[13] Inositol phospholipids and phosphates for investigation of intact cell phospholipase C substrates and products

1991 ◽  
pp. 149-158 ◽  
Author(s):  
Michael R. Hanley ◽  
David R. Poyner ◽  
Phillip T. Hawkins
Keyword(s):  
Phospholipase C ◽  
Intact Cell ◽  
Inositol Phospholipids

scholarly journals Physiological Requirement for Both SH2 Domains for Phospholipase C-γ1 Function and Interaction with Platelet-Derived Growth Factor Receptors

1999 ◽  
Vol 19 (7) ◽  
pp. 4961-4970 ◽  
Author(s):  
Qun-sheng Ji ◽  
Ansuman Chattopadhyay ◽  
Manuela Vecchi ◽  
Graham Carpenter
Keyword(s):  
Growth Factor ◽  
Phospholipase C ◽  
Intact Cell ◽  
Biological Response ◽  
Sh2 Domain ◽  
Site Directed Mutagenesis ◽  
Platelet Derived Growth Factor ◽  
Surface Plasmon Resonance Analysis ◽  
Sh2 Domains ◽  
The Individual

ABSTRACT Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-γ1 (PLC-γ1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) β receptor. To assess SH2 function in the context of the full-length PLC-γ1 molecule as well as within the intact cell, PLC-γ1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed in Plcg1−/− fibroblasts. Under equilibrium incubation conditions (4°C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-γ1 association with the activated PDGF receptor and PLC-γ1 tyrosine phosphorylation. When both SH2 domains in PLC-γ1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+mobilization and IP3 formation), both SH2 domains of PLC-γ1 are essential to growth factor responsiveness.

scholarly journals Quantification of inositol phospholipid breakdown in isolated rat hepatocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 865-872 ◽  
Author(s):  
C J Allan ◽  
J H Exton
Keyword(s):  
Phospholipase C ◽  
Phospholipase D ◽  
Inositol Phosphates ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Phospholipid Hydrolysis ◽  
Inositol Phospholipids ◽  
Inositol Phospholipid ◽  
Inositol Phospholipid Breakdown ◽  
Hydrolysis Of

The hydrolysis of inositol phospholipids induced by vasopressin in hepatocytes during 60 min was quantified chemically. There was a large release of myo-inositol which was abolished by Li+, indicating that it was derived from inositol phosphates and not from phospholipase D action on PtdIns. There was also a large release of inositol phosphates which was increased approx. 2-fold by Li+ at 30 min, but then remained constant, suggesting that inositol phospholipid breakdown declined substantially beyond this time. In cells prelabelled with myo-[3H]inositol and treated with Li+, [3H]PtdIns(4,5)P2 decreased maximally (50%) at 15 s and then recovered to a level at 5 min that was maintained at 25% below control for 40 min. [3H]PtdIns4P and [3H]PtdIns showed slower decreases to approx. 30% below control at 15 min, but with no further changes. Labelled Ins(1,4,5)P3 and Ins(1,3,4)P3 showed 2-4-fold increases within 30 s and then declined to values that were maintained at a constant level above the control, except for [3H]Ins(1,3,4)P3, which showed a second increase. [3H]Ins(1,4)P2 showed a very large increase over 10 min, whereas [3H]Ins4P and [3H]Ins1P showed little change before 6 and 15 min respectively. The total [3H]inositol phosphates showed little further increase after 20 min. These data are consistent with a rapid, but not sustained, hydrolysis of PtdIns-(4,5)P2, but not of PtdIns, by phospholipase C, but do not exclude PtdIns4P as a substrate. Phosphatidate was rapidly increased by vasopressin, whereas diacylglycerol was increased after a 1-2 min lag. Both were maintained at levels 2-3-fold above control for 60 min. The vasopressin-induced increase in inositol phosphates plus myo-inositol (approx. 120 nmol/100 mg) was greater than the increase in diacylglycerol plus phosphatidate (approx. 60 nmol/100 mg) between 10 and 40 min. This indicates that there was substantial further metabolism of these lipids. Addition of 75 mM ethanol resulted in rapid production of phosphatidylethanol in response to vasopressin and a 35% reduction in phosphatidate, but no decrease in diacylglycerol. In summary, the results indicate that inositol phospholipid hydrolysis by phospholipase C can account for most of the diacylglycerol and phosphatidate that accumulate during 60 min of vasopressin action, but that these phospholipids are probably not the major source of the phosphatidate that is formed during the first 2 min by phospholipase D, or of the diacylglycerol and phosphatidate that are formed beyond 30 min.

scholarly journals The affinities of human platelet and Acanthamoeba profilin isoforms for polyphosphoinositides account for their relative abilities to inhibit phospholipase C.

1990 ◽  
Vol 1 (12) ◽  
pp. 937-950 ◽  
Author(s):  
L M Machesky ◽  
P J Goldschmidt-Clermont ◽  
T D Pollard
Keyword(s):  
Phospholipase C ◽  
Human Platelet ◽  
Gel Filtration ◽  
Human Platelets ◽  
Lower Affinity ◽  
High Affinity ◽  
Inositol Phospholipids ◽  
Cytoskeletal Dynamics ◽  
Inositol Phospholipid ◽  
Phosphatidylinositol 4

In light of recent work implicating profilin from human platelets as a possible regulator of both cytoskeletal dynamics and inositol phospholipid-mediated signaling, we have further characterized the interaction of platelet profilin and the two isoforms of Acanthamoeba profilin with inositol phospholipids. Profilin from human platelets binds to phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) with relatively high affinity (Kd approximately 1 microM for PIP2 by equilibrium gel filtration), but interacts only weakly (if at all) with phosphatidylinositol (PI) or inositol trisphosphate IP3) in small-zone gel-filtration assays. The two isoforms of Acanthamoeba profilin both have a lower affinity for PIP2 than does human platelet profilin, but the more basic profilin isoform from Acanthamoeba (profilin-II) has a much higher (approximately 10-microM Kd) affinity than the acidic isoform (profilin-I, 100 to 500-microM Kd). None of the profilins bind to phosphatidylserine (PS) or phosphatidylcholine (PC) in small-zone gel-filtration experiments. The differences in affinity for PIP2 parallel the ability of these three profilins to inhibit PIP2 hydrolysis by soluble phospholipase C (PLC). The results show that the interaction of profilins with PIP2 is specific with respect to both the lipid and the proteins. In Acanthamoeba, the two isoforms of profilin may have specialized functions on the basis of their identical (approximately 10 microM) affinities for actin monomers and different affinities for PIP2.

scholarly journals Exogenous oxidants initiate hydrolysis of endothelial cell inositol phospholipids

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 491-499 ◽  
Author(s):  
DM Shasby ◽  
M Yorek ◽  
SS Shasby
Keyword(s):  
Fatty Acids ◽  
Endothelial Cells ◽  
Protein Kinase C ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Inflammatory Cells ◽  
Kinase C ◽  
Inositol Phospholipids ◽  
Hydrolysis Of

Oxidants released from inflammatory cells contribute to the pathogenesis of acute inflammatory edema in many models. Chemically produced oxidants can reversibly alter the barrier properties of cultured endothelial and epithelial monolayers. This report examines the effects of nonlytic doses of H2O2 on endothelial cell lipids. H2O2 oxidized omega-6 fatty acids in the endothelial cells and initiated hydrolysis of endothelial cell phospholipids. When endothelial cells were exposed to peroxidized linoleic acid, it caused lysis of the cells at doses 1,000-fold lower than effective doses of H2O2. The phospholipid hydrolysis was directed primarily at the inositol phospholipids and consisted of both A and C type phospholipase activity. The phospholipase A hydrolysis resulted in increases in endothelial cell free fatty acids and lysophosphatidylinositol. The phospholipase C hydrolysis resulted in increases in diglycerides, phosphatidic acid, and inositol polyphosphate levels. The phospholipase C hydrolysis of phosphatidylinositol is known to activate protein kinase C in most cells. Stimulation of protein kinase C with phorbol- 12,13-dibutyrate increased albumin flux across endothelial monolayers and altered endothelial cell shape, similar to effects of oxidants. These data are consistent with the hypothesis that oxidant-initiated hydrolysis of endothelial cell inositol phospholipids contributes to oxidant-mediated reversible changes in endothelial monolayer barrier function.

scholarly journals Evidence for a model of integrated inositol phospholipid pools implies an essential role for lipid transport in the maintenance of receptor-mediated phospholipase C activity in 1321N1 cells

1998 ◽  
Vol 330 (3) ◽  
pp. 1069-1077 ◽  
Author(s):  
H. Ian BATTY ◽  
A. Richard CURRIE ◽  
C. Peter DOWNES
Keyword(s):  
Phospholipase C ◽  
Time Course ◽  
Inositol Phosphates ◽  
Specific Radioactivity ◽  
Lipid Transport ◽  
Intact Cells ◽  
Whole Cells ◽  
Metabolic Compartmentation ◽  
Astrocytoma Cells ◽  
Inositol Phospholipids

The compartmentation of inositol phospholipids was examined by using a combination of radiolabelling approaches in intact and permeabilized 1321N1 astrocytoma cells. A ‘chase’ protocol was developed with whole cells in which phosphoinositide (PI) pools were labelled to steady state with [3H]inositol and the cellular [3H]inositol pool was then diluted selectively with non-radioactive inositol. In these cells muscarinic-receptor-stimulated phospholipase C (PLC) hydrolysed [3H]PI at approx. 1-2%/min. However, after the chase procedure the relative specific radioactivity of [3H]Ins(1,3,4)P3, a rapidly metabolized and sensitive marker of PLC activity, decreased only after more than 5 min and over a time course similar to that during which the labelling of each [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 declined by at least 50%. These results demonstrate a large receptor-responsive [3H]PI pool that is accessed by stimulated PLC without apparent metabolic compartmentation, despite its probable distribution between different membrane fractions. Support for this was obtained in intact cells by using an acute [3H]inositol labelling method in which increases in the specific radioactivity of [3H]inositol phosphates stimulated by carbachol occurred only in parallel with similar increases in the labelling of the bulk of cellular [3H]PI. In [3H]inositol-prelabelled cells permeabilized to deplete cytosolic proteins, carbachol and guanosine 5ʹ-[γ-thio]triphosphate stimulated the endogenous PLC to degrade only approx. 5% of [3H]PI. This was increased to approx. 30% in the presence of exogenous PtdIns transfer protein, which, at a concentration approx. 5-10% of that in 1321N1 cell cytosol, was sufficient to support PLC activity comparable with that observed in response to carbachol in whole cells. These and earlier results in 1321N1 cells suggest a model of integrated PI pools involving an obligatory role for lipid transport. Given the multifunctional capacity of PI in cellular signalling mechanisms, this model has important implications, particularly for the hypothesis that the ability of Li+ ions to influence these selectively might account for its therapeutic actions.

scholarly journals Collagen stimulates [3H]inositol trisphosphate formation in indomethacin-treated human platelets

1985 ◽  
Vol 226 (3) ◽  
pp. 831-837 ◽  
Author(s):  
S P Watson ◽  
B Reep ◽  
R T McConnell ◽  
E G Lapetina
Keyword(s):  
Phospholipase C ◽  
Platelet Activation ◽  
Inositol Trisphosphate ◽  
Atp Release ◽  
Human Platelets ◽  
Lag Phase ◽  
Inositol Phospholipids ◽  
Rapid Hydrolysis ◽  
Hydrolysis Of ◽  
Indomethacin Treatment

The present study investigates the pathway of metabolism of inositol phospholipids in human platelets exposed to collagen. Platelet activation by collagen was preceded by a lag phase usually lasting 10-20 s. Formation of [3H]inositol trisphosphate (IP3) was not observed during this period, but occurred in parallel with the onset of aggregation, release of ATP and phosphorylation of a 20 000 Da and a 40 000 Da protein. Indomethacin treatment partially inhibited all of these responses. Aggregation and ATP release, but not IP3 formation, were further inhibited in indomethacin-treated platelets loaded with the fluorescent Ca2+ indicator, quin2. Under these conditions there was no detectable mobilization of Ca2+. These results demonstrate that activation of platelets by collagen is associated with rapid hydrolysis of polyphosphoinositides by phospholipase C, thereby producing IP3. This observation is discussed in relation to IP3 as a possible Ca2+-mobilizing agent.

scholarly journals Evidence that the inositol phospholipids are necessary for exocytosis. Loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATP

1990 ◽  
Vol 268 (1) ◽  
pp. 15-25 ◽  
Author(s):  
D A Eberhard ◽  
C L Cooper ◽  
M G Low ◽  
R W Holz
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Slow Phase ◽  
Rapid Phase ◽  
Permeabilized Cells ◽  
Inositol Phospholipids ◽  
Dependent Component ◽  
Subsequent Activation ◽  
Adrenal Chromaffin ◽  
Tight Correlation

We directly manipulated the levels of PtdIns, PtdInsP and PtdInsP2 in digitonin-treated adrenal chromaffin cells with a bacterial phospholipase C (PLC) from Bacillus thuringiensis and by removal of ATP. The PtdIns-PLC acted intracellularly to cause a large decrease in [3H]inositol- or [32P]phosphate-labelled PtdIns, but did not directly hydrolyse PtdInsP or PtdInsP2. [3H]PtdInsP and [3H]PtdInsP2 levels declined markedly, probably because of the action of phosphatases in the absence of synthesis. Removal of ATP also caused marked decreases in [3H]PtdInsP and [3H]PtdInsP2. The decrease in polyphosphoinositide levels by PtdIns-PLC treatment or ATP removal was reflected by the inhibition of the production of inositol phosphates upon subsequent activation of the endogenous PLC by Ca2(+)-dependent catecholamine secretion from permeabilized cells was strongly inhibited by PtdIns-PLC treatment and by ATP removal. Ca2(+)-dependent secretion was similarly correlated with the sum of PtdInsP and PtdInsP2 when the level of these lipids was changed by either manipulation. PtdIns-PLC inhibited only the ATP-dependent component of secretion and did not affect ATP-dependent secretion. Both PtdIns-PLC and ATP removal inhibited the late slow phase of secretion, but had little effect on the initial rapid phase. Although we found a tight correlation between polyphosphoinositide levels and secretion, endogenous phospholipase C activity (stimulated by Ca2+, guanine nucleotides and related agents) was not correlated with secretion. Additional experiments indicated that neither the products of the PtdIns-PLC reaction (diacylglycerol and InsP1) nor the inability to generate products by subsequent activation of the endogenous PLC is likely to account for the inhibition of secretion. Incubation of permeabilized cells with neomycin in the absence of ATP maintained the level of polyphosphoinositides and more than doubled subsequent Ca2(+)-dependent secretion. The data suggest that: (1) Ca2(+)-dependent secretion has a requirement for the presence of inositol phospholipids; (2) the enhancement of secretion by ATP results in part from increased polyphosphoinositide levels; and (3) the role for inositol phospholipids in secretion revealed in these experiments is independent of their being substrates for the generation of diacylglycerol and InsP3.

scholarly journals Exogenous oxidants initiate hydrolysis of endothelial cell inositol phospholipids

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 491-499 ◽  
Author(s):  
DM Shasby ◽  
M Yorek ◽  
SS Shasby
Keyword(s):  
Fatty Acids ◽  
Endothelial Cells ◽  
Protein Kinase C ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Inflammatory Cells ◽  
Kinase C ◽  
Inositol Phospholipids ◽  
Hydrolysis Of

Abstract Oxidants released from inflammatory cells contribute to the pathogenesis of acute inflammatory edema in many models. Chemically produced oxidants can reversibly alter the barrier properties of cultured endothelial and epithelial monolayers. This report examines the effects of nonlytic doses of H2O2 on endothelial cell lipids. H2O2 oxidized omega-6 fatty acids in the endothelial cells and initiated hydrolysis of endothelial cell phospholipids. When endothelial cells were exposed to peroxidized linoleic acid, it caused lysis of the cells at doses 1,000-fold lower than effective doses of H2O2. The phospholipid hydrolysis was directed primarily at the inositol phospholipids and consisted of both A and C type phospholipase activity. The phospholipase A hydrolysis resulted in increases in endothelial cell free fatty acids and lysophosphatidylinositol. The phospholipase C hydrolysis resulted in increases in diglycerides, phosphatidic acid, and inositol polyphosphate levels. The phospholipase C hydrolysis of phosphatidylinositol is known to activate protein kinase C in most cells. Stimulation of protein kinase C with phorbol- 12,13-dibutyrate increased albumin flux across endothelial monolayers and altered endothelial cell shape, similar to effects of oxidants. These data are consistent with the hypothesis that oxidant-initiated hydrolysis of endothelial cell inositol phospholipids contributes to oxidant-mediated reversible changes in endothelial monolayer barrier function.

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