scholarly journals Characterization of phospholipase activity in Dictyostelium discoideum. Identification of a Ca2+-dependent polyphosphoinositide-specific phospholipase C

1992 ◽  
Vol 283 (2) ◽  
pp. 371-378 ◽  
Author(s):  
A B Cubitt ◽  
R A Firtel
Keyword(s):  
Phospholipase C ◽  
Dictyostelium Discoideum ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Fold Increase ◽  
Total Activity ◽  
Rapid Formation ◽  
Membrane Bound ◽  
Fold Stimulation

We have identified a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C activity in Dictyostelium discoideum. Addition of Ca2+ (20 microM) results in the rapid formation of Ins(1,4,5)P3 within 5 s and leads to sustained inositol phosphate production for up to 40 min in membranes prepared from [3H]inositol-labelled cells. The phospholipase C activity is primarily membrane-bound under the conditions used to lyse the cells. In addition to this activity we also identified a family of Ca(2+)-regulated phospholipase activities active on a range of phospholipid substrates, using [3H]palmitate labelling. Inositol-specific phospholipase C activity is highest in vegetatively growing cells and in starved cells during the first 6 h in development, during which time Ca2+ elicited a 5-fold stimulation of inositol phosphate formation. After this time, total activity decreased progressively until 15 h, after which the activity remained constant up until 24 h. During this period, Ca2+ was able to stimulate a 2-fold increase in inositol phosphates.

scholarly journals G-proteins are not directly involved in the CD3-antigen-mediated production of inositol phosphates in HPB-ALL T-leukaemia cells expressing phospholipase C isoforms γ1 and β3

1993 ◽  
Vol 289 (2) ◽  
pp. 387-394 ◽  
Author(s):  
M Biffen ◽  
M Shiroo ◽  
D R Alexander
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
G Protein ◽  
G Proteins ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cross Linking ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Inositol Phosphate Production

The possible involvement of G-proteins in T cell antigen-receptor complex (TCR)-mediated inositol phosphate production was investigated in HPB-ALL T-cells, which were found to express the phospholipase C gamma 1 and beta 3 isoforms. Cross-linking the CD3 antigen on streptolysin-O-permeabilized cells stimulated a dose-dependent increase in inositol phosphate production, as did addition of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or vanadate, a phosphotyrosine phosphatase inhibitor. It was possible, therefore, that the CD3-antigen-mediated production of inositol phosphates was either via a G-protein-dependent mechanism or by stimulation of protein tyrosine phosphorylation. The CD3-induced inositol phosphate production was potentiated by addition of vanadate, but not by addition of GTP[S]. Guanosine 5′-[beta-thio]diphosphate (GDP[S]) inhibited the rise in inositol phosphates induced by GTP[S], vanadate or cross-linking the CD3 antigen. The increase in protein tyrosine phosphorylation stimulated by vanadate or the OKT3 monoclonal antibody was not observed in the presence of GDP[S], showing that in permeabilized HPB-ALL cells, GDP[S] inhibits the actions of tyrosine kinases as well as G-protein function. Addition of either ADP[S] or phenylarsine oxide inhibited CD3- and vanadate-mediated increases in both tyrosine phosphorylation and inositol phosphate production, but did not inhibit GTP[S]-stimulated inositol phosphate production. On the other hand, pretreatment of cells with phorbol 12,13-dibutyrate inhibited subsequent GTP[S]-stimulated inositol phosphate production but did not inhibit significantly inositol phosphate production stimulated by either OKT3 F(ab')2 fragments or vanadate. Our results are consistent with the CD3 antigen stimulating inositol phosphate production by increasing the level of protein tyrosine phosphorylation, but not by activating a G-protein.

scholarly journals Relationships between the degree of cross-linking of surface immunoglobulin and the associated inositol 1,4,5-trisphosphate and Ca2+ signals in human B cells

1992 ◽  
Vol 284 (2) ◽  
pp. 447-455 ◽  
Author(s):  
F M McConnell ◽  
S B Shears ◽  
P J L Lane ◽  
M S Scheibel ◽  
E A Clark
Keyword(s):  
B Cells ◽  
Tyrosine Kinase ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cross Linking ◽  
Immunoglobulin Receptor ◽  
Surface Immunoglobulin ◽  
Human B Cells ◽  
Inositol 1,4,5 Trisphosphate

Cross-linking of surface immunoglobulin (Ig) receptors on human B cells leads to the activation of a tyrosine kinase. The activated tyrosine kinase subsequently phosphorylates a number of substrates, including phospholipase C-gamma. This enzyme breaks down phosphoinositol bisphosphate to form two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C and the release of intracellular Ca2+ respectively. We have used h.p.l.c. and flow cytometry to measure accurately the inositol phosphate turnover and Ca2+ release in anti-Ig-stimulated human B cells. In particular, we have examined the effect of dose of the cross-linking antibody on the two responses. The identity of putative messenger inositol phosphates has been verified by structural analysis, and the amounts of both inositol phosphates and Ca2+ present have been quantified. In the Ramos Burkitt lymphoma, which is very sensitive to stimulus through its Ig receptors, both inositol phosphate production and Ca2+ release were found to be related to the dose of anti-Ig antibody applied. This suggests that phospholipase C-mediated signal transduction in human B cells converts the degree of cross-linking of the immunoglobulin receptor quantitatively into intracellular signals.

Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats

1990 ◽  
Vol 258 (1) ◽  
pp. H173-H178 ◽  
Author(s):  
M. B. Turla ◽  
R. C. Webb
Keyword(s):  
Phospholipase C ◽  
Vascular Reactivity ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Phosphoinositide Metabolism ◽  
Hypertensive Rats ◽  
Wistar Kyoto

Recent studies suggest that serotonergic receptor activation is coupled to phospholipase C-mediated phosphoinositide hydrolysis, which results in the release of intracellular second messengers. The purpose of this study was to determine whether altered phosphoinositide metabolism is the basis for augmented vascular responsiveness to serotonin in genetic hypertension. Thoracic aortic segments isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) were labeled with myo-[3H]inositol and stimulated with serotonin in the presence of LiCl. Accumulation of [3H]inositol phosphates was then quantitated by column chromatography. Basal inositol phosphate accumulation and basal incorporation of myo-[3H]inositol into aortic cell membranes from SHRSP was not significantly different from WKY values. At 2.6 x 10(-7) to 2.6 x 10(-4) M serotonin, phosphoinositide metabolism was significantly augmented in aortae from SHRSP compared with WKY. Depolarization (100 mM KCl) did not increase phosphoinositide hydrolysis above basal levels in SHRSP or WKY. 2-Nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phospholipase C, prevented the serotonin-induced phosphoinositide metabolism. NCDC also partially inhibited phasic contractions (responses in calcium-free solution) to serotonin in aortas from SHRSP and WKY. In conclusion, abnormal phosphoinositide metabolism may be one mechanism responsible for the characteristic increase in vascular reactivity to serotonin in hypertension.

Ca2+ stores in smooth muscle cells: Ca2+ buffering and coupling to AVP-evoked inositol phosphate synthesis

1994 ◽  
Vol 266 (1) ◽  
pp. C276-C283 ◽  
Author(s):  
D. M. Berman ◽  
T. Sugiyama ◽  
W. F. Goldman
Keyword(s):  
Smooth Muscle ◽  
Inverse Relationship ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Enzyme System ◽  
Similar Treatment ◽  
A7r5 Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Communication Pathway

Cytosolic Ca2+ concentrations ([Ca2+]cyt) and [3H]inositol phosphates ([3H]InsP) were correlated while decreasing the Ca2+ content of sarcoplasmic reticulum (SR) stores in cultured A7r5 cells at rest and after activation with 8-arginine vasopressin (AVP). Decreasing Ca2+ influx by reducing extracellular Ca2+ or by treatment with verapamil had no effect on resting [Ca2+]cyt but significantly inhibited the AVP-evoked Ca2+ transients (delta Ca2+). Neither treatment affected basal [3H]InsP, but both treatments increased AVP-evoked synthesis of [3H]InsP. Likewise, basal [3H]InsP were unaffected by brief (10-30 s) exposures to thapsigargin (TG), while AVP-induced [3H]InsP synthesis was significantly augmented. Similar treatment with TG rapidly increased resting [Ca2+]cyt and decreased SR Ca2+ by 9-25% as manifested by decreased delta Ca2+. By contrast, ryanodine induced slow increases in [Ca2+]cyt that stabilized within 30 min; subsequent AVP-induced delta Ca2+ were attenuated by 50%. Ryanodine had no effect on either basal or stimulated [3H]InsP levels. Agents that elevate adenosine 3',5'-cyclic monophosphate (cAMP) such as caffeine, 8-bromo-cAMP, and forskolin inhibited AVP-evoked [3H]InsP formation. These observations provide further characterization of a communication pathway between the AVP-sensitive Ca2+ stores in the SR and the plasmalemmal enzyme system involved in the synthesis of inositol 1,4,5-trisphosphate. This pathway is manifested by an inverse relationship between the Ca2+ content of an AVP-sensitive, ryanodine-insensitive SR Ca2+ store and evoked [3H]InsP synthesis and may represent an important component in the tonic regulation of resting [Ca2+]cyt and vasoconstrictor- and hormone-evoked SR Ca2+ release.

Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2

1991 ◽  
Vol 11 (4) ◽  
pp. 2018-2025
Author(s):  
L Sultzman ◽  
C Ellis ◽  
L L Lin ◽  
T Pawson ◽  
J Knopf
Keyword(s):  
Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cell Surface Receptor ◽  
Platelet Derived Growth Factor ◽  
Surface Receptor ◽  
Phospholipase C Gamma

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.

scholarly journals Thyrotropin Stimulates the Generation of Inositol 1,4,5-Trisphosphate in Human Thyroid Cells

2006 ◽  
Vol 91 (3) ◽  
pp. 1099-1107 ◽  
Author(s):  
Jacqueline Van Sande ◽  
Didier Dequanter ◽  
Philippe Lothaire ◽  
Claude Massart ◽  
Jacques E. Dumont ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Cell Line ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Human Cells ◽  
Human Thyroid ◽  
Fresh Tissue ◽  
Thyroid Cells ◽  
Thyroid Stimulating Antibodies ◽  
Inositol 1,4,5 Trisphosphate

Abstract Context: Dual activation by TSH of the phospholipase C and cAMP cascades has been reported in human thyroid cells. In contrast, Singh et al. reported convincing data in FRTL-5 thyrocytes arguing against such an effect in this model. Their data in FRTL-5 cells indicated no increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to TSH. Therefore, the authors questioned results previously obtained on human cells by cruder methodology. Objective: We investigated the formation of inositol phosphates by HPLC techniques in human thyroid slices to separate the inositol phosphate isomers. Results: Ins(1,4,5)P3, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate were increased after TSH stimulation. The effect of TSH in human thyroid cells was reproduced by recombinant TSH and prevented by antibodies blocking the TSH receptor. Thyroid-stimulating antibodies at concentrations eliciting a cAMP response equivalent to TSH failed to stimulate inositol phosphate generation. Conclusions: TSH, but not thyroid-stimulating antibodies, activates both cAMP and the phospholipase C cascade in human thyroid as now demonstrated by an increase in Ins(1,4,5)P3 and its inositol phosphate metabolites. Therefore, this effect cannot be extrapolated to the FRTL-5 cell line. The apparent discrepancy may be due to a difference between species (human vs. rat) or to the loss of the fresh tissue properties in a cell line. The dual effect of TSH in human cells, through cAMP on secretion of thyroid hormones and through the diacylglycerol, Ins(1,4,5)P3 Ca2+ pathway on thyroid hormone synthesis, implies the possible separation of these effects in thyroid disease.

scholarly journals Convulxin-induced platelet aggregation is accompanied by a powerful activation of the phospholipase C pathway

1994 ◽  
Vol 298 (1) ◽  
pp. 87-91 ◽  
Author(s):  
A Faili ◽  
J Randon ◽  
I M Francischetti ◽  
B B Vargaftig ◽  
M Hatmi
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Platelet Activating Factor ◽  
Phosphatidyl Inositol ◽  
High Concentration ◽  
Adp Release ◽  
Pathway Formation ◽  
Phosphatidylinositol 4

Platelet aggregation and stimulation of phosphoinositide-specific phospholipase C (PLC) by thrombin and by convulxin (Cvx), a non-enzymic snake venom glycoprotein, were compared. Cvx-stimulated production of inositol phosphates by washed platelets was independent of the cyclo-oxygenase pathway, formation of platelet-activating factor and ADP release, but prostacyclin (prostaglandin I2), a stimulator of cyclic AMP formation, suppressed its effects on platelet and PLC activation. Kinetic analysis showed that inositol 1,4,5-trisphosphate formation reached its maximal value 15 s after platelet stimulation with Cvx and persisted for at least 5 min. Neomycin sulphate (10 mM), which complexes phosphatidylinositol 4-phosphate and phosphatidyl-inositol 4,5-bisphosphate, decreased the production of inositol phosphates, partially prevented platelet aggregation induced by a high concentration of Cvx (10 nM) and abolished both platelet aggregation and inositol phosphate formation induced by thrombin (2 units/ml) and by a stable prostaglandin H2 analogue, U46619 (1 microM). In contrast with neomycin sulphate, Na2SO4 had no significant effect against all agonists tested. It is concluded that platelet activation by Cvx is partially mediated by PLC and involves other mechanisms as well.

Characterization of phospholipase C activity at h5-HT 2C compared with h5-HT 2B receptors: influence of novel ligands upon membrane-bound levels of [ 3 H]phosphatidylinositols

2002 ◽  
Vol 365 (3) ◽  
pp. 242-252 ◽  
Author(s):  
Didier Cussac ◽  
Adrian Newman-Tancredi ◽  
Yann Quentric ◽  
Nathalie Carpentier ◽  
Guillaume Poissonnet ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Membrane Bound

scholarly journals Differences in the regulation of endothelin-1- and lysophosphatidic-acid-stimulated Ins(1,4,5)P3 formation in rat-1 fibroblasts

1991 ◽  
Vol 280 (3) ◽  
pp. 609-615 ◽  
Author(s):  
R Plevin ◽  
E E MacNulty ◽  
S Palmer ◽  
M J O Wakelam
Keyword(s):  
Phospholipase C ◽  
Lysophosphatidic Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Regulatory Protein ◽  
Permeabilized Cells ◽  
Endothelin 1 ◽  
Kinase C ◽  
Guanine Nucleotide Binding ◽  
Dose Dependent

Endothelin-1 (ET-1)- and lysophosphatidic acid (LPA)-stimulated PtdIns(4,5)P2 hydrolysis has been studied in Rat-1 fibroblasts. Although both agonists caused the dose-dependent accumulation of inositol phosphates, a number of differences were observed. LPA induced a transient increase in Ins(1,4,5)P3 mass which returned to basal levels within 90 s, whereas the response to ET-1 did not desensitize, with levels remaining at 3-4 times basal values for up to 15 min. Stimulated decreases in mass levels of PtdIns(4,5)P2 mirrored Ins(1,4,5)P3 formation for both agonists. Experiments with electropermeabilized cells demonstrated that the effects of both agonists are stimulated by a phospholipase C controlled by a guanine-nucleotide-binding regulatory protein; however, there are differences in the nature of these interactions. The inositol phosphate response to ET-1 is poorly potentiated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and markedly inhibited by guanosine 5′-[beta-thio]diphosphate (GDP[S]), whereas that to LPA is potentiated by GTP[S] but is relatively insensitive to GDP[S]. In addition, LPA decreased the lag time for the onset of GTP[S]-stimulated [3H]InsP3 accumulation, whereas ET-1 was without effect. Phorbol 12-myristate 13-acetate treatment of the cells inhibited LPA-stimulated, but not ET-1-stimulated, inositol phosphate formation in both intact and permeabilized cells, suggesting that the site of protein kinase C-mediated phosphorylation may be blocked in ET-1-stimulated Rat-1 cells. The results indicate that the receptor-G-protein-phospholipase C interaction for the two agonists may not conform to the same model.

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