/11)alpha antibody, blunted the inhibitory effect of SKF 38393 on NHE3 activity. It is concluded that dopamine D(1)-mediated inhibition of NHE3 in renal OK cells involves both adenylyl cyclase-PKA and the phospholipase C-PKC pathways, a mechanism similar to that described for Na(+),K(+)-ATPase.
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PMID:Dopamine acutely decreases type 3 Na(+)/H(+) exchanger activity in renal OK cells through the activation of protein kinases A and C signalling cascades. 1504 35

We examined the effects of intracellular Cl- concentration ([Cl-]i) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of Na+ transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120: 71-85, 2002). Treatment with cAMP (100 microM 8-cpt-cAMP plus 100 microM IBMX) stimulated ENaC-mediated Na+ absorption as well as Cl- secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in alpha-toxin-permeabilized monolayers to have the anion selectivity sequence NO3- > Br- > Cl- > I-. With the use of FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane [determined from the amiloride-sensitive short-circuit current (AS-Isc) driven by an apical-to-basolateral Na+ concentration gradient] was progressively inhibited by increasing the [Cl-] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [Cl-] in the apical solution. This inhibitory effect of [Cl-]i occurred regardless of the presence or absence of net Cl- transport. However, from fluorometric measurements using the Cl(-)-sensitive dye 6-methoxy-N-(3-sulfopropyl)-quinolinium in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both Na+ absorption and Cl- secretion, produced a decrease of [Cl-]i from 76 +/- 14 to 36 +/- 8 mM (P = 0.03). Thus it might be expected that activation of Cl- secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase in [Cl-]i from 15 to 145 mM decreased AS-Isc from 24.5 +/- 1.0 to 10.2 +/- 1.6 microA/cm2. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 +/- 0.16 to 1.32 +/- 0.17 fmol/cm2 and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 +/- 1.2 to 8.2 +/- 1.4 microA/fmol.
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PMID:Inhibition of ENaC by intracellular Cl- in an MDCK clone with high ENaC expression. 1516 4

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl- channel that also controls the activity of other membrane transport proteins, such as the epithelial Na+ channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl- ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl- channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl- channel CLC-0 caused inhibition of amiloride-sensitive Na+ currents after cAMP-dependent stimulation and in the presence of a 100 mM bath Cl- concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl- concentration was increased in Xenopus oocytes in the presence of a high bath Cl- concentration, which inhibited ENaC without changing surface expression of alpha beta gammaENaC. In contrast, a 5 mM bath Cl- concentration reduced the cytosolic Cl- concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl- into oocytes and in inside/out macropatches by exposure to high cytosolic Cl- concentrations. The effect of Cl- was mimicked by Br-, Br-, NO3(-), and I-. Inhibition by Cl- was reduced in trimeric channels with a truncated COOH terminus of betaENaC and gammaENaC, and it was no longer detected in dimeric alpha deltaCbeta ENaC channels. Deletion of the NH2 terminus of alpha-, beta-, or gammaENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of betaENaC and gammaEnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl(-)-dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of betaENaC for Cl(-)-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR.
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PMID:Cl- interference with the epithelial Na+ channel ENaC. 1602 56


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